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Quantitative FFPE Histopathology of Wound Healing in Mice using Special Stains

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Complex dermal wounds represent major medical and financial burdens, especially in the context of comorbidities such as diabetes, infection and advanced age. New approaches to accelerate and improve, or "fine tune" the healing process, so as to improve the quality of cutaneous wound healing and management, are the focus of intense investigation. Here, we investigate the topical application of a recombinant immune modulating protein which inhibits the interactions of chemokines with glycosaminoglycans, reducing damaging or excess inflammation responses in a splinted full-thickness excisional wound model in mice. M-T7 is a 37 kDa-secreted, virus-derived glycoprotein that has demonstrated therapeutic efficacy in numerous animal models of inflammatory immunopathology. Topical treatment with recombinant M-T7 significantly accelerated wound healing when compared to saline treatment alone. Healed wounds exhibited properties of improved tissue remodeling, as determined by collagen maturation. M-T7 treatment accelerated the rate of peri-wound angiogenesis in the healing wounds with increased levels of TNF, VEGF and CD31. The immune cell response after M-T7 treatment was associated with a retention of CCL2 levels, and increased abundances of arginase-1-expressing M2 macrophages and CD4 T cells. Thus, topical treatment with recombinant M-T7 promotes a pro-resolution environment in healing wounds, and has potential as a novel treatment approach for cutaneous tissue repair.
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Microtubules (MTs) are intracellular polymers that provide structure to the cell, serve as railways for intracellular transport, and regulate many cellular activities, including cell migration. The dynamicity and function of the MT cytoskeleton are determined in large part by its regulatory proteins, including the recently discovered MT severing enzyme Fidgetin-like 2 (FL2). Downregulation of FL2 expression with small interfering RNA (siRNA) results in a more than twofold increase in cell migration rate in vitro as well as translates into improved wound-healing outcomes in in vivo mouse models. Here we utilized a commercially available surfactant polymer dressing (SPD) as a vehicle to deliver FL2 siRNA. To this end we incorporated collagen microparticles containing FL2 siRNA into SPD (SPD-FL2-siRNA) for direct application to the injury site. Topical application of SPD-FL2 siRNA to murine models of full-thickness excision wounds and full-thickness burn wounds resulted in significant improvements in the rate and quality of wound healing, as measured clinically and histologically, compared with controls. Wound healing occurred more rapidly and with high fidelity, resulting in properly organized collagen substructure. Taken together, these findings indicate that the incorporation of FL2 siRNA into existing treatment options is a promising avenue to improve wound outcomes. © Brian O'Rourke et al, 2018; Published by Mary Ann Liebert, Inc. 2018.
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To develop a flexible method of separation and quantification of immunohistochemical staining by means of color image analysis. An algorithm was developed to deconvolve the color information acquired with red-green-blue (RGB) cameras and to calculate the contribution of each of the applied stains based on stain-specific RGB absorption. The algorithm was tested using different combinations of diaminobenzidine, hematoxylin and eosin at different staining levels. Quantification of the different stains was not significantly influenced by the combination of multiple stains in a single sample. The color deconvolution algorithm resulted in comparable quantification independent of the stain combinations as long as the histochemical procedures did not influence the amount of stain in the sample due to bleaching because of stain solubility and saturation of staining was prevented. This image analysis algorithm provides a robust and flexible method for objective immunohistochemical analysis of samples stained with up to three different stains using a laboratory microscope, standard RGB camera setup and the public domain program NIH Image.
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Mast cells (MCs) are an important part of the innate immune system and are abundant in barrier organs such as the skin. They are known primarily for initiating allergic reactions, but many other biological functions have now been described for these cells. Studies have indicated that during wound repair, MCs enhance acute inflammation, stimulate reepithelialization and angiogenesis and promote scarring. MCs have also been linked to abnormal healing, with high numbers of MCs observed in chronic wounds, hypertrophic scars and keloids. Although MCs have gained attention in the wound healing field, several unique features of MCs have yet to be examined in the context of cutaneous repair. These include the ability of MCs to: (i) produce anti-inflammatory mediators; (ii) release mediators without degranulating; and (iii) change their phenotype. Recent findings highlight the complexity of MCs and suggest that more information is needed to understand their complete range of activities during repair.
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In this paper, we propose a new method to obtain the Euclidean distance transformation and the Voronoi diagram based on the exact Euclidean metric for an n-dimensional picture. We present four algorithms to perform the transformation which are constructed by the serial composition of n-dimensional filters. When performed by a general purpose computer, they are faster than the method by H. Yamada for a two-dimensional picture. Those algorithms require only one n-dimensional array for storing input/output pictures and a single one-dimensional array for a work area, if an input picture needs not be preserved.
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The goal of animal wound healing models is to replicate human physiology and predict therapeutic outcomes. There is currently no model of wound healing in rodents that closely parallels human wound healing. Rodents are attractive candidates for wound healing studies because of their availability, low cost, and ease of handling. However, rodent models have been criticized because the major mechanism of wound closure is contraction, whereas in humans reepithelialization and granulation tissue formation are the major mechanisms involved. This article describes a novel model of wound healing in mice utilizing wound splinting that is accurate, reproducible, minimizes wound contraction, and allows wound healing to occur through the processes of granulation and reepithelialization. Our results show that splinted wounds have an increased amount of granulation tissue deposition as compared to controls, but the rate of reepithelialization is not affected. Thus, this model eliminates wound contraction and allows rodents' wounds to heal by epithelialization and granulation tissue formation. Given these analogies to human wound healing, we believe that this technique is a useful model for the study of wound healing mechanisms and for the evaluation of new therapeutic modalities.
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