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Cold is supposed to be associated with alterations in blood coagulation and a pronounced risk for thrombosis. We studied the effect of clinically encountered systemic hypothermia on microvascular thrombosis in vivo and in vitro. Ferric chloride-induced microvascular thrombus formation was analyzed in cremaster muscle preparations from hypothermic mice. Additionally, flow cytometry and Western blot analysis was used to evaluate the effect of hypothermia on platelet activation. To test whether preceding hypothermia predisposes for enhanced thrombosis, experiments were repeated after hypothermia and rewarming to 37 degrees C. Control animals revealed complete occlusion of arterioles and venules after 742 +/- 150 and 824 +/- 172 s, respectively. Systemic hypothermia of 34 degrees C accelerated thrombus formation in arterioles and venules (279 +/- 120 and 376 +/- 121 s; P0.05 vs. 37 degrees C). This was further pronounced after cooling to 31 degrees C (163 +/- 57 and 281 +/- 71 s; P0.05 vs. 37 degrees C). Magnitude of thrombin receptor activating peptide (TRAP)-induced platelet activation increased with decreasing temperatures, as shown by 1.8- and 3.0-fold increases in mean fluorescence after PAC-1 binding to glycoprotein (GP)IIb-IIIa and 1.6- and 2.9-fold increases of fibrinogen binding on incubation at 34 degrees C and 31 degrees C. Additionally, tyrosine-specific protein phosphorylation in platelets was increased at hypothermic temperatures. In rewarmed animals, kinetics of thrombus formation were comparable to those in normothermic controls. Concomitantly, spontaneous and TRAP-enhanced GPIIb-IIIa activation did not differ between rewarmed platelets and those maintained continuously at 37 degrees C. Moderate systemic hypothermia accelerates microvascular thrombosis, which might be mediated by increased GPIIb-IIIa activation on platelets but does not cause predisposition with increased risk for microvascular thrombus formation after rewarming.

Objective— By heme degradation, heme oxygenase-1 (HO-1) provides endogenous carbon monoxide and bilirubin, both of which play major roles in vascular biology. The current study aimed to examine whether induction of HO-1 and its byproducts modulate the process of microvascular thrombus formation in vivo. Methods and Results— In individual microvessels of mouse cremaster muscle preparations, ferric chloride-induced thrombus formation was analyzed using intravital fluorescence microscopy. When mice were pretreated with an intraperitoneal injection of hemin, a HO-1 inducer, immunohistochemistry and Western blot protein analysis of cremaster muscle tissue displayed a marked induction of HO-1. In these animals, superfusion with ferric chloride solution induced arteriolar and venular thrombus formation, which, however, was significantly delayed when compared with thrombus formation in animals without HO-1 induction. The delay in thrombus formation in hemin-treated mice was completely blunted by tin protoporphyrin-IX, a HO-1 inhibitor, but not by copper protoporphyrin-IX, which does not inhibit the enzyme. Coadministration of the vitamin E analogue Trolox in HO-1–blocked animals almost completely restored the delay in thrombus formation, implying that, besides CO, the antioxidant HO pathway metabolite bilirubin mainly contributes to the antithrombotic property of HO-1. This was further supported by the fact that bilirubin was found as effective as hemin in delay of ferric chloride-induced thrombus formation. Animals with HO-1 induction revealed reduced P-selectin protein expression in cremaster muscle tissue, which most probably presented the molecular basis for delayed thrombus growth. Conclusion— Local induction of HO-1 activity may be of preventive and therapeutic value for clinical disorders with increased risk of thrombotic events.

A powerful replica molding methodology to transfer on-demand functional topographies to the surface of bacterial cellulose nanofiber textures is presented. With this method, termed guided assembly-based biolithography (GAB), a surface-structured polydimethylsiloxane (PDMS) mold is introduced at the gas-liquid interface of an Acetobacter xylinum culture. Upon bacterial fermentation, the generated bacterial cellulose nanofibers are assembled in a three-dimensional network reproducing the geometric shape imposed by the mold. Additionally, GAB yields directional alignment of individual nanofibers and memory of the transferred geometrical features upon dehydration and rehydration of the substrates. Scanning electron and atomic force microscopy are used to establish the good fidelity of this facile and affordable method. Interaction of surface-structured bacterial cellulose substrates with human fibroblasts and keratinocytes illustrates the efficient control of cellular activities which are fundamental in skin wound healing and tissue regeneration. The deployment of surface-structured bacterial cellulose substrates in model animals as skin wound dressing or body implant further proves the high durability and low inflammatory response to the material over a period of 21 days, demonstrating beneficial effects of surface structure on skin regeneration.

Palmar lip injuries of the proximal interphalangeal joint with dorsal fracture-dislocation are difficult to treat and often require major reconstruction. A systematic review was performed and yielded 177 articles. Thirteen articles on hemi-hamate autograft were included in full-text analysis. Results of 71 cases were summarized. Mean follow-up was 36 months and mean proximal interphalangeal joint range of motion was 77°. Overall complication rate was around 35%. Up to 50% of the patients showed radiographic signs of osteoarthritis. However, few of those patients complained about pain or impaired finger motion. Based on this systematic analysis and review, hemi-hamate autograft can be considered reliable for the reconstruction of acute and chronic proximal interphalangeal joint fracture-dislocations with joint involvement >50%, but longer-term follow-up studies are required to evaluate its outcome, especially regarding the rate of osteoarthritis. Level of Evidence: II

A 47-year-old HIV-positive woman presented with fever and a painful swollen right forearm. Clinical presentation and MRI were suggestive for a necrotising fasciitis. Surgical exploration revealed small transparent cystic bodies resembling white caviar, which were identified by their typical morphological features as larval stages (cysticerci) of Taenia crassiceps. Molecular methods, using sequence analysis of the small subunit rRNA gene, definitively confirmed T crassiceps. T crassiceps (Cestodea: Taeniidae) is a tapeworm found in the intestines of red foxes and dogs in the Northern Hemisphere. Human infections are rare and appear to depend on the host's immunocompetence. The eight published cases could not clarify the mode of infection but discuss ingestion of teniid eggs or penetration through a cutaneous wound. The optimal treatment remains unclear. We describe a detailed and successful treatment strategy including extensive surgical interventions, prolonged anthelmintic and antiretroviral treatment.

Background: The radial forearm flap (RFF) is known for its excellent and reliable results regarding defect coverage though donor site morbidity represents a persisting problem. Integra is widely used in reconstructive surgery. This study aims to assess long-term outcomes regarding functionality and cosmesis after donor site coverage with respect to esthetic appearance, scar quality, and wrist function as well as ability to work after donor site coverage with Integra and split-thickness skin. Methods: The prospective follow-up exam, after a mean time of 23.8months, enrolled 13 patients. Identification of patients' comorbidities and evaluation of the subjective esthetic outcome, sensibility, cold intolerance, and scar instability was assessed by a questionnaire. In clinics, designed Pinch test assessed scar tissue mobility over the flexor tendons. The Vancouver Scar Scale (VSS) was calculated; wrist function and grip strength were determined. Results: The satisfaction score for the esthetic appearance of the donor site was in average 3.42 ± 0.44. Two patients experienced an unstable scar and cold intolerance. The VSS resulted in a value of 4.2 representing a good result. The pinch test revealed an average scar mobility of 8mm without any tendon adhesions. Active range of motion of the wrist was equal to the non-operated site as was grip strength. The pinch test showed a significant negative correlation with the VSS making it a reliable tool to measure scar quality. Conclusion: Long-term results show an esthetic and functional successful defect coverage of the RFF donor site by the use of Integra and split-thickness skin. Level of Evidence: Level IV, therapeutic study.

Surplus harvested skin grafts are routinely stored at 4 to 6°C in saline for several days in plastic surgery. The purpose of this study was to evaluate the influence of storage on human skin graft performance in an in vivo intravital microscopic setting after transplantation. Freshly harvested human full-thickness skin grafts and split-thickness skin grafts (STSGs) after storage of 0, 3, or 7 days in moist saline at 4 to 6°C were transplanted into the modified dorsal skinfold chamber, and intravital microscopy was performed to evaluate vessel morphology and angiogenic change of the wound bed. The chamber tissue was harvested 10 days after transplantation for evaluation of tissue integrity and inflammation (hematoxylin and eosin) as well as for immunohistochemistry (human CD31, murine CD31, Ki67, Tdt-mediated dUTP-biotin nick-end labelling). Intravital microscopy results showed no differences in the host angiogenic response between fresh and preserved grafts. However, STSGs and full-thickness skin grafts exhibited a trend toward different timing and strength in capillary widening and capillary bud formation. Preservation had no influence on graft quality before transplantation, but fresh STSGs showed better quality 10 days after transplantation than 7-day preserved grafts. Proliferation and apoptosis as well as host capillary in-growth and graft capillary degeneration were equal in all groups. These results indicate that cells may activate protective mechanisms under cold conditions, allowing them to maintain function and morphology. However, rewarming may disclose underlying tissue damage. These findings could be translated to a new approach for the design of full-thickness skin substitutes.

Background Buttock augmentation is gaining increasing popularity in aesthetic surgery. The relatively high incidence of complications after silicone implant placement lead to the increased use of lipofilling techniques, yielding variable results with respect to graft take rate and long-term stability. Platelet-rich plasma (PRP) has been shown to have beneficial effects on wound healing and angiogenesis in the past. Therefore, we aimed at investigating the long-term results and patient satisfaction after PRP-enriched lipofilling for buttock augmentation. Methods Twenty-four bilateral gluteal augmentations with PRP-enriched autologous fat were performed. Additionally, contour shaping was achieved by liposuction of the adjacent zones. Post-operative results and complications were recorded, and satisfaction with buttock shape was estimated by a patient questionnaire. Results Mean follow-up time was 44 months, and mean amount of transferred fat was 481 cc for both sides. No seroma or hematoma formation, infection or liponecrosis were reported during the post-operative follow-up. Subjective patient satisfaction in general increased from preoperatively to 3 months postoperatively and declined only slightly in the long-term course. Satisfaction levels in general were specific for each patient. Patient recovery was quick, and the majority of patients returned to work within 10 days after surgery. Conclusions PRP-enhanced lipofilling of the buttocks proved to be a safe procedure including a low complication rate and consistent results. However, subjective patient expectations have to be taken into account when choosing the indication. Further large volume studies are needed to elucidate the potential and benefit of PRP in this context. Level of Evidence: Level IV, therapeutic study.

Storage of split-thickness skin grafts (STSGs) represents a standard procedure in burn surgery. The purpose of this study was to evaluate clinical routine of STSG preservation. Further, we aimed at investigating the effect of storage on tissue integrity and cell viability, proliferation, apoptosis and vascularization. A survey was performed among plastic surgery centres in Europe. STSGs were harvested from healthy patients and analysed by histology (HE, Verhoeff's, Masson's Trichrome, Sirius Red) and immunohistochemistry (Ki67, TUNEL, CD31). Cell viability was determined by MTT assay. The survey revealed that storage of STSGs up to 10 days is common practice. STSGs mostly were stored at 4 °C in saline-moisturized gauze. Histology showed no disintegration of the tissue or a decrease of collagen and elastic fibres. Proliferation increased to 22.5% of total cells after 3 days. On day 7 of STSG storage apoptotic cells amounted for 25% of total cells. Cell viability decreased by 50% after day 3 of storage. Even though reportedly superior methods for skin grafts storage exist, most study participants applied the simplest method of storage. Our data underscore this practice. However, a reduced cell viability after 3 days of storage may have an influence on graft healing.

Today split- and full-thickness skin grafting represent standard procedures to close skin defects, i.e. in burn injuries and other acute or chronic wounds (leg ulcers, traumatic skin losses, defects after tumour resections, etc.). Nevertheless, the exact process of skin graft revascularisation remains elusive. A better understanding of this process becomes crucial for the increasingly popular research field of tissue engineering. A variety of skin substitutes have been developed in the last decade and become commercially available. However, in particular, full-thickness substitutes still lack complete successful incorporation into the human body. A better knowledge of the mechanisms of the taking of skin grafts and their acquisition blood supply would aid substantially in their fabrication. By this the composition of dermo-epidermal substitutes could be tailored according to the physiologic process of revascularisation.

This study aims to quantify by intravital microscopy and histological wound scoring the effect of radial pressure wave treatment (RPWT) on murine incisional wound healing. The dorsal skinfold chamber in mice was used for intravital microscopy, whereby an incisional wound was created within the chamber. RPWT to the wound was carried out using a ballistic pressure wave source (EMS Swiss DolorClast). Animals received a dose of 500 pulses at an energy flux rate of 0.1mJ/mm(2) and a frequency of 3Hz at day 1, 3, 5, 7, 9, and 11 post wounding. RPW treated and untreated ApoE depleted mice (ApoE(-/-)) were compared to normal healing wild type animals (WT). The microcirculation of the wound was analyzed quantitatively in vivo using epi-illumination intravital fluorescence microscopy. Tissue samples were examined ex vivo for wound scoring and immunohistochemistry. Upon RPWT total wound score in ApoE(-/-) mice was increased by 13% (not significant) on day 3, by 37% on day 7 (P

This study aims to elucidate the effect of erythropoietin (EPO) on the microcirculation during wound healing in mice genetically depleted of apolipoprotein E (ApoE(-/-)). The skinfold chamber in mice was used for intravital microscopy, whereby an incisional wound was created within the chamber. Animals received Recormon(®) 1000 U kg(-1) body weight (BW) intra-peritoneally (i.p.) at day 1, 3, 5, 7, 9 and 11 post-wounding at a concentration of 100 Uml(-1) (n=42). Normal healing and vehicle-treated wild type animals (WT) served as controls. The microcirculation of the wound was analysed quantitatively in vivo using epi-illumination intravital fluorescence microscopy. Microtomography (micro-CT) analysis of casted wound microvessels was performed allowing three-dimensional (3D) histomorphometric analysis. Tissue samples were examined ex vivo for wound scoring and for expression analysis of EPO-Receptor (Epo-R) and endothelial nitric oxide synthase (eNOS). Upon EPO treatment, the total wound score in ApoE(-/-) mice was increased by 23% on day 3, by 26% on day 7 and by 18% on day 13 when compared to untreated ApoE(-/-) mice (all P

The purpose of this article is to prospectively evaluate early postoperative morphologic and functional changes after deep flexor tendon repair in zone II using ultrasound and to correlate findings from ultrasound with the clinical outcome.Ten patients (mean age, 34 years; range, 19-55 years) with 11 injured deep flexor tendons of the hand underwent surgical tendon repair. Postoperative tendon morphology was assessed with gray-scale and power Doppler ultrasound over a period of 3 months. Tendon excursion over the proximal interphalangeal joint was assessed by sonographic scar tracking. Correlation of ultrasound findings with clinical outcome was performed.Almost all repaired tendons exhibited a spindlelike shape after 1 week, of which 50% developed a normal shape after 12 weeks. A persisting spindlelike shape over 3 months was associated with a significantly increased tendon excursion (p0.05) and a trend toward better active motion of the fingers (p = 0.056). Tendons with increased power Doppler signal showed a significantly better tendon excursion and active motion after 12 weeks (all p0.05). Tendon excursion measurements obtained by scar tracking showed excellent correlation (r = 0.84; p0.05) with total active finger motion.Preliminary data of this study indicate a better clinical outcome if a sutured tendon maintains a spindlelike shape and increased power Doppler signal. This might indicate a predominantly intrinsic healing pattern with reduced adhesion formation. Ultrasound morphology, power Doppler signal, and tendon excursion may be helpful tools to rate tendon healing and to establish individually modified rehabilitation protocols.

Summary We report a rare case of human Taenia crassiceps infection in a 47-year-old female patient with untreated acquired immunodeficiency syndrome and chronic hepatitis C infection. Little experience exists regarding the appropriate treatment of this infection. Usually, a combination of anthelmintic drugs is applied. Whether surgical measures are indicated have not been clarified. In our patient, initial surgery showed an abscess and fluid collection with numerous transparent cysts localised in the subcutaneous tissue of the cubital fossa. Parasitological and pathological examinations identified these structures as larvae of the cestode T. crassiceps . After treatment with anthelmintic medications, the patient was discharged in good condition. However, the patient presented with the clinical symptoms of an acute fasciitis of the right upper extremity 7 days later. The deteriorating general condition entailing a pre-septical state demanded emergency debridement and fasciectomy of the right arm. After the surgery, the patient recovered fully. Conclusions Surgical treatment appears to be an important measure to reduce the tissue parasite load in patients with severe immunodeficiency. It also has to be questioned whether the bioavailability and the penetration of the drugs commonly administered is sufficiently high to treat such a fulminant infection alone.

Introduction Skin substitutes are increasingly produced in tissue engineering, but still the understanding of the physiological skin revascularization process is lacking. To study in vivo conditions we recently introduced a mouse model, in which we already characterized the angiogenic changes within the wound bed and the skin graft. The aim of this study was to identify the origination of the vasculature during skin graft revascularization in vivo and to track vessel development over time. Methods We created a crossover wild-type/GFP skin transplantation model, in which each animal carried the graft from the other strain. The preparation of the modified dorsal skin fold chamber including cross-over skin grafting was performed in male C57BL/6J wild-type mice (n = 5) and C57BL/6-Tg(ACTB-EGFP)1Osb/J mice (n = 5). Intravital microscopy in 12 areas of wild-type and GFP skin grafts was performed daily over a time period of 10 days. Results Graft reperfusion started after 48–72 h. After reperfusion GFP-positive structures from the wound bed were visible in the graft capillaries with the highest density in the center of the graft. Overall, we observed a replacement of existing graft capillaries with vessels from the wound bed in 68% of the vessels. Of note, vessel replacement occurred in almost 100% of graft vessels in the periphery. Additionally, vessels within the graft showed a temporary angiogenic response between days 3–8, which originated predominantly from the autochthonous graft vasculature, but also contained already grown-in vessels from the wound bed. Conclusions These in vivo data indicate an early in-growth of angiogenic bed vessels into the existing vascular channels of the graft and subsequent centripetal replacement. Additionally we observed a temporary angiogenic response of the autochthonous capillaries of the skin graft with contribution from bed vessels. These findings further support the theory that sprouting angiogenesis from the wound bed in combination with the replacement of existing graft vessels are the key factors in skin graft taking. Thus, manufacturing of skin substitutes should be aimed at providing pre-formed vascular channels within the construct to improve vascularization.

Background: This study aims to quantify by intravital microscopy the microhemodynamic response after extracorporeal shock wave application (ESWA) to the physiologic microcirculation of the mouse dorsal skinfold chamber. Materials and Methods: ESWA was carried out using an electrohydraulic shock wave source. Two different shock wave doses of 500 and 1000 pulses at an energy flux rate of 0.08 mJ/mm2 and a frequency of 4 Hz were compared with sham-operated animals. Microcirculatory analyses were performed at baseline (BL) and during a 3 d observation period after ESWA. The expression of caspase-3 (casp-3), proliferating cell nuclear antibody (PCNA), von Willebrand factor (vWF), and endothelial nitric oxide synthase (eNOS) were analyzed semiquantitatively by immunohistochemistry. Results: ESWA provoked a significant and persistent increase of functional capillary density (FCD) throughout the observation period, reaching a maximum (140% ± 5% of BL, P < 0.05 versus sham) after 1 d when animals were treated with 1000 pulses. ESWA induced a slight increase of leukocyte rolling (not, vert, similar2- to not, vert, similar3.5-fold, P < 0.05) and leukocyte adherence (not, vert, similar1.5- to not, vert, similar2-fold, P < 0.05) to the endothelial lining of postcapillary venules. One day following ESWA, we observed enhanced expression of casp-3 (not, vert, similar3- to not, vert, similar4-fold), PCNA (not, vert, similar9- to not, vert, similar14-fold), vWF (not, vert, similar11- to not, vert, similar14-fold), and eNOS (not, vert, similar3-fold), all P < 0.05. Conclusion: This study shows that ESWA provokes a favorable persistent increase of patent capillaries, however accompanied by a transient and slight inflammatory response but also by dose-dependant apoptotic cell death. Our data suggest that ESWA might represent a noninvasive biomechanical tool to treat critically perfused and endangered tissues, but certainly warrants further investigation.

In the present study, we aimed to evaluate whether erythropoietin (EPO) treatment may exert nonhematopoietic endothelial protection against TNF-[alpha]-induced microvascular inflammation and to determine the involvement of the nitric oxide (NO)-producing enzyme isoforms endothelial NO synthase (eNOS) and inducible NO synthase (iNOS). Murine dorsal skinfold chambers of wild-type (WT) animals were topically stimulated with TNF-[alpha] after pretreatment with epoetin beta (1,000 IU/kg body weight i.p.) or physiological saline. Leukocyte behavior, microvascular perfusion, and apoptosis were assessed by in vivo fluorescence microscopy. To study the involvement of NO, we compared eNOS-deficient (eNOS-/-) and iNOS-deficient (iNOS-/-) mice with WT animals. TNF-[alpha]-associated leukocyte activation, perfusion failure, and apoptosis were substantially attenuated in EPO-pretreated WT mice, which was accompanied by marked reduction of perivascular infiltration with F4/80-stained macrophages. The anti-inflammatory protective effects of EPO were abolished in eNOS-/-, but not in iNOS-/- mice, both with unaffected intercellular adhesion molecule 1 expression. However, the antiapoptotic effect of EPO was maintained in both eNOS-/- and iNOS-/- mice, indicating that this mechanism might rather be independent of NO. We conclude that EPO treatment elicits protection against TNF-[alpha]-induced microcirculatory dysfunction, depending on NO derived from endothelial cells, but not on the inducible isoform.

Combined pancreas-kidney transplantation is one therapeutic option of Diabetes mellitus. In the early postoperative period pancreatic venous thrombosis is one of the major complications leading to allograft dysfunction and graft loss. One risk factor is the ischemia-reperfusion(I/R)- injury. Additionally, immunosuppressive drugs, such as the calcineurine inhibitor tacrolimus, have been implicated in the development of microvascular thrombosis. Several studies report a beneficial effect of antithrombin III (ATIII)- and N-acetylcysteine-(NAC)-substitution on the ischemia-reperfusion injury. Aim of this study was to analyse the therapeutic effect of ATIII and NAC on microvascular thrombus formation in post-ischemic tissue under immunosuppressive therapy with tacrolimus in vivo.

BACKGROUND: Models of skin graft revascularization are based mostly on histologic evaluations but lack the possibility of analyzing the vascular biology in vivo. The aim of the present study was therefore to develop an animal model that allows continuous monitoring of the microcirculation during skin graft healing. METHODS: Skin and subcutaneous tissue were removed from the back of dorsal skinfold chamber preparations in mice, leaving one layer of striated muscle and subcutaneous tissue as a wound bed (n = 5). A corresponding full-thickness skin graft was harvested from the groin and sutured into the defect in the back of the chamber. To study graft healing, repetitive intravital microscopy was performed during the first 10 days after engraftment. RESULTS: Capillary widening in the wound bed appeared at day 1 after grafting and increased until day 4. Capillary buds and sprouts first appeared at day 2. Blood filling of autochthonous graft capillaries occurred at day 3, resulting in almost complete restoration of the original skin microcirculation on day 5. This was achieved by interconnections between the microvasculature of the wound bed and the skin graft through a temporary angiogenic response. In principle, angiogenic blood vessel growth originated in the wound bed and was directed toward the graft. CONCLUSIONS: This new model allows for repetitive analysis of the microcirculation during skin graft healing. It provides ideal in vivo conditions to further delineate the exact mechanisms of blood vessel interconnection during the complex process of angiogenesis, and may also allow study of the vascularization of tissue-engineered skin substitutes.

The aim of this study was to investigate the effect of human recombinant erythropoietin (EPO) on the microcirculation and oxygenation of critically ischemic tissue and to elucidate the role of endothelial NO synthase in EPO-mediated tissue protection. Island flaps were dissected from the back skin of anesthetized male Syrian golden hamsters including a critically ischemic, hypoxic area that was perfused via a collateralized vasculature. Before ischemia, animals received an injection of epoetin beta at a dose of 5,000 U/kg body weight with (n = 7) or without (n = 7) blocking NO synthase by 30 mg/kg body weight L-NAME (Nomega-nitro-L-arginine methyl ester hydrochloride). Saline-treated animals served as control (n = 7). Ischemic tissue damage was characterized by severe hypoperfusion and inflammation, hypoxia, and accumulation of apoptotic cell nuclei after 5 h of collateralization. Erythropoietin pretreatment increased arteriolar and venular blood flow by 33% and 37%, respectively (P < 0.05), and attenuated leukocytic inflammation by approximately 75% (P < 0.05). Furthermore, partial tissue oxygen tension in the ischemic tissue increased from 8.2 to 15.8 mmHg (P < 0.05), which was paralleled by a 21% increased density of patent capillaries (P < 0.05) and a 50% reduced apoptotic cell count (P < 0.05). The improved microcirculation and oxygenation were associated with a 2.2-fold (P < 0.05) increase of endothelial NO synthase protein expression. Of interest, L-NAME completely abolished all the beneficial effects of EPO pretreatment. Our study demonstrates that, in critically ischemic and hypoxic collateralized tissue, EPO pretreatment improves tissue perfusion and oxygenation in vivo. This effect may be attributed to NO-dependent vasodilative effects and anti-inflammatory actions on the altered vascular endothelium.

Models of skin graft revascularization are mostly based on histological evaluations, but lack the possibility to analyze the vascular biology in vivo. The aim of the present study was therefore to develop an animal model which allows continuous monitoring of the microcirculation during skin graft healing. Skin and subcutaneous tissue were removed from the back of dorsal skinfold chamber preparations in mice, leaving one layer of striated muscle and subcutaneous tissue as wound bed. A respective full-thickness skin graft was harvested from the groin and sutured into the defect in the back of the chamber. To study graft healing, repetitive intravital microscopy was performed during the first 10 days after engraftment. Capillary widening in the wound bed appeared at day 1 after grafting and increased until day 4. Capillary buds and sprouts firstly appeared at day 2. Blood filling of autochthonous graft capillaries occurred at day 3, resulting in almost complete restoration of the original skin microcirculation at day 5. This was achieved by interconnections between the microvasculature of the wound bed and the skin graft through a temporary angiogenic response. In principle, angiogenic blood vessel growth originated in the wound bed and was directed towards the graft. This new model allows for repetitive analysis of the microcirculation during skin graft healing. It provides ideal in vivo conditions to further delineate the exact mechanisms of blood vessel interconnection during the complex process of angiogenesis, and may also allow to study the vascularization of tissueengineered skin substitutes.

Thrombotic microangiopathy (TMA) is a fatal complication that occurs after combined pancreaskidney transplantation leading to allograft dysfunction and graft loss. Immunosuppressive drugs, such as calcineurin inhibitors, have been implicated in the development of nonimmune TMA. Tacrolimus has been shown to be associated with decreased NO-production, thus contributing to thrombus formation. ADMA acts as an endogenous NO-synthase inhibitor and is considered to be a marker of endothelial dysfunction. Aim of this study was to analyse the influence of different immunosuppressants on microvascular thrombus formation in vivo and to further examine the underlying endothelial function. Using the skin fold chamber in C57BL/6J mice, microvascular thrombus formation was induced photochemically and was quantitatively analyzed by intravital fluorescence microscopy. Mice were treated with tacrolimus (TAC: 10 mg/kg/d; n = 7), cyclosporine (CYA: 5 mg/kg/d) or sirolimus (RAPA: 1,5 mg/kg/d ip) on 3 consecutive days. Control mice received NaCl 0,9 % (10 ml/kg/d ip; n = 5). Drug plasma levels were examined to ensure therapeutic doses. Additionally, the systemic circulation of sP- and sE-selectin as well as ADMA plasma levels were measured by ELISA. Application of immunosuppressants produced clinically relevant plasma levels (TAC: 8 ± 2 ng/ml, CYA: 214 ± 16 ng/ml; RAPA: 9 ± 1 ng/ml). Microvascular thrombus formation was significantly accelerated in mice receiving TAC compared to control mice (arteriolar and venular occlusion: 251 ± 101s and 179 ± 27 s vs. control 818 ± 221 s and 749 ± 231 s; p < 0,05). Application of CYA significantly increased thrombus formation only in venules (276 ± 49s; p < 0,05 vs. control), whereas RAPA had no significant effect on thrombosis induction. Plasma concentrations of sP-Selectin and sE-selectin were slightly reduced after TAC and CYA application. ADMA-levels however were significantly increased in mice receiving TAC (1,28 ± 0,16 µmol/l vs. control: 0,74 ± 0,13 µmol/l; p < 0,05). The present results show that clinically relevant plasma levels of cyclosporine enhanced thrombus formation only in venules whereas tacrolimus significantly accelerates thrombus formation in arterioles and venules. This effect could be explained by increased ADMA-levels in plasma resulting in impaired NO-bioavailability and increased thrombogenicity, thus presenting a new molecular mechanism in the development of transplant-thrombosis.

In the era of intravascular approaches for regenerative cell therapy, the underlying mechanisms of stem cell migration to non-marrow tissue have not been clarified. We hypothesized that next to a local inflammatory response implying adhesion molecule expression, endothelial nitric oxide synthase (eNOS)-dependent signaling is required for stromal- cell-derived factor-1 alpha (SDF-1alpha)-induced adhesion of c-kit+ cells to the vascular endothelium. SDF-1alpha/tumor necrosis factor-alpha (TNF-alpha)-induced c-kit+-cell shape change and migration capacity was studied in vitro using immunohistochemistry and Boyden chamber assays. In vivo interaction of c-kit+ cells from bone marrow with the endothelium in response to SDF-1alpha/TNF-alpha stimulation was visualized in the cremaster muscle microcirculation of wild-type (WT) and eNOS (-/-) mice using intravital fluorescence microscopy. In addition, NOS activity was inhibited with N-nitro-L-arginine-methylester-hydrochloride in WT mice. To reveal c-kit+-specific adhesion behavior, endogenous leukocytes (EL) and c-kit+ cells from peripheral blood served as control. Moreover, intercellular adhesion molecule-1 (ICAM-1) and CXCR4 were blocked systemically to determine their role in inflammation-related c-kit+-cell adhesion. In vitro, SDF-1alpha enhanced c-kit+-cell migration. In vivo, SDF-1alpha alone triggered endothelial rolling-not firm adherence-of c-kit+ cells in WT mice. While TNF-alpha alone had little effect on adhesion of c-kit+ cells, it induced maximum endothelial EL adherence. However, after combined treatment with SDF-1alpha+TNF-alpha, endothelial adhesion of c-kit+ cells increased independent of their origin, while EL adhesion was not further incremented. Systemic treatment with anti-ICAM-1 and anti-CXCR4-monoclonal antibody completely abolished endothelial c-kit+-cell adhesion. In N-nitro-L-arginine-methylester-hydrochloride-treated WT mice as well as in eNOS (-/-) mice, firm endothelial adhesion of c-kit+ cells was entirely abrogated, while EL adhesion was significantly increased. The chemokine SDF-1alpha mediates firm adhesion c-kit+ cells only in the presence of TNF-alpha stimulation via an ICAM-1- and CXCR4-dependent mechanism. The presence of eNOS appears to be a crucial and specific factor for firm c-kit+-cell adhesion to the vascular endothelium.

Erythropoietin-treatment is generally assumed to be associated with an increased risk for thrombosis due to an elevated hematocrit. However, previous in vitro and in vivo studies of our group revealed a reduction of platelet reactivity and no significant effect on microvascular thrombus formation during treatment with darbepoetin-alpha (DPO), an erythropoietin-derivative with a 3-fold longer half life. Aim of the study was therefore to investigate the effect of DPO application on endothelial function and to further characterize the role of eNOS in this context.

Cigarette smoking increases the risk for myocardial diseases and is associated with a higher incidence of postoperative complications. Previous studies showed an increased leukocyte rolling and adhesion following nicotine application. Next to this the use of estrogen containing oral contraceptives is linked to a higher risk for deep vein thrombosis. However, clinical studies investigating the influence of transdermal nicotine application on cardiovascular risk factors could not show a negative effect of nicotine. Therefore transdermal nicotine replacement is regarded as a safe aid to smoking cessation. It is still not clear whether nicotine or other substances in cigarette smoke are responsible for the prothrombotic risk associated with cigarette smoking. Aim of this study was therefore to investigate the influence of nicotine on microvascular thrombus formation in vivo and to further characterize the underlying mechanisms. In microvessels of dorsal skin fold chambers of mice, light-dye-induced thrombus formation was analyzed using intravital fluorescence microscopy. Male and female C57BL/ 6J mice received nicotine chronically via the drinking water (100 µg/ml) for 8 weeks. Control animals of both gender received normal drinking water. Subsequently plasma levels of cotinine, a stable metabolite of nicotine, were determined. To study the influence of nicotine on endothelial cell function immunohistochemical tissue sections for the vascular localization of P-selectin, PAI-1, PAF-R and ICAM-1 were performed. Furthermore the plasma concentrations of soluble P-selectin, E-selectin, ICAM-1 and VCAM-1 were quantified by ELISA. To characterize the influence of nicotine on platelet activation, flowcytometric analyses of the expression of P-selectin, GPIIb/IIIa and CD107a on murine platelets were carried out. Chronic nicotine exposition caused cotinine plasma levels similar to those seen in chronic smokers (> 100 ng/ml vs. controls: < 10 ng/ml). In male animals no significant differences in microvascular thrombus formation were detected following chronic nicotine application, when compared to controls (complete arteriolar and venular occlusion: 640 ± 96 s and 427 ± 51 s vs. control: 577 ± 78 s and 531 ± 102 s). Likewise in female animals no significant differences were found, following long-term nicotine application (complete arteriolar and venular occlusion: 715 ± 130 s and 493 ± 79 s vs. control: 866 ± 24 s and 481 ± 70 s). In line with this, flow cytometric analyses did not show significant effects of nicotine on P-selectin, GPIIb/IIIa and CD107a expression on resting platelets or upon stimulation with thrombin (20 U/ml). In contrast to this chronic nicotine application caused a marked reduction of vascular expression of P-selectin, PAF-R and PAI-1 in male mice, while in female mice only PAI-1 was found significantly reduced. Additionally chronic nicotine treatment lead to a significant reduction of sP-selectin and sE-selectin circulation in male mice. The present study shows no significant influence of chronic nicotine treatment on microvascular thrombus formation and platelet adhesion molecule expression despite elevated cotinine levels in mice of both gender. However, chronic nicotine treatment resulted in a significant reduction of endothelial adhesion molecule expression in male mice, while there was only a slight effect in female mice.

Hypothermia during sepsis significantly impairs patient outcome in clinical practice. Severe sepsis is closely linked to activation of the coagulation system, resulting in microthrombosis and subsequent organ failure. Herein, we studied whether systemic hypothermia accelerates microvascular thrombus formation during lipopolysacharide (LPS)-induced endotoxemia in vivo, and characterized the low temperature-induced endothelial and platelet dysfunctions.Ferric-chloride induced microvascular thrombus formation was analyzed in cremaster muscles of hypothermic endotoxemic mice. Flow cytometry, ELISA and immunohistochemistry were used to evaluate the effect of hypothermia on endothelial and platelet function.Control animals at 37 degrees C revealed complete occlusion of arterioles and venules after 759 +/- 115 s and 744 +/- 112 s, respectively. Endotoxemia significantly (p0.05) accelerated arteriolar and venular occlusion in 37 degrees C animals (255 +/- 35 s and 238 +/- 58 s, respectively). This was associated with an increase of circulating endothelial activation markers, agonist-induced platelet reactivity, and endothelial P-selectin and plasminogen activator inhibitor (PAI)-1 expression. Systemic hypothermia of 34 degrees C revealed a slight but not significant reduction of arteriolar (224 +/- 35 s) and venular (183 +/- 35 s) occlusion times. Cooling of the endotoxemic animals to 31 degrees C core body temperature, however, resulted in a further acceleration of microvascular thrombus formation, in particular in arterioles (127 +/- 29 s, p0.05 versus 37 degrees C endotoxemic animals). Of interest, hypothermia did not affect endothelial receptor expression and platelet reactivity, but increased endothelial PAI-1 expression and, in particular, soluble PAI-1 antigen (sPAI-Ag) plasma levels.LPS-induced endotoxemia accelerates microvascular thrombus formation in vivo, most probably by generalized endothelial activation and increased platelet reactivity. Systemic hypothermia further enhances microthrombosis in endotoxemia. This effect is associated with increased endothelial PAI-1 expression and sPAI-Ag in the systemic circulation rather than further endothelial activation or modulation of platelet reactivity.

In der vorliegenden Arbeit konnte unter Nutzung zweier unterschiedlicher Versuchsmodelle eindrucksvoll gezeigt werden, dass bei septischen Bedingungen der hochdosierte Einsatz einer alleinigen AT-Gabe (250 U/kg) der klassischerweise mit Heparin durchgefuhrten Thromboseprophylaxe uberlegen ist. Allerdings kam es unter diesem Regime zu einer Verlangerung der Blutungszeit. Erstmalig konnte somit in zwei gut etablierten experimentellen Sepsismodellen die antithrombotische Potenz von Antithrombin zur Verhinderung der mikrovaskularen Thrombosierung charakterisiert werden. Damit konnte in der adjuvanten Therapie septischer Patienten auf die Heparingabe verzichtet werden zugunsten von AT und dessen anti-inflammatorischen Potenz.

Die vorliegende Studie zeigt bei masig erhohten, Darbepoetin-α induzierten Hamatokritwerten von ca. 55 % bereits eine reduzierte Aktivierbarkeit von Thrombozyten im Sinne einer Zelldysfunktion, welche sich jedoch nicht in einer Blutungsneigung mit Verlangerung der mikrovaskularen Thrombusbildung ausert. Zur endgultigen Klarung des Effektes von Erythropoietin bzw. seiner Derivate auf die Hamostase bedarf es weiterfuhrender Analysen endothelialer und plasmatischer Faktoren.

Diabetic macro- and microangiopathy are associated with a high risk of vascular complications. The diabetic patient exhibits a pathological coagulation state, with an increased synthesis of coagulation factors and plasminogen activator inhibitor 1 (PAI-1) as well as an enhanced aggregation of platelets. Previous studies have shown that C-peptide can reduce leucocyte-endothelial cell interaction and improve microvascular blood flow in patients with type 1 diabetes. In the present study, we examined in vivo whether C-peptide is able to reduce platelet activation and through that microvascular thrombus formation.In the microvessels of cremaster muscle preparations taken from normal and diabetic mice, ferric chloride-induced thrombus formation was analysed using intravital fluorescence microscopy.I.V. administration of C-peptide in high dose (70 nmol/kg), but not in low dose (7 nmol/kg), caused a significant delay in arteriolar and venular thrombus growth in normal and diabetic mice. This effect was repressed by cremaster muscle superfusion with insulin (100 microU/ml) in diabetic animals, but particularly in normal animals. In parallel, immunohistochemistry demonstrated a higher number of PAI-1-expressing vessels in cremaster muscle tissue from control animals and from animals treated with C-peptide and insulin compared with tissue from animals with C-peptide treatment application alone.We conclude that C-peptide possesses antithrombotic actions in vivo. A causal role of PAI-1 in this scenario needs to be further addressed. However, the reversal of C-peptide action by insulin may invalidate the use of this peptide as a treatment option to improve rheology and microcirculation in diabetic patients.

Strategies for myocardial regeneration focus on intravascular administration or mobilisation of stem cells (SC). SDF-1 is discussed as homing factor for SC to injured myocardium. We investigated the role of SDF-1 for mediating SC homing using different in vitro and in vivo approaches in the mouse. In hearts of C57/BL6J mice the LAD coronary artery was ligated (n=4). After 24 h SDF-1 cardiac mRNA expression was measured by RT-PCR. Chemoattractant activity of SDF-1 (100–500 ng/ml) for murine c-kit+ bone marrow SC was studied using Boyden chamber assays (0.2 × 106 SC in upper chamber) (n=3). The microvascular bed of right cremaster muscles was topically exposed to SDF-1 (200 ng/ml) and studied by intravital fluorescence microscopy upon intra-arterial injection of green-fluorescently labelled SC (2 × 106 c-kit+) (n=5). SC rolling was recorded during first pass, while firm adherence of SC to the venular endothelium was assessed after 1 h. Values were compared to those in controls without SDF-1-exposure. SDF-1 mRNA expression was significantly increased 24 h after myocardial infarction. In vitro, SC migrated against an SDF-1 concentration gradient in a dose-dependent fashion (20%–75%). In vivo, the fraction of rolling SC increased significantly upon SDF-1 exposure (14 ± 2% vs. control: 6 ± 1% p

Diabetic macro- and microangiopathy is associated with a high risk of vascular complications and in consequence a high morbidity and mortality rate. The diabetic patient exhibits a pathological coagulation state, which is represented by both an increased synthesis of factor VII, thrombin, tissue factor and plasminogen activator inhibitor 1 (PAI-1) as well as by platelet aggregation enhanced due to elevated GP IIb/IIIa expression and altered calcium homeostasis. Former studies showed improved microvascular blood flow of skin and muscle after C-peptide administration in type 1 diabetic patients. Additionally, positive effects of C-peptide application on leukocyte-endothelial-interaction with increased production of nitric oxide and decreased expression of endothelial P-selectin and ICAM-1 have been reported. The aim of this study was therefore to examine whether the administration of C-peptide has anti-thrombotic effects in vivo. In microvessels of mouse cremaster muscle preparations of diabetic and non-diabetic mice, ferric chloride-induced thrombus formation was analyzed using intravital fluorescence microscopy. C-peptide was applied systemically in low (7 nmol/kg) and high (70 nmol/kg) dose, while control groups received equivalent amounts of heat-inactivated C-peptide. Diabetes was induced by application of streptozotocin (40 mg/kg) on 5 consecutive days. The diabetic metabolic state was confirmed by repeated positive testing for hypergly-cemia and glucosuria. Additionally, flow cytometric analysis of human platelet P-selectin and GP IIb/IIIa expression and immunohistochemistry of endothelial PAI-1 expression within the cremaster muscle tissue were conducted. Administration of C-peptide in high, but not in low dose, caused a significant delay in venular and arteriolar thrombus growth in diabetic mice (complete occlusion: 1171 ± 233 s and 1317 ± 218 s vs. control: 496 ± 50 s and 488 ± 50 s; p

A randomized controlled study (KyberSept-Trial [1]) in patients with severe sepsis showed that anti-thrombin-III significantly reduced lethality in a subgroup of septic patients not receiving concomitant heparin for thrombosis prophylaxis. Therefore, the present study investigated the antithrombotic property of ATIII in an animal study as potential monotherapy in order to omit concomitant heparin therapy. In a light/dye injury model of microvascular thrombosis at the ear of the mouse, we investigated a total of 30 homozygous (SKH1-hr) hairless mice of either sex (10–12 weeks old with a body weight (bw) of 22–40 g) under ip anaesthesia with ketamine/xylazine (90/25 mg/kg bw). Intravital fluorescence microscopy allowed quantitative analysis of the kinetics of thrombus formation in arterioles and venules. Prior to thrombus induction all animals received a single intravenous bolus of hirudin (1 mg/kg bw) or ATIII in dosages of 25, 50, 100 or 250 U/kg bw. Control animals received equivalent volumes of physiologic saline (n=5 animals per group). In saline-treated controls, complete thrombotic occlusion could be induced in 100% of all arterioles (9/9) and venules (40/40) studied. Hirudin prevented thrombotic vessel occlusion in 56% of the arterioles (5/9) and 39% of the venules (9/23). ATIII-250 was found more effective than hirudin, as thrombus formation was banned in all arterioles (13/13) and venules (24/24) (p

Thromboembolism is still a major problem in almost all fields of clinical medicine. The inducible enzyme heme oxygenase 1 (HO-1) provides endogenous iron, vasodilative carbon monoxide (CO) and anti-oxidant biliverdin by decomposition of heme. The aim of this study was to examine whether the expression of HO-1 increases the anti-thrombotic properties of the endothelium and subsequently modulates the process of microvascular thrombus formation in vivo. In micro-vessels of mouse cremaster muscle preparations ferric chloride-induced thrombus formation was analyzed using intravital fluorescence microscopy. When mice were pretreated with an intraperi-toneal injection of hemin, a HO-1 inducer, Western blot protein analysis of cremaster muscle tissue revealed a marked induction of HO-1. Immunohistochemistry displayed a preferential expression of HO-1 in the vascular endothelium of arterioles and venules. In these animals, superfusion with ferric chloride caused arteriolar and venular thrombus growth, which however was significantly delayed (complete occlusion: 670 ± 96 s and 647 ± 129 s) when compared with thrombus formation in control animals (complete occlusion: 143 ± 18 s and 130 ± 25 s). The delay in thrombus formation in hemin-treated mice was abolished by tin protoporphyrin-IX, a HO-1 inhibitor, but not by copper protoporphyrin-IX, which does not inhibit the enzyme. Application of the vitamin E analogue Trolox in HO-1-blocked animals almost completely restored the delay in thrombus formation, implying that - beside CO - the anti-oxidant biliverdin mainly contributes to the anti-thrombotic effect of HO-1. Animals with HO-1 induction exhibited a reduced P-selectin protein expression in cremaster muscle tissue, most probably presenting the molecular basis for delayed thrombus growth. Local induction of HO-1 activity may therefore be of preventive and therapeutic value for clinical disorders with an increased risk of thrombotic events.

As a reaction to reported adverse outcomes after lidocaine infiltration in tumescent liposuction, prilocaine has gained increasing popularity. Previous studies investigating large-volume liposuction procedures found maximum prilocaine levels and methemoglobinemia up to 12 h postoperatively, suggesting that liposuction should be performed as a hospital procedure only. The aim of this study was to determine prilocaine plasma levels and methemoglobinemia in patients after low- to average-volume liposuction for the purpose of defining the required postoperative surveillance period.In 25 patients undergoing liposuction involving less than 2,000 ml prilocaine levels and methemoglobinemia were measured over 4 h postoperatively. Liposuction was conducted after the tumescent technique using a 0.05% hypotonic prilocaine solution with epinephrine.The average prilocaine dose was 6.8 + 0.8 mg/kg, with a maximum dose of 15 mg/kg. The peak prilocaine plasma level of 0.34 mug/ml occurred 3 h after the infiltration. The mean methemoglobinemia at this time point was 0.65%. Only one patient demonstrated a slightly elevated methemoglobin level of 1.4%, but lacked any clinical signs of methemoglobinemia. The prilocaine recovery in the aspirate averaged 36 +/- 4%, indicating that a large amount is removed by suctioning.The patients did not experience high plasma levels of prilocaine or methemoglobinemia undergoing liposuction involving less than 2,000 ml using a 0.05% hypotonic prilocaine solution. The authors therefore conclude that this procedure can be performed safely with a monitoring period of 12 h.

SummaryEbselen, a seleno-organic compound showing glutathione peroxidase-like activity, has potent anti-inflammatory and anti-oxidant effects. Since selenium deficiency is thought to be associated with an increased incidence of vascular thrombosis, we studied the effect of ebselen on blood cell aggregate formation and vessel occlusion in vivo. In individual microvessels of rat cremaster muscle preparations, photochemically induced thrombus formation was analyzed in detail using intravital fluorescence microscopy. In ebselen-pretreated animals (30mg/kg ip), venular thrombus formation was significantly delayed (50% vessel occlusion: 535±34s; initial stasis: 872±82s; complete occlusion: 908±87s) as compared to vehicle-treated controls (416±42; 612±49; 647±51). Moreover, ebselen significantly prolonged the kinetics of arteriolar thrombus formation and even completely prevented blood cell aggregate and thrombus formation in 88.9% of all arterioles studied (p

Ebselen, a seleno-organic compound showing glutathione peroxidase-like activity, has potent anti-inflammatory and anti-oxidant effects. Since selenium deficiency is thought to be associated with an increased incidence of vascular thrombosis, we studied the effect of ebselen on blood cell aggregate formation and vessel occlusion in vivo. In individual microvessels of rat cremaster muscle preparations photochemically induced thrombus formation was analyzed in detail using intravital fluorescence microscopy. In ebselen-pretreated animals (30 mg/kg ip), venular thrombus formation was significantly delayed (50% vessel occlusion: 535 ± 34s; initial stasis: 872 ± 82s; complete occlusion: 908 ± 87s) as compared to vehicle-treated controls (416 ± 42s; 612 ± 49s; 647 ± 51s). Moreover, ebselen significantly prolonged the kinetics of arteriolar thrombus formation and even completely prevented blood cell aggregate and thrombus formation in 88.9% of all arterioles studied (p < 0.05 vs controls: 37.5%). As assessed by flow cytometry, oxidant stress-induced upregulation of CD62P on isolated platelets was found dose-dependently inhibited by increasing concentrations of ebselen (10 – 100 μM), suggesting that the antithrombotic effect of ebselen is achieved by attenuation of CD62P-dependent platelet/leukocyte aggregation. Thus, ebselen represents preventive and therapeutic value for disorders with increased risk for oxidant stress-associated thrombotic events.

Nicole Lindenblatt,1 Karem El-Rabadi,2 Thomas H Helbich,2 Heinrich Czembirek,3 Maria Deutinger,4 Heike Benditte-Klepetko5 1Division of Plastic and Hand Surgery, Department of Surgery, University Hospital Zurich, Zurich, Switzerland; 2Department of Biomedical Imaging and Image-guided Therapy, Division of Molecular and Gender Imaging, Medical University of Vienna - General Hospital Vienna, 3Department of Radiology, Hospital Wiener Privatklinik, 4Department of Plastic and Reconstructive Surgery, Hospital Rudolfstiftung, Vienna, Austria; 5Department of Plastic and Reconstructive Surgery, Erasmus Medical Center, Rotterdam, the Netherlands Background: Silicone gel breast implants may silently rupture without detection. This has been the main reason for magnetic resonance imaging (MRI) of the augmented or reconstructed breast. The aim of the present study was to investigate the accuracy of MRI for implant rupture. Methods: Fifty consecutive patients with 85 silicone gel implants were included in the study. The mean age of the patients was 51 (range 21–72) years, with a mean duration of implantation of 3.8 (range 1–28) years. All patients underwent clinical examination and breast MRI. Intraoperative implant rupture was diagnosed by the operating surgeon. Results: Nineteen of the 50 patients suffered from clinical symptoms. An implant rupture was diagnosed by MRI in 22 of 85 implants (26%). In seven of 17 removed implants (41%), the intraoperative diagnosis corresponded with the positive MRI result. However, only 57% of these patients were symptomatic. Ultrasound imaging of the harvested implants showed signs of interrupted inner layers of the implant despite integrity of the outer shell. By microsurgical separation of the different layers of the implant shell, we were able to reproduce this phenomenon and to produce signs of implant rupture on MRI. Conclusion: Our results show that rupture of only the inner layers of the implant shell with integrity of the outer shell leads to a misdiagnosis on MRI. Correlation with clinical symptoms and the specific wishes of the patient should guide the indication for implant removal. Keywords: breast implant, rupture, silicone gel, magnetic resonance imaging, linguine, keyhole

Thrombotic microangiopathy (TMA) is a fatal complication that occurs after combined pancreaskidney transplantation leading to allograft dysfunction and graft loss. Immunosuppressive drugs, such as calcineurin inhibitors, have been implicated in the development of nonimmune TMA. Tacrolimus has been shown to be associated with decreased NO-production, thus contributing to thrombus formation. ADMA acts as an endogenous NO-synthase inhibitor and is considered to be a marker of endothelial dysfunction. Aim of this study was to analyse the influence of different immunosuppressants on microvascular thrombus formation in vivo and to further examine the underlying endothelial function. Using the skin fold chamber in C57BL/6J mice, microvascular thrombus formation was induced photochemically and was quantitatively analyzed by intravital fluorescence microscopy. Mice were treated with tacrolimus (TAC: 10 mg/kg/d; n = 7), cyclosporine (CYA: 5 mg/kg/d) or sirolimus (RAPA: 1,5 mg/kg/d ip) on 3 consecutive days. Control mice received NaCl 0,9 % (10 ml/kg/d ip; n = 5). Drug plasma levels were examined to ensure therapeutic doses. Additionally, the systemic circulation of sPand sE-selectin as well as ADMA plasma levels were measured by ELISA. Application of immunosuppressants produced clinically relevant plasma levels (TAC: 8 ± 2 ng/ml, CYA: 214 ± 16 ng/ml; RAPA: 9 ± 1 ng/ml). Microvascular thrombus formation was significantly accelerated in mice receiving TAC compared to control mice (arteriolar and venular occlusion: 251 ± 101s and 179 ± 27 s vs. control 818 ± 221 s and 749 ± 231 s; p < 0,05). Application of CYA significantly increased thrombus formation only in venules (276 ± 49s; p < 0,05 vs. control), whereas RAPA had no significant effect on thrombosis induction. Plasma concentrations of sP-Selectin and sE-selectin were slightly reduced after TAC and CYA application. ADMA-levels however were significantly increased in mice receiving TAC (1,28 ± 0,16 μmol/l vs. control: 0,74 ± 0,13 μmol/l; p < 0,05). The present results show that clinically relevant plasma levels of cyclosporine enhanced thrombus formation only in venules whereas tacrolimus significantly accelerates thrombus formation in arterioles and venules. This effect could be explained by increased ADMA-levels in plasma resulting in impaired NO-bioavailability and increased thrombogenicity, thus presenting a new molecular mechanism in the development of transplant-thrombosis.

Introduction Hypothermia during sepsis significantly impairs patient outcome in clinical practice. Severe sepsis is closely linked to activation of the coagulation system, resulting in microthrombosis and subsequent organ failure. Herein, we studied whether systemic hypothermia accelerates microvascular thrombus formation during lipopolysacharide (LPS)-induced endotoxemia in vivo, and characterized the low temperature-induced endothelial and platelet dysfunctions.