Project description:The cells of the mononuclear phagocyte system are essential for the correct healing of adult skin wounds, but their specific functions remain ill-defined. The absence of granulation tissue immediately after skin injury makes it challenging to study the role of mononuclear phagocytes at the initiation of this inflammatory stage. To study their recruitment and migratory behavior within the wound bed, we developed a new model for real-time in vivo imaging of the wound, using transgenic mice that express green and cyan fluorescent proteins and specifically target monocytes. Within hours after the scalp injury, monocytes invaded the wound bed. The complete abrogation of this infiltration in monocyte-deficient CCR2(-/-) mice argues for the involvement of classical monocytes in this process. Monocyte infiltration unexpectedly occurred as early as neutrophil recruitment did and resulted from active release from the bloodstream toward the matrix through microhemorrhages rather than transendothelial migration. Monocytes randomly scouted around the wound bed, progressively slowed down, and stopped. Our approach identified and characterized a rapid and earlier than expected wave of monocyte infiltration and provides a novel framework for investigating the role of these cells during early stages of wound healing.
Project description:Defects in apoptotic cell clearance, or efferocytosis, can cause inflammatory diseases and prevent tissue repair due in part to inducing a pro-repair transcriptional program in phagocytic cells like macrophages. While the cellular machinery and metabolic pathways involved in efferocytosis have been characterized, the precise efferocytic response of macrophages is dependent on the identity and macromolecular cues of apoptotic cells, and the complex tissue microenvironment in which efferocytosis occurs. Here, we find that macrophages undergoing active efferocytosis in mid-stage mouse skin wounds in vivo display a pro-repair gene program, while efferocytosis of apoptotic skin fibroblasts in vitro also induces an inflammatory transcription response. These data provide a resource for understanding how the skin wound environment influences macrophage efferocytosis and will be useful for future investigations that define the role of efferocytosis during tissue repair.
Project description:Non-invasive fluorescence retinal imaging in small animals is an important requirement for an array of translational vision applications. The in vivo two-photon imaging of the mouse retina may enable the long-term investigation of the structure and function of healthy and diseased retinal tissue. However, to date, this has only been possible using relatively complex adaptive-optics systems. Here, the optical modeling of the murine eye and of the imaging system is used to achieve correction-free two-photon microscopy through the pupil of a mouse eye to yield high-quality, optically sectioned fundus images. By remotely scanning the focus using an electronically tunable lens, high-resolution three-dimensional fluorescein angiograms and cellular-scale images are acquired, thus introducing a correction-free baseline performance level for two-photon in vivo retinal imaging. Moreover, the system enables functional calcium imaging of repeated retinal responses to light stimulation using the genetically encoded indicator, GCaMP6s. These results and the simplicity of the new add-on optics are an important step toward several structural, functional, and multimodal imaging applications that will benefit from the tight optical sectioning and the use of near-infrared light.
Project description:Microporous annealed particle (MAP) scaffolds are injectable granular materials comprised of micron sized hydrogel particles (microgels). The diameter of these microgels directly determines the size of the interconnected void space between particles where infiltrating or encapsulated cells reside. This tunable porosity allows the authors to use MAP scaffolds to study the impact of spatial confinement (SC) on both cellular behaviors and the host response to biomaterials. Despite previous studies showing that pore size and SC influence cellular phenotypes, including mitigating macrophage inflammatory response, there is still a gap in knowledge regarding how SC within a biomaterial modulates immune cell recruitment in vivo in wounds and implants. Thus, the immune cell profile within confined and unconfined biomaterials is studied using small (40 µm), medium (70 µm), and large (130 µm) diameter spherical microgels, respectively. This work uncovered that MAP scaffolds impart regenerative wound healing with an IgG1-biased Th2 response. MAP scaffolds made with large microgels promote a balanced pro-regenerative macrophage response, resulting in enhanced wound healing with mature collagen regeneration and reduced inflammation levels.
Project description:The signal and resolution during in vivo imaging of the mouse brain is limited by sample-induced optical aberrations. We find that, although the optical aberrations can vary across the sample and increase in magnitude with depth, they remain stable for hours. As a result, two-photon adaptive optics can recover diffraction-limited performance to depths of 450 μm and improve imaging quality over fields of view of hundreds of microns. Adaptive optical correction yielded fivefold signal enhancement for small neuronal structures and a threefold increase in axial resolution. The corrections allowed us to detect smaller neuronal structures at greater contrast and also improve the signal-to-noise ratio during functional Ca(2+) imaging in single neurons.
Project description:Understanding macrophage heterogeneity in tissue repair is a major challenge. Here, we describe a protocol that combines isolation of immune cells from skin wounds with subsequent flow-cytometry-based sorting of wound macrophages and single-cell RNA sequencing. We use a modified version of the original Smart-seq2 protocol to increase speed and accuracy. This protocol is useful for analyzing the pronounced heterogeneity of activation phenotypes in wound macrophages and might be adapted to other experimental models of skin inflammation. For complete details on the use and execution of this protocol, please refer to Willenborg et al. (2021).
Project description:Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) frequently causes skin and soft tissue infections, including impetigo, cellulitis, folliculitis, and infected wounds and ulcers. Uncomplicated CA-MRSA skin infections are typically managed in an outpatient setting with oral and topical antibiotics and/or incision and drainage, whereas complicated skin infections often require hospitalization, intravenous antibiotics, and sometimes surgery. The aim of this study was to develop a mouse model of CA-MRSA wound infection to compare the efficacy of commonly used systemic and topical antibiotics. A bioluminescent USA300 CA-MRSA strain was inoculated into full-thickness scalpel wounds on the backs of mice and digital photography/image analysis and in vivo bioluminescence imaging were used to measure wound healing and the bacterial burden. Subcutaneous vancomycin, daptomycin, and linezolid similarly reduced the lesion sizes and bacterial burden. Oral linezolid, clindamycin, and doxycycline all decreased the lesion sizes and bacterial burden. Oral trimethoprim-sulfamethoxazole decreased the bacterial burden but did not decrease the lesion size. Topical mupirocin and retapamulin ointments both reduced the bacterial burden. However, the petrolatum vehicle ointment for retapamulin, but not the polyethylene glycol vehicle ointment for mupirocin, promoted wound healing and initially increased the bacterial burden. Finally, in type 2 diabetic mice, subcutaneous linezolid and daptomycin had the most rapid therapeutic effect compared with vancomycin. Taken together, this mouse model of CA-MRSA wound infection, which utilizes in vivo bioluminescence imaging to monitor the bacterial burden, represents an alternative method to evaluate the preclinical in vivo efficacy of systemic and topical antimicrobial agents.
Project description:Monocytes and macrophages (Mo/MΦ) play critical roles in all phases of skin wound healing. The majority of these cells are thought to be recruited from blood Mo; however, the role local proliferation of Mo/MΦ in the wound has not been defined. Therefore, we tested the hypothesis that local proliferation of Mo and/or MΦ contributes to their accumulation during wound healing. Male C57Bl/6 mice (N = 4-9/group) were subjected to excisional skin wounding. Proliferating Mo/MΦ (F4/80+Ki67+) were observed in wound cryosections, peaking on day 5 post-wounding. Cell cycle analysis on cells isolated from skin tissue revealed that wounding increased both the number and percentage of inflammatory Ly6C+F4/80lo/- Mo/MΦ in the S/G2/M phases, peaking on day 6 post-wounding. In contrast, more mature Ly6C-F4/80+ cells were found predominantly in the G0 phase with less than 1% cells in S/G2/M phase following injury. In peripheral blood, Mo were very rarely found in the S/G2/M phase, suggesting that the wound environment triggered the Ly6C+F4/80lo/- Mo proliferative response. Furthermore, injury induced several potential regulators of proliferation in wounds, including IL-1β and IL-6, and wound Mo/MΦ expressed surface receptors for these cytokines. However, wound Mo/MΦ proliferation was not altered in IL-1R1 knockout (KO) or IL-6 KO mice. In summary, our findings indicate that proliferation contributes to Mo/MΦ accumulation in wounds and, contrary to findings in other pathophysiologic conditions, Ly6C+/F4/80lo/- Mo/MΦ proliferate during skin wound healing whereas mature Ly6C-F4/80+ MΦ do not.
Project description:Proper skin barrier function is paramount for our survival, and, suffering injury, there is an acute need to restore the lost barrier and prevent development of a chronic wound. We hypothesize that rapid wound closure is more important than immediate perfection of the barrier, whereas specific treatment may facilitate perfection. The aim of the current project was therefore to evaluate the quality of restored tissue down to the molecular level. We used Göttingen minipigs with a multi-technique approach correlating wound healing progression in vivo over three weeks, monitored by classical methods (e.g., histology, trans-epidermal water loss (TEWL), pH) and subsequent physicochemical characterization of barrier recovery (i.e., small and wide-angle X-ray diffraction (SWAXD), polarization transfer solid-state NMR (PTssNMR), dynamic vapor sorption (DVS), Fourier transform infrared (FTIR)), providing a unique insight into molecular aspects of healing. We conclude that although acute wounds sealed within two weeks as expected, molecular investigation of stratum corneum (SC) revealed a poorly developed keratin organization and deviations in lipid lamellae formation. A higher lipid fluidity was also observed in regenerated tissue. This may have been due to incomplete lipid conversion during barrier recovery as glycosphingolipids, normally not present in SC, were indicated by infrared FTIR spectroscopy. Evidently, a molecular approach to skin barrier recovery could be a valuable tool in future development of products targeting wound healing.