Hypoxia-induced nuclear translocation of β-catenin in the healing process of frostbite

Frostbite occurs when the skin is exposed to localized low temperatures. The main causes of frostbite are thought to be direct cell injury due to freezing of cells and tissue ischemia due to abnormal blood circulation. However, the molecular mechanism of frostbite has not been elucidated. This study aims to explain the molecular dynamics of frostbite using a mouse frostbite model and keratinocyte cell culture. Comprehensive gene expression analysis performed on mouse skin samples revealed that β-catenin signaling is activated by frostbite. Immunohistochemistry showed nuclear translocation of β-catenin in the skin of frostbite model mice that was not observed in mice subjected to a mechanical skin damage model induced by tape stripping. Tissue hypoxia, as detected by pimonidazole staining, coexisted with nuclear expression of β-catenin. In keratinocyte cell cultures, nuclear translocation of β-catenin was induced by hypoxia, but not by low temperature. Hypoxia induced epithelial-mesenchymal transition - an important biological event in the healing process of skin - and in vitro wound-healing activity, both of which were suppressed by β-catenin inhibition. Our results suggest that during frostbite, impaired blood flow causes hypoxia, which in turn activates β-catenin that promotes keratinocyte motility and tissue repair.     

Evidence for an early free radical-mediated reperfusion injury in frostbite

Frostbite is characterized by acute tissue injury induced by freezing and thawing. Initial complete ischemia is followed by reperfusion and later, tissue necrosis. These vascular events support the hypothesis that free radical-mediated reperfusion injury at thawing might contribute to tissue necrosis after frostbite in a manner similar to that seen after normothermic ischemia. To test this hypothesis, rabbit ears were frozen at -21 degrees C for 30, 60, 90, or 120 s and rewarmed at room temperature (22 degrees C). Rabbits were treated "blindly" with saline alone, highly purified, pharmaceutical grade superoxide dismutase (SOD), allopurinol, or deferoxamine. The area of ear necrosis was determined 3 weeks after frostbite by "blinded" morphometry. The administration of SOD at the time of thawing significantly improved viability in ears frozen for 60 and 90 s, but not in those frozen for 30 or 120 s. Deferoxamine also improved viability in ears frozen for 60 s. Allopurinol did not significantly affect ear survival. Electron micrographs showed the appearance of severe endothelial cell injury beginning during freezing and extending through early reperfusion. Later, neutrophil adhesion, erythrocyte aggregation, and microvascular stasis were seen. These findings suggest that free radical-mediated reperfusion injury has a role in frostbite, and quantitate the proportion of the injury that is due to this mechanism.     

A 3D thermal model to analyze the temperature changes of digits during cold stress and predict the danger of frostbite in human fingers

The existing computational models of frostbite injury are limited to one and two dimensional schemes. In this study, a coupled thermo-fluid model is applied to simulate a finger exposed to cold weather. The spatial variability of finger-tip temperature is compared to experimental ones to validate the model. A semi-realistic 3D model for tissue and blood vessels is used to analyze the transient heat transfer through the finger. The effect of heat conduction, metabolic heat generation, heat transport by blood perfusion, heat exchange between tissues and large vessels are considered in energy balance equations. The current model was then tested in different temperatures and air speeds to predict the danger of frostbite in humans for different gloves. Two prevalent gloves which are commonly used in cold climate are considered for investigation. The endurance time and the fraction of necrotic tissues are two main factors suggested for obtaining the response of digit tissues to different environmental conditions.     

Frostbite Protection in Mice Expressing an Antifreeze Glycoprotein

Ectotherms in northern latitudes are seasonally exposed to cold temperatures. To improve survival under cold stress, they use diverse mechanisms to increase temperature resistance and prevent tissue damage. The accumulation of anti-freeze proteins that improve cold hardiness occurs in diverse species including plants, arthropods, fish, and amphibians. We previously identified an Ixodes scapularis anti-freeze glycoprotein, named IAFGP, and demonstrated its cold protective function in the natural tick host and in a transgenic Drosophila model. Here we show, in a transgenic mouse model expressing an anti-freeze glycoprotein, that IAFGP protects mammalian cells and mice from cold shock and frostbite respectively. Transgenic skin samples showed reduced cell death upon cold storage ex vivo and transgenic mice demonstrated increased resistance to frostbite injury in vivo. IAFGP actively protects mammalian tissue from freezing, suggesting its application for the prevention of frostbite, and other diseases associated with cold exposure.     

缺氧条件下冻伤对大鼠微循环液血灌流量的影响

本文采用体重２００±２０ｇ健康雄性Ｗｉｓｔａｒ大鼠,随机分为平原冻伤（ＦＮ）组,急性缺氧冻伤（ＦＡＨ）组和缺氧习服缺氧冻伤（ＦＨＡＣ）组,实验观察了大鼠右后肢重度冻伤前后各组大鼠双后肢皮肤微循环灌流量的改变,结果表明,平原冻伤使大鼠以后肢微循环灌流量明显减少,提示局部重度冻伤对微循环的影响不只局限于冻区也涉及到对侧肢体,冻冻前ＦＡＨ组大鼠微循环灌流量已明显低于ＦＮ组,表明生缺氧时血容量进行代偿性的     

Radioactive albumin, red blood cells, and sodium as indicators of tissue damage after frostbite

Determination of tissue damage soon after frostbite would permit an evaluation of treatment. We reported a preliminary study using ¹³¹I-RISA to determine tissue loss soon after frostbite. We now present further studies with ¹³¹I-RISA, ⁵¹Cr-tagged red blood cells and ²⁴NaCl.     

Acute systemic changes in blood cells, proteins, coagulation, fibrinolysis, and platelet aggregation after frostbite injury in the rabbit

Acute systemic blood changes were measured in New Zealand white rabbits after severe and mild frostbite injury to the foot. There were observed after 72 hr, in the severely frostbitten rabbits, a decrease in erythrocytes, hematocrit, lymphocytes, and albumin, and an increase in total leukocytes, neutrophils, platelets, fibrinogen, and antithrombin III. Mildly frostbitten rabbits showed similar changes except for no changes in the platelets, albumin, and antithrombin III. In severely frostbitten rabbits, after 72 hr, the changes in the plasma coagulation tests were a prolonged partial thromboplastin time, an accelerated prothrombin time, and increased activities of Factors VII, IX, X, and XI. In mildly frostbitten rabbits there were a prolonged partial thromboplastin time and an increased activity of Factor VII. No changes in fibrinolysis were seen in either group of rabbits. Platelet aggregation, studied only in the severely frostbitten rabbits, showed a change only by an increase in the slope of the collagen-induced platelet aggregation. The blood changes observed in the rabbit model are different than those reported in human frostbite cases. No disseminated intravascular coagulation was apparent in the rabbit model after frostbite injury.     

An evaluation of the ability of the peripheral vasodilator buflomedil to improve vascular patency after acute frostbite

The extent of microvascular damage from frostbite can be accurately demonstrated by vascular microcorrosion casting techniques (P. S. Daum, W. D. Bowers, Jr., J. Tejada, and M. P. Hamlet, Cryobiology 24, 65-73, 1987). In the present investigation, the peripheral vasodilator buflomedil was evaluated for its ability to ameliorate microcirculatory damage from acute experimentally induced freeze injury. This drug has been reported to decrease tissue loss in human frostbite patients when given intravenously during thawing (J. Foray, P. E. Baisse, J. P. Mont, and Cl. Cahen, Sem. Hop. Paris 56, 490-497, 1980). In seven groups of anesthetized rats, left hindpaws were cooled to heat of fusion; cooling continued until the temperature in the footpads fell to -15 degrees C. Prior to cooling, group 1 received a tail vein injection of 1 ml saline/kg, while group 2 received 10 mg buflomedil/kg. Immediately following cooling, group 3 received an injection of 10 mg buflomedil/kg. Hindpaws were rapidly rewarmed in a 40 degree C bath. During rewarming, left hindpaws from group 4 were immersed in deionized water, from group 5 in 24 mg buflomedil in deionized water, from group 6 in 30% dimethyl sulfoxide (Me2SO), and from group 7 in 24 mg buflomedil in 30% Me2SO. Right hindpaws served as controls. Vascular microcorrosion casts were made from left and right hindpaws of all groups. There was no significant difference in mean cast weights when frozen hindpaws of the seven groups were compared, although treatment with buflomedil increased the mean cast weight of control hindpaws from groups 3 and 7. It therefore appears that, in this acute model for frostbite, buflomedil does not improve vascular patency.     

蝮蛇抗栓酶对冻伤家兔血凝系统某些指标的影响

本研究观察了家兔双后足重度冻伤后用蝮蛇抗栓酶（０．２５Ｕ／ｋｇ体重加入２０ｍｌ／ｋｇ体重生理盐水中,耳缘静脉点滴）进行治疗后其血凝系统某些指标的改变。结果表明,重度冻伤后家兔的出血时间及凝血时间明显缩短,血小板功能呈不同程度的改变,血浆纤维蛋白原含量增加,血液处于高凝状态;用蝮蛇抗栓酶治疗后,上述各项指标均有不同程度的改善,结果提示,蝮蛇抗栓酶可明显缓解冻伤所致的血凝增强的病变过程,这对重度冻伤的治疗将起到重要作用     

Morphologic characterization of acute injury to vascular endothelium of skin after frostbite

Cyclo-oxygenase inhibitors and free-radical scavengers protect the skin against necrosis induced by frostbite. However, the tissue component(s) that determine the evolution of skin necrosis and the mechanism of this pharmacologic protection are not precisely defined. We have studied freezing injury to rabbit ears by serial biopsies examined by light and electron microscopy. The morphologic evidence of skin injury due to freezing was localized exclusively in the endothelial cells, particularly in the arterioles. Within 1 hour, the entire microvasculature demonstrated endothelial damage. Intravascular platelet aggregation occurred just after thawing and closely paralleled the endothelial cell injury. Very few neutrophils were seen initially (at 10 minutes). By 1 hour, leukocyte aggregates were present, and they further increased at 6 hours. Swelling of the interstitium started 10 minutes after thawing, while extravasation of erythrocytes began to appear by 6 hours. Parenchymal elements of skin were relatively free of damage. In the ear cartilage, the chondrocytes showed evidence of damage immediately after freezing. The administration of superoxide dismutase (SOD) during thawing (reperfusion) did not qualitatively alter any of the initial morphologic changes induced by freezing. We conclude that the endothelial cell is the initial target of injury induced by freezing, an initial injury that is mediated by a non-free-radical-mediated mechanism. It is likely that this acute injury ultimately compromises blood flow and leads to skin necrosis.     

三种大鼠肝损伤模型中细胞角蛋白异常表达及其机制──免疫组化及免疫电镜研究

本研究应用ＡＢＣ免疫组化技术显示,奶油黄、液氮致局部冻伤及ＣＣｌ＿４所致的三种肝损伤中也有细胞角蛋白（ＣＫ）异常表达肝细胞。（１）在局部肝冻伤及奶油黄性肝损伤中表明不伴脂肪变性的肝细胞坏死不能直接引起肝细胞ＣＫ表达的改变;（２）在奶油黄性肝损伤中显示了卵圆细胞对肝细胞ＣＫ异常表达的诱导作用,表明层粘连蛋白（ＬＮ）可能是这种作用的媒介;（３）在ＣＣｌ＿４致慢性肝损伤中表明肝细胞ＣＫ异常表达和ＬＮ异常沉积无论在位相上还是在时相上都一致,提出肝小叶结构破坏可能也是通过ＬＮ异常沉积而影响肝细胞的ＣＫ表达;（４）应用电镜及免疫电镜技术表明ＣＣｌ＿４性肝损伤中肝细胞中间丝细胞骨架结构的改变伴随着ＣＫ１９阳性抗原决定簇的出现;（５）设计了一种局部肝冻伤模型,利用这种模型表明,ＣＫ１９阳性肝细胞在肝小叶结构完整性遭到破坏且伴纤维组织增生时出现,随小叶结构的恢复而消失。这是对关于肝细胞ＣＫ异常表达是肝小叶结构修复过程中局部肝细胞的修复性反应这一假说的有力支持。讨论了这种改变的意义。     

Explicit formula of finite difference method to estimate human peripheral tissue temperatures during exposure to severe cold stress

During cold exposure, peripheral tissues undergo vasoconstriction to minimize heat loss to preserve the maintenance of a normal core temperature. However, vasoconstricted tissues exposed to cold temperatures are susceptible to freezing and frostbite-related tissue damage. Therefore, it is imperative to establish a mathematical model for the estimation of tissue necrosis due to cold stress. To this end, an explicit formula of finite difference method has been used to obtain the solution of Pennes' bio-heat equation with appropriate boundary conditions to estimate the temperature profiles of dermal and subdermal layers when exposed to severe cold temperatures. The discrete values of nodal temperature were calculated at the interfaces of skin and subcutaneous tissues with respect to the atmospheric temperatures of 25 °C, 20 °C, 15 °C, 5 °C, −5 °C and −10 °C. The results obtained were used to identify the scenarios under which various degrees of frostbite occur on the surface of skin as well as the dermal and subdermal areas. The explicit formula of finite difference method proposed in this model provides more accurate predictions as compared to other numerical methods. This model of predicting tissue temperatures provides researchers with a more accurate prediction of peripheral tissue temperature and, hence, the susceptibility to frostbite during severe cold exposure.     

Paradox: increased blood perfusion to the face <Emphasis Type="BoldItalic">enhances</Emphasis> protection against frostbite while it <Emphasis Type="BoldItalic">lowers</Emphasis> wind chill equivalent temperatures

A model of facial heat exchange in cold and windy environments is presented. The tissue is depicted as a hollow cylinder and the model includes heat conduction and heat transport by blood circulation from the warmer core. A steady-state solution facilitating the estimation of wind chill equivalent temperature (WCET) as a function of the effective wind velocity, air temperature and blood perfusion rate was obtained. The results quantify and demonstrate the elevation of skin temperatures caused by increased flow of warmer blood from the inner core to the face. Elevated facial temperatures, while enhancing protection against frostbite and other cold-related injuries, also increase heat loss to the colder environment. Paradoxically, such elevated facial temperatures cause WCETs, as estimated by the prevailing definition, to attain lower rather than higher values, indicating, in fact, increased risk of frostbite. The results of this study should be useful in understanding and quantifying the effects of blood perfusion in protection against cold-related injuries. They should also be considered in the re-evaluation and re-formulation of the concept of wind chill, which has been a useful cold weather indicator for decades.     

Rapid analysis of fatty acid-binding proteins with immunosensors and immunotests for early monitoring of tissue injury

Fatty acid-binding protein (FABP) holds promise for early detection of tissue injury. This small protein (15 kD) appears earlier in the blood than large proteins after cell damage. Combined its characteristics of high concentration tissue contents and low normal plasma values provide the possibility of a rapid rise above the respective reference values, and thus an early indication of the appearance of tissue injury. A general review was presented on the current status of different types of FABP for the detection of tissue injury in patients with myocardial injury, brain injury and also in athletes or horses with skeletal muscle injury.

To take full advantage of the characteristics of the early marker FABP, rapid analysis is a crucial parameter. In this review, an overview of the development of immunoassay for the quantification of FABP in buffer, plasma or whole blood was outlined. The characteristics of different FABP immunosensors and immunotests were described. The feasibility of these immunoassays to be used in routine clinical practice and in emergency case was also discussed.

Nowadays, the improved automated immunoassays (e.g. a microparticle-enhanced turbidimetric immunoassay), less time-consuming bedside immunosensors and immunotests (e.g. a one-step FABP lateral flow immunotest), are the main advance technology in point-of-care testing. With these point-of-care tests, the application of FABP as an early tissue injury marker has a great potential for many clinical purposes.     

Face cooling by cold wind in walking subjects

The effects of low to moderate wind speeds on face temperature, thermal and pain sensations while subjects walked on a treadmill during cold exposure were studied in eight healthy men. The purpose of the study was to evaluate further the risk of frostbite at different activity levels. The walking speed was 2.8 km h(-1) and two inclination levels were used, 0 degrees and 6 degrees. The subjects were exposed to -10 degrees C and 0, 1 or 5 m s(-1) wind for 60 min dressed in cold-protective clothing with only the face unprotected. Results from previous experiments with the same subjects standing for 30 min were included in the analysis of the data. Each individual was exposed to all combinations of air velocity and activity level. The exposure to -10 degrees C and the highest wind speed used would carry no risk of frostbite according to the wind chill index. Cold lowered the skin temperature of the face significantly and wind further increased skin cooling. The activity level did not affect forehead and cheek temperatures, but the average nose skin temperature was higher and pain sensations were reduced at a higher work rate. The predicted risk of frostbite in the nose, based on average responses, would thus be less at a higher work rate. However, the results indicate that exercise does not necessarily protect all individuals from frostbite at moderate air speeds, since the nose skin temperature of 25% of the subjects dropped to 0 degrees C at 5 m s(-1) during both standing and walking. Thus the potential individual risk of frostbite in the nose is similar during light exercise and standing. Moreover, the risk of frostbite seems to be underestimated by the wind chill index under the conditions tested in this study.     

Coagulation and coagulation signalling in fibrosis

Following tissue injury, a complex and coordinated wound healing response comprising coagulation, inflammation, fibroproliferation and tissue remodelling has evolved to nullify the impact of the original insult and reinstate the normal physiological function of the affected organ. Tissue fibrosis is thought to result from a dysregulated wound healing response as a result of continual local injury or impaired control mechanisms. Although the initial insult is highly variable for different organs, in most cases, uncontrolled or sustained activation of mesenchymal cells into highly synthetic myofibroblasts leads to the excessive deposition of extracellular matrix proteins and eventually loss of tissue function. Coagulation was originally thought to be an acute and transient response to tissue injury, responsible primarily for promoting haemostasis by initiating the formation of fibrin plugs to enmesh activated platelets within the walls of damaged blood vessels. However, the last 20 years has seen a major re-evaluation of the role of the coagulation cascade following tissue injury and there is now mounting evidence that coagulation plays a critical role in orchestrating subsequent inflammatory and fibroproliferative responses during normal wound healing, as well as in a range of pathological contexts across all major organ systems. This review summarises our current understanding of the role of coagulation and coagulation initiated signalling in the response to tissue injury, as well as the contribution of uncontrolled coagulation to fibrosis of the lung, liver, kidney and heart. This article is part of a Special Issue entitled: Fibrosis: Translation of basic research to human disease.     

冻伤对大鼠血凝系统某些因素的影响

本研究观察了大鼠双后足局部冻伤后其血凝系统某些因素的改变。结果表明,冻伤后大鼠的出血时间及凝血时间明显缩短,血小板数增加,血小板聚集率增高,血浆血栓素Ａ２、纤维蛋白原及钙离子浓度均明显增加,且上述指标的改变与冻伤程度有密切关系,冻伤愈重,改变愈明显,恢复亦愈慢。结果提示,冻伤可使血凝系统发生改变,血液处于高凝状态,这对浆伤后发生的血液循环障碍最终导致冻伤组织坏死的病理过程起重要作用。     

Chromosomale Mosaike in der klinischen Zytogenetik

In contrast to molecular genetics, in cytogenetic analyses single cells are analyzed individually. This affords an opportunity to detect cells with chromosomal mosaicism. This article provides an overview on problems arising in the detection and interpretation of chromosomal mosaicism in cytogenetic diagnostics. A particular challenge in the diagnostics is to distinguish between clinically relevant genuine mosaicism on the one hand and cultured artifacts, pseudomosaics, age effects, maternal contamination and chimerism on the other. The probability of overlooking mosaicism in cytogenetic routine diagnostics is very high, as on average only 15 of the 10¹² cells in the body are examined and usually only a single tissue is analyzed. However, some cytogenetic mosaics are typical for certain syndromes, such as Pallister-Killian syndrome, cat eye syndrome or Ullrich-Turner syndrome and others are characteristic for certain disorders including some hematological malignancies.     

Haemodynamic changes in patients with whiplash injury measured by transcranial Doppler sonography (TCD)

The daily increasing number of cervical whiplash injuries presents ever-greater requirement for vertebrobasilar diagnostics. A cervical spine injury, which is quite frequent injury, may occur during a fall, or industrial, traffic, sport or war injury. Transcranial Doppler (TCD) sonography with Transcan 3-D EME device and 2 MHz probe was used for the assessment of vertebrobasilar circulation in patients with a whiplash injury of the cervical spine, that occurred mostly in car accident. This study includes 47 patients with clinically verified cervical spine trauma with x-ray evidence of no bone lesion. The patients were examined by TCD within a month, and then six months following the accident. The obtained values were compared to normal blood flow velocities and correlated with the severity of clinical picture. During the first month after the injury, statistically significant disturbances in the vertebrobasilar circulation were recorded, such as the increase in mean blood flow velocities in AVL (68%), AVR (62%) and BA (51%) (mostly as spasam). Six months later, normal findings were obtained in about 50% of the vessels, whereas in rest of the patients vasospasam persisted in one, two or all examined blood vessels. TCD of the vertebrobasilar circulation was found to be a very useful method in the diagnostics and follow-up of patients with a whiplash injury.