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Summary: Background: To determine the adherence to supervised exercise training and underlying reasons for non-adherence amongst patients with inpatient treatment of symptomatic lower extremity peripheral arterial disease (PAD). Patients: This was a prospective questionnaire-based survey study of all consecutively treated inpatients with treatment for either intermittent claudication or chronic limb-threatening ischaemia (CLTI) surveyed at sixteen participating centres in Germany. Results: A total of 235 patients (median age 70 years) were included, thereof 29.4% females and 34.6% with CLTI. The median time from first PAD diagnosis was 4 years (IQR: 1–8). Only 11.4% have previously participated in any walking exercise programme before the index treatment, thereby 10.0% in the IC subgroup and 12.0% with CLTI. Amongst all patients, 35.6% responded they were appropriately informed about the necessity and benefits of walking exercise programmes by their hospital physicians (25.8% by general practitioners), and 65.3% agreed that adherence to supervised exercise may improve their pain-free walking distance. A total of 24.5% responded they had access to necessary information concerning local walking exercise programmes. Amongst 127 free text comments on the reasons for non-adherence to supervised exercise training, 64% of the comments contained lack of information or consent on such measures. Conclusions: Less than 12% of the patients enrolled in the current study have ever participated in a walking exercise programme during their life course. Although all practice guidelines contain corresponding class I recommendations, especially for patients suffering from IC, most patients responded that they were not appropriately informed about the necessity of exercise training along with the fact that 65% agreed that exercise may increase the pain-free walking distance. Taken all together, these results emphasise that we miss an important opportunity in the patient-physician communication. Efforts should be made to improve acceptance and application of structured walking-exercise for patients with PAD.

Background: Tympanic membrane perforations (TMPs) are a common complication of trauma and infection. Persisting perforations result from the unique location of the tympanic membrane. The wound is surrounded by air of the middle ear and the external auditory canal. The inadequate wound bed, growth factor, and blood supply lead to circular epithelialization of the perforation’s edge and premature interruption of defect closure. Orthotopic animal models use mechanical or chemical tympanic membrane laceration to identify bioactive wound dressings and overcome premature epithelialization. However, all orthotopic models essentially lack repetitive visualization of the biomaterial-wound interface. Therefore, recent progress in 3D printing of customized wound dressings has not yet been transferred to the unique wound setup of the TMP. Here, we present a novel application for the mice dorsal skinfold chamber (DSC) with an epithelialized full-thickness defect as TMP model. Methods: A circular 2-mm defect was cut into the extended dorsal skinfold using a biopsy punch. The skinfold was either perforated through both skin layers without prior preparation or perforated on 1 side, following resection of the opposing skin layer. In both groups, the wound was sealed with a coverslip or left unclosed (n = 4). All animals were examined for epithelialization of the edge (histology), size of the perforation (planimetry), neovascularization (repetitive intravital fluorescence microscopy), and inflammation (immunohistology). Results: The edge of the perforation was overgrown by the cornified squamous epithelium in all pre­parations. Reduction in the perforation’s size was enhanced by application of a coverslip. Microsurgical preparation before biopsy punch perforation and sealing with a coverslip enabled repetitive high-quality intravital fluorescence microscopy. However, spontaneous reduction of the perforation occurred frequently. Therefore, the direct biopsy punch perforation without microsurgical preparation was favorable: spontaneous reduction did not occur throughout 21 days. Moreover, the visualization of the neovascularization was sufficient in intravital microscopy. Conclusions: The DSC full-thickness defect is a valuable supplement to orthotopic TMP models. Repetitive intravital microscopy of the epithelialized edge enables investigation of the underlying pathophysiology during the transition from the inflammation to the proliferation phase of wound healing. Using established analysis procedures, the present model provides an effective platform for the screening of bioactive materials and transferring progress in tissue engineering to the special conditions of tympanic membrane wound healing.

INTRODUCTION: The volatile endogenous mediator hydrogen sulfide (H2S) is known to impair thrombus formation by affecting the activity of human platelets. Beside platelets and coagulation factors the endothelium is crucial during thrombogenesis. OBJECTIVE: This study evaluates the effect of the H2S donor GYY4137 (GYY) on human umbilical vein endothelial cells (HUVECs) in vitro. METHODS: Flow cytometry of resting, stimulated or GYY-treated and subsequently stimulated HUVECs was performed to analyse the expression of E-selectin, ICAM-1 and VCAM-1. To study a potential reversibility of the GYY action, E-selectin expression was further assessed on HUVECs that were stimulated 24 h after GYY exposure. A WST-1 assay was performed to study toxic effects of the H2S donor. By using the biotin switch assay, protein S-sulfhydration of GYY-exposed HUVECs was assessed. Further on, the effects of GYY on HUVEC migration and von Willebrand factor (vWF) secretion were assessed. RESULTS: GYY treatment significantly reduced the expression of E-selectin and ICAM-1 but not of VCAM-1. When HUVECs were stimulated 24 h after GYY treatment, E-selectin expression was no longer affected. The WST-1 assay revealed no effects of GYY on endothelial cell viability. Furthermore, GYY impaired endothelial migration, reduced vWF secretion and increased protein S-sulfhydration. CONCLUSIONS: Summarizing, GYY dose dependently and reversibly reduces the activity of endothelial cells.

Postoperative quality of life is an important outcome parameter after treatment of abdominal aortic aneurysms. The aim of this retrospective single-center study was to assess and compare the health-related quality of life (HRQoL) of patients after open repair (OR) or endovascular treatment (EVAR), and furthermore to investigate the effect of incisional hernia (IH) formation on HRQoL. Patients who underwent OR or EVAR for treatment of an abdominal aortic aneurysm between 2008 and 2016 at a University Medical Center were included. HRQoL was assessed using the SF-36 questionnaire. The incidence of IH was recorded from patient files and by telephone contact. SF-36 scores of 83 patients (OR: n = 36; EVAR: n = 47) were obtained. The mean follow-up period was 7.1 years. When comparing HRQoL between OR and EVAR, patients in both groups scored higher in one of the eight categories of the SF36 questionnaires. The incidence of IH after OR was 30.6%. In patients with postoperative IH, HRQoL was significantly reduced in the dimensions “physical functioning”, “role physical” and “role emotional” of the SF-36. Based on this data, it can be concluded that neither OR nor EVAR supply a significant advantage regarding HRQoL. In contrast, the occurrence of IH has a relevant impact on the HRQoL of patients after OR.

INTRODUCTION: Neointima formation is closely linked to vascular stenosis and occurs after endothelial damage. Hydrogen sulfide is an endogenous pleiotropic mediator with numerous positive effects on the cardio vascular system. OBJECTIVE: This study evaluates the effect of the slow releasing hydrogen sulfide donor GYY4137 (GYY) on neointimal formation in vivo. METHODS: The effect of GYY on neointimal formation in the carotid artery was studied in the FeCl3 injury model in GYY- or vehicle-treated mice. The carotid arteries were studied at days 7 and 21 after treatment by means of histology and immunohistochemistry for proliferating cell nuclear antigen (PCNA) and alpha smooth muscle actin (α-SMA). RESULTS: GYY treatment significantly reduced the maximal diameter and the area of the newly formed neointima on both days 7 and 21 when compared to vehicle treatment. GYY additionally reduced the number of PCNA- and α-SMA-positive cells within the neointima on day 21 after FeCl3 injury of the carotid artery. CONCLUSIONS: Summarizing, single treatment with the slow releasing hydrogen sulfide donor GYY reduced the extent of the newly formed neointima by affecting the cellular proliferation at the site of vascular injury.

Journal of Clinical Medicine 11(3), (2022). doi:10.3390/jcm11030498 special issue: "Topical Collection "Peripheral Artery Disease: Epidemiology and Global Perspectives" / Editors: Dr. Christian-Alexander Behrendt, Collection Editor; Dr. Frederik Peters, Collection Editor; Dr. Ulrich Rother, Collection Editor", Published by MDPI, Basel

Inguinal hernia repair, according to Desarda, is a pure tissue surgical technique using external oblique fascia to reinforce the posterior wall of the inguinal canal. This has provided an impetus for the rethinking of guideline adherence toward minimally invasive and mesh-based surgery of inguinal hernia. In this study, a retrospective analysis of this technique was conducted in two German hospitals. Between 6/2013 and 12/2020, 120 operations were performed. Analysis included patient characteristics, duration of operation, length of hospital stay, and perioperative complications. Data were used to achieve a matched-pair analysis comparing Desarda to laparoscopic transabdominal preperitoneal (TAPP) hernia repair. Propensity scores were calculated based on five preoperative variables, including sex, age, American Society of Anesthesiology classification, localization, and width of the inguinal hernia in order to achieve comparability. Additionally, we assessed pain level and quality of life (QoL) 12 months postoperatively. The focus of our study was a comparison of QoL to a reference population and TAPP cohort. The study population consisted of 106 male and 14 female patients, and the median age was 37.5 years. The median operation time was 50 min, and the median length of hospital stay was 2 days. At a follow-up of 17 months, the median recurrence rate was 0.8%, and two cases of chronic postoperative pain were recorded. Postoperative QoL does not significantly differ between Desarda and TAPP. In contrast, Desarda patients had a significantly higher QoL compared with the reference population. In summary, Desarda’s procedure is a good option as a pure tissue method for inguinal hernia repair.

Background: Assessment of tissue oxygenation is an important aspect of detection and monitoring of patients with peripheral artery disease (PAD). Hyperspectral imaging (HSI) is a non-contact technology for assessing microcirculatory function by quantifying tissue oxygen saturation (StO2). This study investigated whether HSI can be used to monitor skin oxygenation in patients with PAD after appropriate treatment of the lower extremities. Methods: For this purpose, 37 patients with PAD were studied by means of ankle–brachial index (ABI) and HSI before and after surgical or endovascular therapy. Thereby, the oxygenation parameter StO2 and near infrared (NIR) perfusion index were quantified in seven angiosomes on the diseased lower leg and foot. In addition, the effects of skin temperature and physical activity on StO2 and the NIR perfusion index and the respective inter-operator variability of these parameters were investigated in 25 healthy volunteers. Results: In all patients, the ABI significantly increased after surgical and endovascular therapy. In parallel, HSI revealed significant changes in both StO2 and NIR perfusion index in almost all studied angiosomes depending on the performed treatment. The increase in tissue oxygenation saturation was especially pronounced after surgical treatment. Neither heat nor cold, nor physical activity, nor repeated assessments of HSI parameters by independent investigators significantly affected the results on StO2 and the NIR perfusion index. Conclusions: Tissue oxygen saturation data obtained with HSI are robust to external confounders, such as temperature and physical activity, and do not show inter-operator variability; therefore, can be used as an additional technique to established methods, such as the ABI, to monitor peripheral perfusion in patients with PAD.

The dorsal skinfold chamber is one of the most important in vivo models for repetitive longitudinal assessment of microcirculation and inflammation. This study aimed to refine this model by introducing a new lightweight chamber made from polyetheretherketone (PEEK). Body weight, burrowing activity, distress, faecal corticosterone metabolites and the tilting angle of the chambers were analysed in mice carrying either a standard titanium chamber or a PEEK chamber. Data was obtained before chamber preparation and over a postoperative period of three weeks. In the early postoperative phase, reduced body weight and increased faecal corticosterone metabolites were found in mice with titanium chambers. Chamber tilting and tilting-related complications were reduced in mice with PEEK chambers. The distress score was significantly increased in both groups after chamber preparation, but only returned to preoperative values in mice with PEEK chambers. In summary, we have shown that light chambers reduce animal distress and may extend the maximum dorsal skinfold chamber observation time. Chambers made of PEEK are particularly suitable for this purpose: They are autoclavable, sufficiently stable to withstand rodent bites, inexpensive, and widely available through 3D printing.

Bacterial infections of vascular grafts represent a major burden in cardiovascular medicine, which is related to an increase in morbidity and mortality. Different factors that are associated with this medical field such as patient frailty, biofilm formation, or immunosuppression negatively influence antibiotic treatment, inhibiting therapy success. Thus, further treatment strategies are required. Bacteriophage antibacterial properties were discovered 100 years ago, but the focus on antibiotics in Western medicine since the mid-20th century slowed the further development of bacteriophage therapy. Therefore, the experience and knowledge gained until then in bacteriophage mechanisms of action, handling, clinical uses, and limitations were largely lost. However, the parallel emergence of antimicrobial resistance and individualized medicine has provoked a radical reassessment of this approach and cardiovascular surgery is one area in which phages may play an important role to cope with this new scenario. In this context, bacteriophages might be applicable for both prophylactic and therapeutic use, serving as a stand-alone therapy or in combination with antibiotics. From another perspective, standardization of phage application is also required. The ideal surgical bacteriophage application method should be less invasive, enabling highly localized concentrations, and limiting bacteriophage distribution to the infection site during a prolonged time lapse. This review describes the latest reports of phage therapy in cardiovascular surgery and discusses options for their use in implant and vascular graft infections.

Introduction: Sufficient tissue oxygenation is essential for anastomotic healing in visceral surgery. Hyperspectral imaging (HSI) is a noncontact, noninvasive technique for clinical assessment of tissue oxygenation in real time. Methods: In this case series, HSI was used in 4 patients who were admitted for either esophageal cancer or cardiac carcinoma (AEG type I or II). Thoraco-abdominal surgical esophageal resection was performed after staging and neoadjuvant therapy. Intraoperative oxygenation of superficial (StO2) and underlying tissue (NIR perfusion index) of the gastric sleeve were studied intrathoracic by means of the TIVITA® Tissue HSI camera. This was performed prior to esophagogastric anastomosis. The postoperative course, especially in view of surgical complications, was recorded. Results: Assessment of StO2 and NIR perfusion index was performed in 4 regions of interest per gastric sleeve, aboral and oral of the clinically determined resection line. It allowed the fast quantification of gastric oxygenation prior gastroesophageal anastomosis. Median StO2 aboral of the determined resection line was 69%, while median StO2 in the oral part of the gastric sleeve was found at 53%. In contrast, the median NIR perfusion index was similar aboral (80) and oral (82) of the resection line. In none of the 4 studied patients, an anastomotic failure appeared. Discussion/Conclusion: This report suggests that HSI is a feasible technique for intraoperative assessment of tissue oxygenation before gastroesophageal anastomosis and might reduce the incidence of anastomotic failure in the gastrointestinal tract.

This study estimates the prevalence and mortality of diseases of the deep veins of the legs such as deep vein thrombosis (DVT), postthrombotic syndrome (PTS), and venous leg ulceration (VLU). We used a random sample of 250 000 patients at age 50+ years of the register of the Allgemeine Ortskrankenkasse from 2004 to 2015. Selected manifestations of venous diseases assumed as risk factors for mortality were analyzed using Cox models while adjusting for various basic demographic and health characteristics. The prevalence in 2004 was 0.05% for DVT of the femoral veins, 0.50% for DVT of any deep veins, 0.86% for PTS, and 0.91% for VLU. The mortality rate in 2004 to 2015 was 20.40 deaths/100 person-years for DVT of the femoral veins, 10.69 for DVT of any deep veins, 4.34 for PTS, and 7.02 for VLU. The model revealed a 35% higher risk ( p < .001) in patients with any DVT, an 88% higher mortality ( p < .001) for femoral DVT, a 23% higher risk ( p < .001) for VLU, and no health disadvantage in persons with PTS. Our study revealed an increased mortality for patients with VLU and DVT. Even after adjustment for embolic events and infections of the venous ulcers mortality remained significantly higher.

Background/purpose Improval of flap viability is one of the main purposes in reconstructive surgery. The endogenous volatile mediator hydrogen sulfide (H2S) exerts plenty beneficial effects on blood rheology and protects from ischemia reperfusion injury. Methods Therefore this study analyzed whether H2S affects tissue necrosis and skin microcirculation during impaired perfusion in the axial pattern ear flap model in hairless mice. Necrosis and microcirculatory parameters were assessed by means of stereo- and intravital fluorescence microscopy before and immediately after flap creation as well as on days 2 and 4 after surgery. Treatment with H2S donor GYY4137 or its vehicle was performed either one-time after flap preparation or two-times with an additional injection on day 2. After 4 days ear tissues were taken and further analyzed histologically to study tissue edema. Results Compared to vehicle treatment, both one- and two-time treatment with GYY4137 significantly reduced tissue necrosis and improved microcirculation in the ear flap, apparently through capillary dilatation and increased functional capillary density. Histological analysis revealed a significantly reduced tissue edema formation in GYY4137-treated animals. While microcirculatory parameters in parts significantly improved by two-time GYY4137 treatment, ear thickness and edema formation were independent from treatment regime. Conclusions In summary H2S treatment is a promising attempt to improve microcirculation in critically perfused skin flaps and consequently to preserve flap viability.

Objective:Oxytocin (OXY) has significant effects on mammalian behavior. Next to its role in lactation and social interactions, it is described to support better wound healing as well. However, direct OXY effects on wound healing and the regeneration of the microvascular network are still not clarified. We therefore examined the effects of OXY and an OXY receptor antagonist [atosiban (ATO)] on skin wound healing, focusing on epithelialization and neovascularization.Methods:Skin wound healing has been assessed using intravital fluorescence microscopy in a model of full dermal thickness wounds in the dorsal skin fold chamber of hairless mice. Animals received repetitive low or high doses of OXY or ATO. Morphological and cellular characterization of skin tissue repair was performed by histology and in vitro cell assays.Results:The assessment of skin tissue repair using this therapy regimen showed that OXY and ATO had no major influence on epithelialization, neovascularization, wound cellularity, or inflammation. Moreover, OXY and ATO did neither stimulate nor deteriorate keratinocyte or fibroblast migration and proliferation.Conclusion:In summary, this study is the first to demonstrate that OXY application does not impair skin wound healing or cell behavior. However, until now, the used transmitter system seems not to be clarified in detail, and it might be proposed that it is associated with the stress response of the organism to various stimuli.

Recently, the potential use of cold atmospheric pressure plasma (CAP) in cancer treatment has gained increasing interest. Especially the enhanced selective killing of tumor cells compared to normal cells has prompted researchers to elucidate the molecular mechanisms for the efficacy of CAP in cancer treatment. This review summarizes the current understanding of how CAP triggers intracellular pathways that induce growth inhibition or cell death. We discuss what factors may contribute to the potential selectivity of CAP towards cancer cells compared to their non-malignant counterparts. Furthermore, the potential of CAP to trigger an immune response is briefly discussed. Finally, this overview demonstrates how these concepts bear first fruits in clinical applications applying CAP treatment in head and neck squamous cell cancer as well as actinic keratosis. Although significant progress towards understanding the underlying mechanisms regarding the efficacy of CAP in cancer treatment has been made, much still needs to be done with respect to different treatment conditions and comparison of malignant and non-malignant cells of the same cell type and same donor. Furthermore, clinical pilot studies and the assessment of systemic effects will be of tremendous importance towards bringing this innovative technology into clinical practice.

Aim of the study was to compare the evolvement of vascularization over time of collagen membranes (CMs) of dermal and pericardial origin in an in vivo animal study. Twenty-eight mice underwent implantation of three commercially available CM derived from porcine dermis (homogenous structure: CM1 (Control 1) and bilayer structure: CM2 [Control 2]), from porcine pericardium (CM3; Test 1) as well as CM3 sprayed with silica-enhanced nanostructured hydroxyapatite (CM4, Test 2). After 3, 6, 9, and 12 days, intravital fluorescence microscopy was conducted for determination of capillary diameter, density, flow, and length. At Day 12, samples were examined immunohistologically for expression of fibroblast growth factor receptor 4 (FGFR4), CD11b, CD68, αSMA, and CD34. In all CM, intravital fluorescence microscopy over time showed increasing values for all parameters with the highest levels in CM4 and the lowest values in CM1. Significant lower amounts of FGFR4, CD11b, and CD68 were detected in CM4 when compared to CM2 (p < .05). In contrast to CM3, lower values of αSMA and higher numbers of CD34 positive-marked vessels were observed in CM4 (p < .05). In conclusion, dermal bilayer as well as pericardial CM seem to have a higher vascularization rate than dermal homogenous CM. Additional coating of pericardial CM with a silica-enhanced hydroxyapatite increases the speed of vascularization as well as biological remodeling processes.

Background Objective, reliable and easy screening for peripheral artery disease (PAD) is essential to confirm the diagnosis and initiate the respective treatment. Therefore, a new non-invasive hyperspectral camera (TIVITA® Tissue) was tested in patients with and without PAD. Objective It was hypothesized that the oxygenation parameters of the TIVITA® Tissue correlate to established modalities for detection of PAD and allow differentiation between individuals with and without PAD. Methods Evaluation of tissue oxygenation was performed in the angiosome of the medial plantar artery in 25 healthy young people and in 24 patients with and 25 patients without PAD in comparable age. Thereby, superficial oxygenation (StO2) and near-infrared (NIR) perfusion index were measured with the TIVITA® Tissue. Additionally, the ankle-brachial-index (ABI), the complaint free walking distance and the vascular quality of life were assessed and demographic data were obtained from all participants. Results TIVITA® Tissue analysis revealed significantly reduced StO2 and NIR perfusion index in PAD compared to healthy young participants and patients without PAD. StO2 and NIR perfusion index positively correlated with ABI, the complaint free walking distance and the vascular quality of life score. Conclusions In summary, this new hyperspectral imaging camera bears great potential for PAD screening as well as for follow up.

Simple Summary Skin cancer still poses a great burden for patients. One of the most promising therapy approaches in recent years is cold atmospheric pressure plasma. The aim of the study was to assess in vivo the effect of cold atmospheric pressure plasma on human squamous cell carcinoma as well as malignant melanoma tumour cell lines in a longitudinal and non-invasive manner with multimodal imaging techniques such as MRI and [18F]FDG PET. By using these techniques and chemiluminescence imaging, we present in the current study that reactive species increase in vivo upon treatment with cold plasma and consequently decrease tumour growth. Therefore, we propose cold atmospheric pressure plasma may be a potential adjuvant therapy option to established standard therapies of skin cancer. Abstract Background: Skin cancer is the most frequent cancer worldwide and is divided into non-melanoma skin cancer, including basal cell carcinoma, as well as squamous cell carcinoma (SCC) and malignant melanoma (MM). Methods: This study evaluates the effects of cold atmospheric pressure plasma (CAP) on SCC and MM in vivo, employing a comprehensive approach using multimodal imaging techniques. Longitudinal MR and PET/CT imaging were performed to determine the anatomic and metabolic tumour volume over three-weeks in vivo. Additionally, the formation of reactive species after CAP treatment was assessed by non-invasive chemiluminescence imaging of L-012. Histological analysis and immunohistochemical staining for Ki-67, ApopTag®, F4/80, CAE, and CD31, as well as protein expression of PCNA, caspase-3 and cleaved-caspase-3, were performed to study proliferation, apoptosis, inflammation, and angiogenesis in CAP-treated tumours. Results: As the main result, multimodal in vivo imaging revealed a substantial reduction in tumour growth and an increase in reactive species after CAP treatment, in comparison to untreated tumours. In contrast, neither the markers for apoptosis, nor the metabolic activity of both tumour entities was affected by CAP. Conclusions: These findings propose CAP as a potential adjuvant therapy option to established standard therapies of skin cancer.

The volatile transmitter hydrogen sulfide (H2S) is known for its various functions in vascular biology. This study evaluates the effect of the H2S-donor GYY4137 (GYY) on thrombus stability and microvascular thrombolysis. Human whole blood served for all in vitro studies and was analyzed in a resting state, after stimulation with thrombin-receptor activating peptide (TRAP) and after incubation with 10 or 30 mM GYY or its vehicle DMSO following TRAP-activation, respectively. As a marker for thrombus stability, platelet-leukocyte aggregation was assessed using flow cytometry after staining of human whole blood against CD62P and CD45, respectively. Furthermore, morphology and quantity of platelet-leukocyte aggregation were studied by means of scanning electron microscopy (scanning EM). Therefore, platelets were stained for CD62P followed by immuno gold labeling. In vivo, the dorsal skinfold chamber preparation was performed for light/dye induction of thrombi in arterioles and venules using intravital fluorescence microscopy. Thrombolysis was assessed 10 and 22 h after thrombus induction and treatment with the vehicle, GYY, or recombinant tissue plasminogen activator (rtPA). Flow cytometry revealed an increase of CD62P/CD45 positive aggregates after TRAP stimulation of human whole blood, which was significantly reduced by preincubation with 30 mM GYY. Scanning EM additionally showed a reduced platelet-leukocyte aggregation and a decreased leukocyte count within the aggregates after preincubation with GYY compared to TRAP stimulation alone. Further on, morphological signs of platelet activation were found markedly reduced upon treatment with GYY. In mice, both GYY and rtPA significantly accelerated arteriolar and venular thrombolysis compared to the vehicle control. In conclusion, GYY impairs thrombus stability by reducing platelet-leukocyte aggregation and thereby facilitates endogenous thrombolysis.

This study analysed the impact of anandamide, cannabidiol (CBD), and WIN55,212-2 on platelet activity and thrombogenesis for the first time. The effects of the cannabinoids on venular thrombosis were studied in the ear of hairless mice. Cannabinoid treatment was performed either once or repetitive by a once-daily administration for three days. To assess the role of cyclooxygenase metabolites in the putative action of anandamide, in vivo studies likewise included a combined administration of anandamide with indomethacin. In vitro, the effect of the cannabinoids on human platelet activation was studied by means of P-selectin expression using flow cytometry. Platelets were analysed under resting or thrombin receptor activating peptide (TRAP)-stimulated conditions, both after cannabinoid treatment alone and after TRAP stimulation and subsequent cannabinoid exposure. Finally, platelet count was assessed after treatment with high concentrations of anandamide. Anandamide, but not CBD and WIN55,212-2, significantly accelerated thrombus growth after one-time treatment as compared to vehicle control. Co-administration with indomethacin neutralized this effect. However, thrombogenesis was not altered by repeated treatment with the cannabinoids. In vitro, anandamide was shown to elicit a concentration-dependent activation of resting human platelets. However, at higher concentrations anandamide reduced the response to TRAP activation associated with a decrease of platelet count. CBD and WIN55,212-2 neither increased nor reduced activation of platelets. Acute exposure to anandamide elicits a cyclooxygenase-dependent prothrombotic effect in vivo. Anandamide seems to affect human platelet activation by a concentration-dependent toxic effect. By contrast, CBD and WIN55,212-2 were not associated with induction of thrombosis or activation of platelets. © 2016 BioFactors, 42(6):581-590, 2016.

Background Duplex ultrasound is the first choice in diagnostics and surveillance of stenoses of the internal carotid arteries before and even after surgery. Therefore, the quality of duplex ultrasound is crucial to investigate these vascular pathologies. Objective Aim of this study was the evaluation whether different surgical techniques affect the postoperative quality of duplex ultrasound. Methods In a time period from January to May 2015 duplex ultrasound of the cervical vessels was performed in 75 patients after unilateral endarterectomy of the internal carotid artery at our department between 2006 and 2012. Thereby, the non-operated contralateral side served as a control. Study groups were defined by the surgical techniques of eversion- or thrombendarterectomy with patch plasty using different patch materials and/or a haemostatic sealant. Duplex ultrasound analysis included acoustic impedance, extinction of ultrasound, thickness of skin and individual anatomic aspects of the patients. Results Carotid endarterectomy itself reduced intravascular grey levels, skin thickness and increased extinction of duplex ultrasound when compared to the non-operated side of the neck. In contrast, neither the kind of chosen operative technique nor the use of different patch materials or the application of a haemostatic sealant showed an effect in this regards. Conclusions Whereas carotid endarterectomy per se worsens the quality of postoperative duplex ultrasound, the different analysed surgical techniques as well as used patches and the application of a haemostatic sealant can be assumed to be equal regarding the quality of postoperative ultrasound.

Background/purpose Objective, reliable and easy monitoring of microvascular tissue perfusion is a goal that was achieved for many years with limited success. Therefore, a new non-invasive hyperspectral camera system (TIVITA™) was tested for this purpose in an in vivo animal model. Methods Evaluation of tissue oxygenation during ischemia and upon reperfusion was performed in left hind limb in a rat model (n = 20). Ischemia was induced by clamping and dissection of the superficial femoral artery. Reperfusion of the limb was achieved by microsurgical anastomosis of the dissected artery. Oxygenation parameters of the hind limb were assessed via TIVITA™ before and immediately after clamping and dissection of the artery, 3 and 30 min after reperfusion as well as on postoperative days 1 and 2. Thereby, the non-operated hind limb served as control. As clinical parameters, the refill of the anastomosis as well as the progress of the affected leg were assessed. Results In 12 from 20 cases, TIVITA™ recorded a sufficient reperfusion with oxygenation parameters comparable to baseline or control condition. However, in 8 from 20 cases oxygenation was found impaired after reperfusion causing a re-assessment of the microvascular anastomosis. Thereby, technical problems like stenosis or local thrombosis were found in all cases and were surgically treated leading to an increased tissue oxygenation. Conclusions The TIVITA™ camera system is a valid non-invasive tool to assess tissue perfusion after microvascular anastomosis. As it safely shows problems in oxygenation, it allows the clinician a determined revision of the site in time in order to prevent prolonged ischemia.

Blood perfusion as the supply of tissue with blood and therefore oxygen is a key factor in clinical practice. Especially in the field of flap surgery, a reduced perfusion of transplanted skin or operated areas is often cause of various complications. The success of microvascular reconstructions is directly related to the flap perfusion. The intraoperative and postoperative assessment of the anastomoses is of great importance in order to recognize possible complications at an early stage and to revise them in good time. Is the affected tissue located on the face, successful treatment and rapid healing is even more important since aesthetic aspects play a not insignificant role. A poor perfusion is often concealed, since methods are missing for an objective assessment of the perfusion status. A method with increasing importance for clinical practice is given by hyperspectral imaging. We developed a new hyperspectral imaging system that can be used to observe tissue oxygenation and other tissue parameters and present the technical background and the parameter validation.

Thrombotic complications of vascular diseases are one leading cause of morbidity and mortality in industrial nations. Due to the complex interactions between cellular and non-cellular blood components during thrombus formation, reliable studies of the physiology and pathophysiology of thrombosis can only be performed in vivo. Therefore, this article presents an ear model in hairless mice and focuses on the in vivo analysis of microcirculation, thrombus formation, and thrombus evolution. By using intravital fluorescence microscopy and the intravenous (iv) application of the respective fluorescent dyes, a repetitive analysis of microcirculation in the auricle can easily be performed, without the need for surgical preparation. Furthermore, this model can be adapted for in vivo studies of different issues, including wound healing, reperfusion injury, or angiogenesis. In summary, the ear of hairless mice is an ideal model for the in vivo study of skin microcirculation in physiological or pathophysiological conditions and for the evaluation of its reaction to different systemic or topical treatments.

Background: Patients with liver cirrhosis have an increased risk of developing hepatocellular carcinoma (HCC). Implantation metastasis following diagnostic biopsy is a well-known complication. Therefore, primary resection of a hypervascularised tumour suspicious for HCC is often performed with curative intent. Case Report: An exophytically growing mass was diagnosed between liver segments III and IVb by means of ultrasound in a 53-year old male patient with decompensated liver cirrhosis. Computed tomography confirmed a 3.5 cm large hypervascularised tumour with given resectability. Intraoperatively, the tumour appeared like a HCC. Thus, an atypical resection was performed. Histopathology revealed ectopic spleen tissue without any signs of malignancy. As enquiries revealed, the patient had undergone splenectomy after a blunt abdominal trauma 9 years prior to admission. Conclusion: In the present patient, hepatic splenosis in a cirrhotic liver was misinterpreted as HCC. In patients with a history of traumatic rupture of the spleen or splenectomy, splenosis has to be considered as a potential differential diagnosis of a hypervascularised tumour. Specific diagnostics should be performed to rule out splenosis.

Unmethylated CpG oligodeoxynucleotides (ODN) bind to the Toll-like receptor 9, thus stimulating the immune system. To study the effects of systemic pretreatment with CpG ODN on dermal regeneration, C57BL6/J Tyr mice were treated with CpG or control ODN 6 days prior to implantation of a dorsal skinfold chamber and skin wounding. Wound epithelialization was analyzed by planimetric microscopy. On day 18, wound tissues were taken for (immuno)histochemical staining. CpG ODN increased epithelialization compared with control ODN treatment. Histological analysis revealed reduced capillary density, reduced wound cellularity, and reduced numbers of infiltrating leukocytes, as well as reduced F4/80-positive macrophages, but increased numbers of RELM-α-positive M2 macrophages after CpG ODN treatment, reflecting a better quality of wound healing on day 18 compared with control ODN treatment. Reverse transcription-polymerase chain reaction analysis of Toll-like receptor 9 showed the receptor expression on both fibroblasts and keratinocytes. Fibroblasts showed an increase of migration upon increasing dosages of CpG and not control ODN, reaching ∼50% of the response of basic fibroblast growth factor-exposed cells. Keratinocytes dose-dependently responded to both CpG and control ODN up to values found in keratinocyte growth factor-exposed cells. In summary, CpG ODN support late tissue-remodeling processes that contribute to resolution of inflammation and solid wounds during skin regeneration.

OBJECTIVE Sepsis, a leading cause of mortality in critically ill patients, is closely linked to the excessive activation of coagulation and inflammation. Protein Z, a cofactor for the protein Z-dependent protease inhibitor, enhances the inhibition of coagulation factor Xa, and protein Z-dependent protease inhibitor inhibits factor XIa in a protein Z-independent fashion. The functions of protein Z and protein Z-dependent protease inhibitor in the inflammatory and coagulant responses to septic illness have not been evaluated. DESIGN For induction of generalized Shwartzman reaction, dorsal skinfold chamber-equipped mice were challenged twice with lipopolysaccharide (0.05 mg/kg on day -1 and 5 mg/kg body weight 24 hr later). Time-matched control animals received equal volumes of saline. SETTING University research laboratory. SUBJECTS, INTERVENTIONS, AND MEASUREMENTS Using intravital fluorescence microscopy in protein Z-dependent protease inhibitor deficient (ZPI) and protein Z deficient (PZ) mice, as well as their wild-type littermates (ZPI, PZ), kinetics of light/dye-induced thrombus formation and microhemodynamics were assessed in randomly chosen venules. Plasma concentrations of chemokine (C-X-C motif) ligand 1, interleukin-6, and interleukin-10 were measured. Liver and lung were harvested for quantitative analysis of leukocytic tissue infiltration and thrombus formation. MAIN RESULTS After induction of generalized Shwartzman reaction, all mice showed significant impairment of microhemodynamics, including blood flow velocity, volumetric blood flow, and functional capillary density, as well as leukocytopenia and thrombocytopenia. Thrombus formation time was markedly prolonged after induction of generalized Shwartzman reaction in all mice, except of ZPI mice, which also had a significantly higher fraction of occluded vessels in liver sections. PZ mice developed the highest concentrations of interleukin-6 and interleukin-10 in response to generalized Shwartzman reaction and showed greater leukocytic tissue infiltration than their wild-type littermates. CONCLUSIONS In this murine model of generalized Shwartzman reaction, protein Z-dependent protease inhibitor deficiency enhanced the thrombotic response to vascular injury, whereas protein Z deficiency increased inflammatory response.

Introduction Hydrogen sulfide (H 2 S) known as a gasotransmitter is increasingly recognized for its anti-adhesive, anti-inflammatory and vasoactive properties. Due to these properties, we analysed anti-thrombotic effects of H 2 S and the participation of the nitric oxide synthase (NOS)-pathway. Materials and Methods In individual venules of the ear of hairless SKH1-hr mice, thrombus formation was induced using a phototoxic light/dye-injury model and intravital fluorescence microscopy. Animals were treated intravenously with the H 2 S donor Na 2 S or NaCl as control. In a second setting, the NOS inhibitor L-NAME was applied intraperitoneally as a bolus 12 h prior to Na 2 S treatment and thrombus induction. Blood and ear tissue were sampled after microscopy for assessment of plasma concentrations of soluble (s)P-selectin, sE-selectin, sVCAM-1 and sICAM-1 and expression of endothelial (e)NOS and inducible (i)NOS, respectively. Results When mice were treated with Na 2 S, venular thrombus formation was significantly delayed versus that in animals of the NaCl-treated control group. While plasma levels of pro-thrombotic adhesion molecules were not affected by Na 2 S, immunohistochemistry of the vessel walls showed a significant up-regulation of eNOS and iNOS expression within the Na 2 S-treated group. The delay of thrombus formation in the Na 2 S-group was partly but significantly reverted by application of L-NAME. Conclusions The anti-thrombotic efficacy of H 2 S involves the NOS-pathway and may be of preventive and therapeutic value for clinical disorders with increased risk of thrombotic events.

This study evaluates the effect of the H2S donor GYY4137 (GYY) on adhesion molecule expression, protein S-sulfhydration and morphology of platelets in vitro and on kinetics of microvascular thrombus formation in vivo. Using flowcytometry, untreated resting, TRAP-activated, or TRAP-activated and GYY-exposed human platelets were studied for expression of P-selectin, GPIb and GPIIb/IIIa as well as for fibrinogen binding. By means of electron microscopy, platelet morphology and intracellular granule numbers were assessed. Platelet shape change was studied using immunohistochemistry for P-selectin, NSF and F-actin by SR-SIM. Biotin switch assay served for the analysis of platelet protein S-sulfhydration by GYY. Using the FeCl3 and the light/dye model in dorsal skinfold chamber-equipped mice, the effect of GYY and its vehicle DMSO was studied on venular thrombus formation and tail-vein bleeding time. Soluble (s)P-selectin plasma concentrations were measured in GYY- or DMSO-treated animals. Exposure to GYY increased the S-sulfhydration of platelet proteins. GYY reduced dose-dependently the TRAP-induced adhesion molecule expression and attenuated the morphological signs of TRAP-associated platelet activation. In mice, GYY caused a significant prolongation of venular thrombus formation and tail-vein bleeding time. Application of an anti-P-selectin antibody in DMSO-exposed animals prolonged thrombosis formation comparably as GYY did. GYY reversed the TRAP-induced distribution of P-selectin at the plasma membrane of platelets. This indicates reduced exocytosis and shedding of P-selectin, which is supported by significantly lower sP-selectin concentrations in GYY- vs. DMSO-treated mice. H2S acts anti-thrombotic and seems to regulate thrombogenesis by interference with platelet activation and adhesion molecule-mediated aggregation.

Unmethylated CpG oligodeoxynucleotides (ODN) bind to the Toll-like receptor 9, thus stimulating the immune system. To study the effects of systemic pretreatment with CpG ODN on dermal regeneration, C57BL6/J Tyr mice were treated with CpG or control ODN 6 days prior to implantation of a dorsal skinfold chamber and skin wounding. Wound epithelialization was analyzed by planimetric microscopy. On day 18, wound tissues were taken for (immuno)histochemical staining. CpG ODN increased epithelialization compared with control ODN treatment. Histological analysis revealed reduced capillary density, reduced wound cellularity, and reduced numbers of infiltrating leukocytes, as well as reduced F4/80-positive macrophages, but increased numbers of RELM-α-positive M2 macrophages after CpG ODN treatment, reflecting a better quality of wound healing on day 18 compared with control ODN treatment. Reverse transcription-polymerase chain reaction analysis of Toll-like receptor 9 showed the receptor expression on both fibroblasts and keratinocytes. Fibroblasts showed an increase of migration upon increasing dosages of CpG and not control ODN, reaching ∼50% of the response of basic fibroblast growth factor-exposed cells. Keratinocytes dose-dependently responded to both CpG and control ODN up to values found in keratinocyte growth factor-exposed cells. In summary, CpG ODN support late tissue-remodeling processes that contribute to resolution of inflammation and solid wounds during skin regeneration.

We could recently report that erythropoietin (EPO) accelerates skin wound healing in mice. Now, we provide insight into the molecular mechanisms of this non-hematopoietic property of EPO analyzing the transforming growth factor (TGF)-β signaling pathway. EPO receptor was found expressed in both non-wounded and wounded skin tissue as well as in fibroblasts and keratinocytes. In saline-treated control animals, wounds exhibited a significant upregulation of TGF-β1 and of α-smooth muscle actin (α-SMA) compared with non-wounded skin. EPO treatment accelerated wound epithelialization and induced mRNA expression of TGF-β1 and α-SMA. In addition, EPO significantly enhanced phosphorylation of Smad2 and Smad3 in fibroblasts and also elevated phosphorylation of Smad3 in wound tissue. Blockade of TGF-β using a neutralizing anti-TGF-β antibody attenuated EPO-induced acceleration of wound epithelialization in vivo and markedly reversed EPO effects on mRNA expression of TGF-β1 and α-SMA. In conclusion, EPO caused activation of the Smad-dependent TGF-β signaling pathway, enhanced differentiation of myofibroblasts, and accelerated skin wound closure.