The Dorsal Skinfold Chamber as a New Tympanic Membrane Wound Healing Model: Intravital Insights into the Pathophysiology of Epithelialized Wounds

<b><i>Background:</i></b> 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. <b><i>Methods:</i></b> 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 (<i>n</i> = 4). All animals were examined for epithelialization of the edge (histology), size of the perforation (planimetry), neovascularization (repetitive intravital fluorescence microscopy), and inflammation (immunohistology). <b><i>Results:</i></b> 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. <b><i>Conclusions:</i></b> 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.

Systemic low-dose erythropoietin administration improves the vascularization of collagen-glycosaminoglycan matrices seeded with adipose tissue-derived microvascular fragments

Adipose tissue-derived microvascular fragments (MVF) are used as vascularization units in tissue engineering. In this study, we investigated whether the vascularization capacity of MVF can be improved by systemic low-dose erythropoietin (EPO) administration. MVF were isolated from the epididymal fat of donor mice and seeded onto collagen-glycosaminoglycan matrices, which were implanted into full-thickness skin defects within dorsal skinfold chambers of recipient mice. Both donor and recipient mice were treated daily with either EPO (500 IU/kg) or vehicle (0.9% NaCl). The implants were analyzed by stereomicroscopy, intravital fluorescence microscopy, histology, and immunohistochemistry. EPO-treated MVF contained a comparable number of proliferating Ki67+ but less apoptotic cleaved caspase-3+ endothelial cells when compared to vehicle-treated controls. Moreover, EPO treatment accelerated and improved the in vivo vascularization, blood vessel maturation, and epithelialization of MVF-seeded matrices. These findings indicate that systemic low-dose EPO treatment is suitable to enhance the viability and network-forming capacity of MVF.

University of Wisconsin solution for the xeno-free storage of adipose tissue-derived microvascular fragments

Aim: Adipose tissue-derived microvascular fragments (ad-MVF) are vascularization units for regenerative medicine. We investigated whether University of Wisconsin (UW) solution is suitable for their xeno-free storage. Materials & methods: Murine ad-MVF were cultivated for 24 h in 4°C or 20°C UW solution and 20°C endothelial cell growth medium (control). The ad-MVF were seeded onto collagen–glycosaminoglycan scaffolds, which were analyzed in dorsal skinfold chambers by intravital fluorescence microscopy and histology. Results: All implants exhibited microvascular networks on day 14 with the highest functional microvessel density in controls. Ad-MVF cultivation in UW solution at 4°C resulted in an improved scaffold vascularization compared with cultivation at 20°C. Conclusion: UW solution is suitable for the hypothermic storage of ad-MVF.

The Coronary Microcirculation in Hamster-to-Rat Cardiac Xenografts

Background: The aim of this study was to establish a new experimental model to directly analyse the coronary microcirculation in cardiac xenografts. Methods: Intravital fluorescence microscopy (IVM) of the subepicardial microcirculation in heterotopically transplanted hamster-to-rat cardiac xenografts was performed at 30 and 90 min of reperfusion. We quantitatively assessed the microcirculatory perfusion characteristics as well as the interactions of leukocytes and platelets with the endothelium of postcapillary coronary venules in non-sensitised as well as sensitised recipients. Results: In this first experimental IVM study of cardiac xenografts, we successfully visualised the subepicardial microcirculation, i.e. feeding arterioles, nutritive capillaries and draining postcapillary venules, during reperfusion. Leukocyte-endothelial and platelet-endothelial cell interactions could be quantified. In the non-sensitised group, the myocardial microcirculation remained stable during the observation period of 90 min, whereas in the sensitised group, xenografts were rejected immediately. Conclusions: We established a model for the assessment of the microcirculatory dysfunction and inflammation during ischaemia/reperfusion injury in hamster-to-rat cardiac xenografts.

Novel Mouse Hemostasis Model for Real-Time Determination of Bleeding Time and Hemostatic Plug Composition

Introduction: Hemostasis is a rapid response by the body to stop bleeding at sites of vessel injury. Both platelets and fibrin are important for the formation of a hemostatic plug. Mice have been used to uncover the molecular mechanisms that regulate the activation of platelets and coagulation under physiological conditions. However, measuring hemostasis in mice is quite variable and current methods do not quantify platelet adhesion or fibrin formation at the site of injury. Methods: We describe a novel hemostasis model that uses intravital fluorescence microscopy to quantify platelet adhesion, fibrin formation, and time to hemostatic plug formation in real-time. Repeated vessel injuries of ~50-100µm in diameter were induced using laser ablation technology in the saphenous vein of mice. Results: Hemostasis in this model was strongly impaired in mice deficient in glycoprotein (GP)Ibα (IL4R/GPIb-tg) or talin-1 (talin1f/f PF4-Cre), important regulators of platelet adhesiveness. In contrast, the time to hemostatic plug formation was only minimally affected in mice defective in the extrinsic (tissue factor (TF)low) or the intrinsic (FIX-/-) coagulation pathways, even though platelet adhesion was significantly reduced. Interestingly, fibrin accumulation was markedly increased in lesions of talin1f/f PF4-Cre, IL4R/GPIb-tg and FIX-/- mice, while it was decreased in TFlow mice. These findings suggest that prolonged plasma exposure to TF leads to increased thrombin and fibrin generation in the surrounding tissue. A partial reduction of platelet adhesiveness using clopidogrel led to instability within the hemostatic plug, especially when combined with impaired coagulation in TFlow mice. Conclusions: In summary, we present a novel, highly sensitive method to quantify hemostatic plug formation in mice. This new model has several defining characteristics, including the use of intravital fluorescence microscopy to monitor the hemostatic process, the novel use of laser ablation technology to generate vascular lesions with a defined diameter, the ability to repeatedly disrupt the hemostatic process at the same site of injury, and the possibility to perform multiple injuries along the exposed saphenous vein. Based on its sensitivity towards platelet adhesion defects and its real-time imaging capability, we propose this model as an ideal tool to study the efficacy and safety of antiplatelet agents. Disclosures No relevant conflicts of interest to declare.

Distinct patterns of leukocyte recruitment in the pulmonary microvasculature in response to local and systemic inflammation

The mechanisms of leukocyte recruitment in the pulmonary microvasculature in response to local and systemic inflammation remain elusive. Male C57BL/6 mice received lipopolysaccharide (LPS) intrapulmonary (intratracheally, it) or systemically (intravenously, iv) for 1–18 h. Leukocyte responses in lung were analyzed by use of intravital fluorescence microscopy. Plasma and lung levels of CXC chemokines as well as Mac-1 and F-actin expression in leukocytes and bronchoalveolar leukocytes were quantified. Venular leukocyte rolling was markedly increased in response to local LPS but only marginally after systemic LPS. Leukocyte adhesion in venules was enhanced in both groups although adhesion was higher in mice receiving LPS intratracheally compared with LPS intravenously. Systemic LPS caused more leukocytes trapping in capillaries compared with local LPS. The ratio of adherent leukocytes in venules compared with capillaries was higher in response to local LPS, suggesting that leukocytes were more prone to accumulate in venules in local inflammation and in capillaries in systemic inflammation. Systemic LPS triggered higher F-actin formation and Mac-1 expression in leukocytes compared with local LPS. Local and systemic LPS caused similar increases in CXC chemokines in the lung whereas intravenous endotoxin provoked higher levels of CXC chemokines in the circulation. Interestingly, intratracheal LPS increased recruitment of leukocytes in the alveolar space whereas intravenous LPS was ineffective in promoting leukocyte accumulation in the bronchoalveolar space. In conclusion, our data demonstrate that pulmonary microvascular recruitment of leukocytes differs in local and systemic inflammation, which might be related to premature activation and stiffening of circulating leukocytes in endotoxemia.