Skinfold Chamber Research Articles

Surface Modification of Porous Polyethylene Implants with an Albumin-Based Nanocarrier-Release System.

Background: Porous polyethylene (PPE) implants are used for the reconstruction of tissue defects but have a risk of rejection in case of insufficient ingrowth into the host tissue. Various growth factors can promote implant ingrowth, yet a long-term gradient is a prerequisite for the mediation of these effects. As modification of the implant surface with nanocarriers may facilitate a long-term gradient by sustained factor release, implants modified with crosslinked albumin nanocarriers were evaluated in vivo. Methods: Nanocarriers from murine serum albumin (MSA) were prepared by an inverse miniemulsion technique encapsulating either a low- or high-molar mass fluorescent cargo. PPE implants were subsequently coated with these nanocarriers. In control cohorts, the implant was coated with the homologue non-encapsulated cargo substance by dip coating. Implants were consequently analyzed in vivo using repetitive fluorescence microscopy utilizing the dorsal skinfold chamber in mice for ten days post implantation. Results: Implant-modification with MSA nanocarriers significantly prolonged the presence of the encapsulated small molecules while macromolecules were detectable during the investigated timeframe regardless of the form of application. Conclusions: Surface modification of PPE implants with MSA nanocarriers results in the alternation of release kinetics especially when small molecular substances are used and therefore allows a prolonged factor release for the promotion of implant integration.

An adapted dorsal skinfold model used for 4D intravital followed by whole-mount imaging to reveal endothelial cell\u2013pericyte association

Endothelial cells and pericytes are highly dynamic vascular cells and several subtypes, based on their spatiotemporal dynamics or molecular expression, are believed to exist. The interaction between endothelial cells and pericytes is of importance in many aspects ranging from basic development to diseases like cancer. Identification of spatiotemporal dynamics is particularly interesting and methods to studies these are in demand. Here we describe the technical details of a method combining the benefits of high resolution intravital imaging and whole-mount histology. With intravital imaging using an adapted light weight dorsal skinfold chamber we identified blood flow patterns and spatiotemporal subtypes of endothelial cells and pericytes in a 4D (XYZ, spatial+T, time dimension) manner as representative examples for this model. Thereafter the tissue was extracted and stained as a whole-mount, by which the position and volumetric space of endothelial cells as well as pericytes were maintained, to identify molecular subtypes. Integration of the two imaging methods enabled 4D dissection of endothelial cell–pericyte association at the molecular level.

Connexin Hemichannel Activation by S-Nitrosoglutathione Synergizes Strongly with Photodynamic Therapy Potentiating Anti-Tumor Bystander Killing.

Simple SummaryBystander effects depend on direct cell-cell communication and/or paracrine signaling mediated by the release of soluble factors into the extracellular environment and may greatly influence therapy outcome. Although the limited data available suggest a role for intercellular gap junction channels, far less is known about the role of connexin hemichannels. Here, we investigated bystander effects induced by photodynamic therapy in syngeneic murine melanoma models in vivo. We determined that (i) photoactivation of a photosensitizer triggered calcium-dependent cell death pathways in both irradiated and bystander tumor cells; (ii) hemichannel activity and adenosine triphosphate release were key factors for the induction of bystander cell death; and (iii) bystander cell killing and antitumor response elicited by photodynamic therapy were greatly enhanced by combination treatment with S-nitrosoglutathione, which promoted hemichannel opening in these experimental conditions. Therefore, these findings in a preclinical model have important translational potential.In this study, we used B16-F10 cells grown in the dorsal skinfold chamber (DSC) preparation that allowed us to gain optical access to the processes triggered by photodynamic therapy (PDT). Partial irradiation of a photosensitized melanoma triggered cell death in non-irradiated tumor cells. Multiphoton intravital microscopy with genetically encoded fluorescence indicators revealed that bystander cell death was mediated by paracrine signaling due to adenosine triphosphate (ATP) release from connexin (Cx) hemichannels (HCs). Intercellular calcium (Ca2+) waves propagated from irradiated to bystander cells promoting intracellular Ca2+ transfer from the endoplasmic reticulum (ER) to mitochondria and rapid activation of apoptotic pathways. Combination treatment with S-nitrosoglutathione (GSNO), an endogenous nitric oxide (NO) donor that biases HCs towards the open state, greatly potentiated anti-tumor bystander killing via enhanced Ca2+ signaling, leading to a significant reduction of post-irradiation tumor mass. Our results demonstrate that HCs can be exploited to dramatically increase cytotoxic bystander effects and reveal a previously unappreciated role for HCs in tumor eradication promoted by PDT.

Vascularization of Microvascular Fragment Isolates from Visceral and Subcutaneous Adipose Tissue of Mice

Background:Adipose tissue-derived microvascular fragments (MVF) represent effective vascularization units for tissue engineering. Most experimental studies in rodents exclusively use epididymal adipose tissue as a visceral fat source for MVF isolation. However, in future clinical practice, MVF may be rather isolated from liposuctioned subcutaneous fat tissue of patients. Therefore, we herein compared the vascularization characteristics of MVF isolates from visceral and subcutaneous fat tissue of murine origin.Methods:MVF isolates were generated from visceral and subcutaneous fat tissue of donor mice using two different enzymatic procedures. For in vivo analyses, the MVF isolates were seeded onto collagen-glycosaminoglycan scaffolds and implanted into full-thickness skin defects within dorsal skinfold chambers of recipient mice.Results:By means of the two isolation procedures, we isolated a higher number of MVF from visceral fat tissue when compared to subcutaneous fat tissue, while their length distribution, viability and cellular composition were comparable in both groups. Intravital fluorescence microscopy as well as histological and immunohistochemical analyses revealed a significantly reduced vascularization of implanted scaffolds seeded with subcutaneous MVF isolates when compared to implants seeded with visceral MVF isolates. Light and scanning electron microscopy showed that this was due to high amounts of undigested connective tissue within the subcutaneous MVF isolates, which clogged the scaffold pores and prevented the interconnection of individual MVF into new microvascular networks.Conclusion:These findings indicate the need for improved protocols to generate connective tissue-free MVF isolates from subcutaneous fat tissue for future translational studies.

Time-Dependent Dynamics Required for the Degradation and Restoration of the Vascular Endothelial Glycocalyx Layer in Lipopolysaccharide-Treated Septic Mice.

The endothelial glycocalyx (GCX) plays a key role in the development of organ failure following sepsis. Researchers have investigated GCX degradation caused by pathological conditions. Nonetheless, the GCX restoration process remains poorly understood. Herein, we developed a model in which GCX restoration could be reproduced in mice using in vivo imaging and a dorsal skinfold chamber (DSC). The severity of sepsis was controlled by adjusting the dose of lipopolysaccharide (LPS) used to trigger GCX degradation in BALB/c mice. We evaluated the GCX thickness, leukocyte-endothelial interactions, and vascular permeability using in vivo imaging through DSC under intravital microscopy. The plasma concentration of syndecan-1(Sdc-1), a GCX structural component, was also determined as a marker of GCX degradation. Thus, we developed a reproducible spontaneous GCX recovery model in mice. Degraded GCX was restored within 24 h by the direct visualization of the endothelial GCX thickness, and leukocyte-endothelial interactions. In contrast, indirectly related indicators of recovery from sepsis, such as body weight and blood pressure, required a longer recovery time. This model can be used to study intractable angiopathy following sepsis.

Targeting sphingosine kinase-1 with the low MW inhibitor SKI-5C suppresses the development of endometriotic lesions in mice.

Limited evidence suggests that the sphingosine-1-phosphate/sphingosine kinase 1 (S1P/SPHK1) signalling pathway is involved in the pathogenesis of endometriosis. Therefore, we analyzed in this study whether the inhibition of SPHK1 and, consequently, decreased levels of S1P affected the vascularization and growth of endometriotic lesions. Endometriotic lesions were surgically induced in the peritoneal cavity and the dorsal skinfold chamber of female BALB/c mice. The animals received a daily dose of the SPHK1 inhibitor SKI-5C or vehicle (control). Analyses involved the determination of lesion growth, cyst formation, microvessel density and cell proliferation within peritoneal endometriotic lesions by means of high-resolution ultrasound imaging, caliper measurement, histology and immunohistochemistry. In the dorsal skinfold chamber model the development of newly formed microvascular networks and their microhemodynamic parameters within endometriotic lesions were investigated by means of intravital fluorescence microscopy. SKI-5C significantly inhibited the development and vascularization of peritoneal endometriotic lesions, as indicated by a reduced growth and cyst formation, a lower microvessel density and a suppressed cell proliferation, when compared to vehicle-treated controls. Endometriotic lesions in dorsal skinfold chambers of SKI-5C-treated animals exhibited a significantly smaller lesion size, lower functional microvessel density, smaller microvessel diameters and a reduced blood perfusion of the newly developing microvascular networks. SPHK1/S1P signalling promotes the establishment and progression of endometriotic lesions. The inhibition of this pathway suppresses the development of endometriotic lesions, suggesting SPHK1 as a potential novel target for future endometriosis therapy.

Erythropoietin exposure of isolated pancreatic islets accelerates their revascularization after transplantation

AimsThe exposure of isolated pancreatic islets to pro-angiogenic factors prior to their transplantation represents a promising strategy to accelerate the revascularization of the grafts. It has been shown that erythropoietin (EPO), a glycoprotein regulating erythropoiesis, also induces angiogenesis. Therefore, we hypothesized that EPO exposure of isolated islets improves their posttransplant revascularization.MethodsFlow cytometric, immunohistochemical and quantitative real-time (qRT)-PCR analyses were performed to study the effect of EPO on the viability, cellular composition and gene expression of isolated islets. Moreover, islets expressing a mitochondrial or cytosolic H2O2 sensor were used to determine reactive oxygen species (ROS) levels. The dorsal skinfold chamber model in combination with intravital fluorescence microscopy was used to analyze the revascularization of transplanted islets.ResultsWe found that the exposure of isolated islets to EPO (3 units/mL) for 24 h does not affect the viability and the production of ROS when compared to vehicle-treated and freshly isolated islets. However, the exposure of islets to EPO increased the number of CD31-positive cells and enhanced the gene expression of insulin and vascular endothelial growth factor (VEGF)-A. The revascularization of the EPO-cultivated islets was accelerated within the initial phase after transplantation when compared to both controls.ConclusionThese findings indicate that the exposure of isolated islets to EPO may be a promising approach to improve clinical islet transplantation.

Aircraft noise exposure drives the activation of white blood cells and induces microvascular dysfunction in mice

Epidemiological studies showed that traffic noise has a dose-dependent association with increased cardiovascular morbidity and mortality. Whether microvascular dysfunction contributes significantly to the cardiovascular health effects by noise exposure remains to be established. The connection of inflammation and immune cell interaction with microvascular damage and functional impairment is also not well characterized. Male C57BL/6J mice or gp91phox−/y mice with genetic deletion of the phagocytic NADPH oxidase catalytic subunit (gp91phox or NOX-2) were used at the age of 8 weeks, randomly instrumented with dorsal skinfold chambers and exposed or not exposed to aircraft noise for 4 days. Proteomic analysis (using mass spectrometry) revealed a pro-inflammatory phenotype induced by noise exposure that was less pronounced in noise-exposed gp91phox−/y mice. Using in vivo fluorescence microscopy, we found a higher number of adhesive leukocytes in noise-exposed wild type mice. Dorsal microvascular diameter (by trend), red blood cell velocity, and segmental blood flow were also decreased by noise exposure indicating microvascular constriction. All adverse effects on functional parameters were normalized or improved at least by trend in noise-exposed gp91phox−/y mice. Noise exposure also induced endothelial dysfunction in cerebral microvessels, which was associated with higher oxidative stress burden and inflammation, as measured using video microscopy. We here establish a link between a pro-inflammatory phenotype of plasma, activation of circulating leukocytes and microvascular dysfunction in mice exposed to aircraft noise. The phagocytic NADPH oxidase was identified as a central player in the underlying pathophysiological mechanisms.

GBT1118 Diminishes Vaso-Occlusion in Sickle Cell Disease Mice

Recurrent, painful vaso-occlusive events are one of the most debilitating complications of sickle cell disease (SCD). The process involves occlusion of blood flow through small vessels by heterogeneous cell clusters comprised of red blood cells (RBCs), neutrophils, and platelets. Activated endothelial cells localize and facilitate vaso-occlusion. The central triggering event in the process is hemoglobin S (HbS) polymerization and the consequent damage to RBCs. Many recent clinical efforts to prevent vaso-occlusive events have focused on downstream events mediated by inflammatory molecules. The present work however studies the triggering event by assessing the direct impact of allosteric modulators of hemoglobin oxygen (Hb-O2) affinity on vaso-occlusion in a murine model of SCD.In this study, we used GBT1118 (a structural analog of GBT440) in a modified dorsal skin fold model in an HbSS knock-in mouse model of SCD (Townes; Wu et al., Blood 2006). GBT1118 binds covalently and reversibly via an imine intermediate to the N-terminal valine of the hemoglobin α chain, and allosterically increases the intracellular hemoglobin affinity for oxygen (Dufu et al., Am J Physiol Heart Circ Physiol 2017). The dorsal skin fold chamber model has been used previously to study vaso-occlusion in SCD mice (Kalambur et al., Am J Hematol 2004). In this present study, mice were treated acutely by oral gavage with GBT1118 or vehicle control in a blinded fashion. Two hours after drug treatment, a baseline of vascular patency at ambient air was established using intravital microscopy to count blood vessels with blood flow. Subsequently animals were exposed to 1 h hypoxia (7.0% oxygen) and 1 h reoxygenation at ambient air. At that time the previously observed blood vessels were monitored again, and the percentage of occluded vessels was recorded. We observed a significant reduction of vaso-occlusion in animals that had been treated with GBT1118 (p<0.05).In summary, while improved survival has been described in hypoxia-challenged SCD mice treated with allosteric modulators of hemoglobin (Abdulmalik et al., Br J Haematol 2005), our study demonstrates for the first time that a hemoglobin allosteric modulator can reduce vaso-occlusion in a mouse model of SCD. The protective effect elicited by GBT1118 may either be attributed to improved oxygenation or allosteric modulation of HbS. Both mechanisms may directly delay hypoxia-triggered polymerization of HbS, sufficiently for RBCs to transit the microcirculation without sickling, without adhering to vessel wall, and therefore without causing vaso-occlusion.The results of this study expand on the potential mechanisms by which allosteric modulators of Hb-O2 affinity may contribute to the treatment of SCD, in addition to previously described hematological benefits. DisclosuresAlt:Bayer: Equity Ownership; Global Blood Therapeutics: Employment, Equity Ownership. Liu:Global Blood Therapeutics: Employment. Li:Global Blood Therapeutics: Employment, Equity Ownership. Silva:Global Blood Therapeutics: Employment, Equity Ownership. Rademacher:Global Blood Therapeutics: Employment, Equity Ownership. Zou:Global Blood Therapeutics: Employment, Equity Ownership. Patel:Global Blood Therapeutics: Employment, Equity Ownership. Dufu:Global Blood Therapeutics: Employment, Equity Ownership. Oksenberg:Global Blood Therapeutics: Employment, Equity Ownership. Kauser:Global Blood Therapeutics: Employment, Equity Ownership.

Differences in growth and vascularization of ectopic menstrual and non-menstrual endometrial tissue in mouse models of endometriosis.

Is there a difference in the growth and vascularization between murine endometriotic lesions originating from menstrual or non-menstrual endometrial fragments? Endometriotic lesions developing from menstrual and non-menstrual tissue fragments share many similarities, but also exhibit distinct differences in growth and vascularization, particularly under exogenous estrogen stimulation. Mouse models are increasingly used in endometriosis research. For this purpose, menstrual or non-menstrual endometrial fragments serve for the induction of endometriotic lesions. So far, these two fragment types have never been directly compared under identical experimental conditions. This was a prospective experimental study in a murine peritoneal and dorsal skinfold chamber model of endometriosis. Endometrial tissue fragments from menstruated (n = 15) and non-menstruated (n = 21) C57BL/6 mice were simultaneously transplanted into the peritoneal cavity or dorsal skinfold chamber of non-ovariectomized (non-ovx, n = 17), ovariectomized (ovx, n = 17) and ovariectomized, estrogen-substituted (ovx+E2, n = 17) recipient animals and analyzed throughout an observation period of 28 and 14 days, respectively. The engraftment, growth and vascularization of the newly developing endometriotic lesions were analyzed by means of high-resolution ultrasound imaging, intravital fluorescence microscopy, histology and immunohistochemistry. Menstrual and non-menstrual tissue fragments developed into peritoneal endometriotic lesions without differences in growth, microvessel density and cell proliferation in non-ovx mice. Lesion formation out of both fragment types was markedly suppressed in ovx mice. In case of non-menstrual tissue fragments, this effect could be reversed by estrogen supplementation. In contrast, endometriotic lesions originating from menstrual tissue fragments exhibited a significantly smaller volume in ovx+E2 mice, which may be due to a reduced hormone sensitivity. Moreover, menstrual tissue fragments showed a delayed vascularization and a reduced blood perfusion after transplantation into dorsal skinfold chambers when compared to non-menstrual tissue fragments, indicating different vascularization modes of the two fragment types. To limit the role of chance, the experiments were conducted under standardized laboratory conditions. Statistical significance was accepted for a value of P < 0.05. N/A. Endometriotic lesions were induced by syngeneic tissue transplantation into recipient mice without the use of pathological endometriotic tissue of human nature. Therefore, the results obtained in this study may not fully relate to human patients with endometriosis. The present study significantly contributes to the characterization of common murine endometriosis models. These models represent important tools for studies focusing on the basic mechanisms of endometriosis and the development of novel therapeutic strategies for the treatment of this frequent gynecological disease. The presented findings indicate that the combination of different experimental models and approaches may be the most appropriate strategy to study the pathophysiology and drug sensitivity of a complex disease such as endometriosis under preclinical conditions. There was no specific funding of this study. The authors have no conflicts of interest to declare.

Evaluation of a fluorescence endoscope in murine in-vivo auto-fluorescence glioma models

IntroductionThe value of extended and radical resection of high grade gliomas remains controversial, but the neurosurgical procedure is still vital for effective cancer treatment. Fluorescence guided surgery provides aggressive resection within the tumor margins even on microscopic levels. Aim of this study was to evaluate if a new developed fluorescence endoscope can improve intraoperative vision and tumor delineation. MethodsAn autofluoresence C6 glioma cell line was established via GFP-transfection. These GFP-C6 glioma cells were transplanted both in a dorsal skinfold chamber of the mouse and orthotopically in a cranial window chamber of the mouse. After five days, tumors were examinated by intravital fluorescence microscopy, a standard fluorescence operation microscope and a fluorescence endoscope. Images were compared in terms of visualization, magnification and delineation of tumor cells from host tissue. ResultsThe fluorescence endoscope showed improved image quality and higher magnifications compared to the operation microscope. Even smallest tumor extensions were visualized by the fluorescence endoscope nearly reaching the quality of an intravital fluorescence microscope. ConclusionsIn summary better visualization can improve the intraoperative decision making of the surgeons. So endoscopic assistance can be seen as a promising tool for the fluorescence guided resection of high grade gliomas in the next years.

Feasibility study of incident dark-field video microscope for measuring microcirculatory variables in the mouse dorsal skinfold chamber model.

BackgroundDespite the importance of microcirculation in organ function, monitoring microcirculation is not a routine practice. With developments in microscopic technology, incident dark field (IDF) microscopy (Cytocam) has allowed visualization of the microcirculation. Dorsal skinfold chamber (DSC) mouse model has been used to investigate microcirculation physiology. By employing Cytocam-IDF imaging with DSC model to assess microcirculatory alteration in lipopolysaccharide (LPS)-induced endotoxemia, we attempted to validate availability of Cytocam-IDF imaging of microcirculation.MethodsDSC was implanted in eight BALB/c mice for each group; control and sepsis. Both groups were given 72 hours to recover from surgery. The sepsis group had an additional 24-hour period of recovery post-LPS injection (4 mg/kg). Subsequently, a video of the microcirculation was recorded using Cytocam. Data on microcirculatory variables were obtained. Electron microscopy was implemented using lanthanum fixation to detect endothelial glycocalyx degradation.ResultsThe microcirculatory flow index was significantly lower (control, 2.8±0.3; sepsis, 2.1±0.8; P=0.033) and heterogeneity index was considerably higher (control, 0.10±0.15; sepsis, 0.53±0.48; P=0.044) in the sepsis group than in the control group. Electron microscopy revealed glycocalyx demolishment in the sepsis group.ConclusionsCytocam showed reliable ability for observing changes in the microcirculation under septic conditions in the DSC model. The convenience and good imaging quality and the automatic analysis software available for Cytocam-IDF imaging, along with the ability to perform real-time in vivo experiments in the DSC model, are expected to be helpful in future microcirculation investigations.

TNF-\u03b1-Inhibition Improves the Biocompatibility of Porous Polyethylene Implants In Vivo

Background: To improve the biocompatibility of porous polyethylene (PPE) implants and expand their application range for reconstructive surgery in poorly vascularized environments, implants were coated with tumor necrosis factor α (TNFα) inhibitor Etanercept. While approved for systemic application, local application of the drug is a novel experimental approach. Microvascular and mechanical integration as well as parameters of inflammation were analyzed in vivo.Methods: PPE implants were coated with Etanercept and extracellular matrix (ECM) components prior to implantation into dorsal skinfold chambers of C57BL/6 mice. Fluorescence microscopy analyses of angiogenesis and local inflammatory response were thrice performed in vivo over a period of 14 days to assess tissue integration and biocompatibility. Uncoated implants and ECM-coated implants served as controls.Results: TNFα inhibition with Etanercept led to a reduced local inflammatory response: leukocyte-endothelial cell adherence was significantly lowered compared to both control groups (n = 6/group) on days 3 and 14, where the lowest values were reached: 3573.88 leukocytes/mm-2 ± 880.16 (uncoated implants) vs. 3939.09 mm-2 ± 623.34 (Matrigel only) vs. 637.98 mm-2 + 176.85 (Matrigel and Etanercept). Implant-coating with Matrigel alone and Matrigel and Etanercept led to significantly higher vessel densities 7 and 14 days vs. 3 days after implantation and compared to uncoated implants. Mechanical implant integration as measured by dynamic breaking strength did not differ after 14 days.Conclusion: Our data show a reduced local inflammatory response to PPE implants after immunomodulatory coating with Etanercept in vivo, suggesting improved biocompatibility. Application of this tissue engineering approach is therefore warranted in models of a compromised host environment.

Lingering Dynamics in Microvascular Blood Flow

The microvascular networks in the body of vertebrates consist of the smallest vessels such as arterioles, capillaries, and venules. The flow of red blood cells (RBCs) through these networks ensures the gas exchange in as well as the transport of nutrients to the tissues. Any alterations in this blood flow may have severe implications on the health state. Because the vessels in these networks obey dimensions similar to the diameter of RBCs, dynamic effects on the cellular scale play a key role. The steady progression in the numerical modeling of RBCs, even in complex networks, has led to novel findings in the field of hemodynamics, especially concerning the impact and the dynamics of lingering events when a cell meets a branch of the network. However, these results are yet to be matched by a detailed analysis of the lingering experiments in vivo. To quantify this lingering effect in in vivo experiments, this study analyzes branching vessels in the microvasculature of Syrian golden hamsters via intravital microscopy and the use of an implanted dorsal skinfold chamber. It also presents a detailed analysis of these lingering effects of cells at the apex of bifurcating vessels, affecting the temporal distribution of plasmatic zones of blood flow in the branches and even causing a partial blockage in severe cases.

Leptin promotes adipocytes survival in non-vascularized fat grafting via perfusion increase

BackgroundThough autologous fat transplantation is regularly and successfully performed in plastic surgery, little is known about the factors that contribute to the rise of preadipocytes and how the viability of adipocytes is regulated. As sufficient blood supply is a key parameter for the transplant's survival, we opted to analyse the development of preadipocytes within the fat transplant via stimulation of tissue perfusion with the angiogenesis enhancing hormone leptin. MethodsIn a murine (C57BL/6N) model inguinal fat was autologously transplanted into a dorsal skinfold chamber. In the intervention group the fat transplant was treated with local administration of leptin (3 μg/ml) at days 3, 7 and 10 after transplantation. Saline solution was administered respectively in the control group. On the postoperative days 3, 7, 10, and 15 intra vital microscopy was done to assess the functional vessel density, vessel diameter, adipocyte survival and preadipocyte development. The study was completed by histological tissue analysis on days 15 after transplantation. ResultsLeptin administration leads to an increase of angiogenesis, which starts from day 7 after implantation and elevates perfusion as well as functional vessel density FVD at days 10 and 15 after transplantation. Perfusion develops first from the border zones of the transplant. Histological evaluation showed that the percentage of perilipin positive adipocytes increased markedly in the study group of mice. Moreover, fat transplants of mice of the leptin group disclosed significantly higher Pref-1 positive cells than fat transplants of the control group. The findings reported in this study indicate that the leptin can enhance the survival and the quality of grafted fat tissue, which may be due to induction of angiogenesis. ConclusionLeptin administration to fat transplants induced an increase in angiogenesis in the transplanted tissue and may play a role in reducing the resorption rate of lipoaspirates.

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.

Longitudinal Intravital Imaging of Tumor-Infiltrating Lymphocyte Motility in Breast Cancer Models.

Immunoreactive dynamics of tumor-infiltrating lymphocytes (TILs) within the tumor microenvironment in breast cancer are not well understood. This study aimed to investigate the spatiotemporal cellular dynamics of TILs in breast cancer models. Breast cancer cells were implanted into the dorsal skinfold chamber of BALB/c nude mice, and T lymphocytes were adoptively transferred. Longitudinal intravital imaging was performed, and the spatiotemporal dynamics of TILs were assessed. In the 4T1 model, TILs progressively exhibited increased motility, and their motility inside the tumor was significantly higher than that outside the tumor. In the MDA-MB-231 model, the motility of TILs progressively decreased after an initial increase. TIL motility in the MDA-MB-231 and MCF-7 models differed significantly, suggesting an association between programmed death-ligand 1 expression levels and TIL motility, which warrants further investigation. Furthermore, intravital imaging of TILs can be a useful method for addressing dynamic interactions between TILs and breast cancer cells.

Microvascular fragment spheroids: Three-dimensional vascularization units for tissue engineering and regeneration

Adipose tissue-derived microvascular fragments (MVF) serve as vascularization units in tissue engineering and regenerative medicine. Because a three-dimensional cellular arrangement has been shown to improve cell function, we herein generated for the first time MVF spheroids to investigate whether this further increases their vascularization potential. These spheroids exhibited a morphology, size, and viability comparable to that of previously introduced stromal vascular fraction (SVF) spheroids. However, MVF spheroids contained a significantly higher number of CD31-positive endothelial cells and α-smooth muscle actin (SMA)-positive perivascular cells, resulting in an enhanced angiogenic sprouting activity. Accordingly, they also exhibited an improved in vivo vascularization and engraftment after transplantation into mouse dorsal skinfold chambers. These findings indicate that MVF spheroids are superior to SVF spheroids and, thus, may be highly suitable to improve the vascularization of tissue defects and implanted tissue constructs.

In vivo analysis of noise dependent activation of white blood cells and microvascular dysfunction in mice

This article contains supporting information on data collection for the research article entitled “Aircraft noise exposure drives the activation of white blood cells and induces microvascular dysfunction in mice” by Eckrich et al. We found that noise-induced stress triggered microvascular dysfunction via involvement of innate immune-derived reactive oxygen species. In this article, we present the instrumentation of mice with dorsal skinfold chambers for in vivo microscopic imaging of blood flow, interaction of leukocytes with the vascular wall (also by fluorescent labelling of blood cells) and vessel diameter. In addition, we explain the preparation of cerebral arterioles for measurement of vascular reactivity in vitro.•visualization of noise-dependent effects in dorsal skinfold chamber.•in vivo microscopy of noise-dependent activation of white blood cells.•analysis of noise-dependent microvascular dysfunction in dorsal skinfold chamber and cannulated cerebral arterioles.

New model in diabetic mice to evaluate the effects of insulin therapy on biofilm development in wounds.

Objective: Diabetic patients suffer more frequently from biofilm-associated infections than normoglycemic patients. Well described in the literature is a relationship between elevated blood glucose levels in patients and the occurrence of biofilm-associated wound infections. Nevertheless, the underlying pathophysiological pathways leading to this increased infection vulnerability and its effects on biofilm development still need to be elucidated. We developed in our laboratory a model to allow the investigation of a biofilm-associated wound infection in diabetic mice under controlled insulin treatment. Methods: A dorsal skinfold chamber was used on 16 weeks old BKS.Cg-Dock7m +/+ Leprdb/J mice and a wound within the observation field of the dorsal skinfold chamber was created. These wounds were infected with Staphylococcus aureus ATCC 49230 (106 cells/mL). Simultaneously, we implanted implants for sustained insulin release into the ventral subcutaneous tissue (N=5 mice). Mice of the control group (N=5) were treated with sham implants. Serum glucose levels were registered before intervention and daily after the operation. Densitometrical analysis of the wound size was performed at day 0, 3, and 6 after intervention. Mice were sacrificed on day 6 and wound tissue was submitted to fluorescence in situ hybridization (FISH) and colony forming unit (CFU) analysis in addition to immunohistochemical staining to observe wound healing. Experiments were carried out in accordance with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals (protocol number 05/19).Results: The insulin implants were able to reduce blood glucose levels in the mice. Hence, the diabetic mice in the intervention group were normoglycemic after the implantation. The combination with the dorsal skinfold chamber allowed for continuous, in vivo measurements of the infection development. Implantation of the insulin implant and the dorsal skinfold chamber was a tolerable condition for the diabetic mice. We succeeded to realize reproducible biofilm infections in the animals. Discussion: We developed a novel model to assess interactions between blood glucose level and S. aureus-induced biofilm-associated wound infections. The combination of the dorsal skinfold chamber model with a sustained insulin treatment has not been described so far. It allows a broad field of glucose and insulin dependent studies of infection.