A novel mouse-driven ex vivo flow chamber for the study of leukocyte and platelet function

Various in vitro and in vivo techniques exist for study of the microcirculation. Whereas in vivo systems impress with their physiological fidelity, in vitro systems excel in the amount of reduction that can be achieved. Here we introduce the autoperfused ex vivo flow chamber designed to study murine leukocytes and platelets under well-defined hemodynamic conditions. In our model, the murine heart continuously drives the blood flow through the chamber, providing a wide range of physiological shear rates. We used a balance of force approach to quantify the prevailing forces at the chamber walls. Numerical simulations show the flow characteristics in the chamber based on a shear-thinning fluid model. We demonstrate specific rolling of wild-type leukocytes on immobilized P-selectin, abolished by a blocking MAb. When uncoated, the surfaces having a constant shear rate supported individual platelet rolling, whereas on areas showing a rapid drop in shear platelets interacted in previously unreported grapelike conglomerates, suggesting an influence of shear rate on the type of platelet interaction. In summary, the ex vivo chamber amounts to an external vessel connecting the arterial and venous systems of a live mouse. This method combines the strengths of existing in vivo and in vitro systems in the study of leukocyte and platelet function. autoperfused flow chamber; intravital microscopy; inflammation; thrombus formation     

A novel environmental chamber for neuronal network multisite recordings

Environmental stability is a critical issue for neuronal networks in vitro. Hence, the ability to control the physical and chemical environment of cell cultures during electrophysiological measurements is an important requirement in the experimental design. In this work, we describe the development and the experimental verification of a closed chamber for multisite electrophysiology and optical monitoring. The chamber provides stable temperature, pH and humidity and guarantees cell viability comparable to standard incubators. Besides, it integrates the electronics for long‐term neuronal activity recording. The system is portable and adaptable for multiple network housings, which allows performing parallel experiments in the same environment. Our results show that this device can be a solution for long‐term electrophysiology, for dual network experiments and for coupled optical and electrical measurements. Biotechnol. Bioeng. 2012; 109: 2553–2566. © 2012 Wiley Periodicals, Inc.     

A novel TK-NOG based humanized mouse model for the study of HBV and HCV infections

The immunodeficient mice transplanted with human hepatocytes are available for the study of the human hepatitis viruses. Recently, human hepatocytes were also successfully transplanted in herpes simplex virus type-1 thymidine kinase (TK)-NOG mice. In this study, we attempted to infect hepatitis virus in humanized TK-NOG mice and urokinase-type plasminogen activator-severe combined immunodeficiency (uPA–SCID) mice. TK-NOG mice were injected intraperitoneally with 6 mg/kg of ganciclovir (GCV), and transplanted with human hepatocytes. Humanized TK-NOG mice and uPA/SCID mice were injected with hepatitis B virus (HBV)- or hepatitis C virus (HCV)-positive human serum samples. Human hepatocyte repopulation index (RI) estimated from human serum albumin levels in TK-NOG mice correlated well with pre-transplantation serum ALT levels induced by ganciclovir treatment. All humanized TK-NOG and uPA–SCID mice injected with HBV infected serum developed viremia irrespective of lower replacement index. In contrast, establishment of HCV viremia was significantly more frequent in TK-NOG mice with low human hepatocyte RI (<70%) than uPA–SCID mice with similar RI. Frequency of mice spontaneously in early stage of viral infection experiment (8 weeks after injection) was similar in both TK-NOG mice and uPA–SCID mice. Effects of drug treatment with entecavir or interferon were similar in both mouse models. TK-NOG mice thus useful for study of hepatitis virus virology and evaluation of anti-viral drugs.     

A model for the study of epithelial migration in wound healing.

A model is described which enables the detailed study of epithelial regeneration in experimentally produced lesions in the common bile duct of the rabbit. The circular on slightly oval defect of 1 mm diameter produced by a specially developed apparatus has a perfectly smooth base. Epithelial migration in this model has been investigated using light microscopy of transverse sections and scanning electron microscopy of whole preparations. Typical changes in the border cells, characterised by the formation of tapered protusions, can be observed as early as two hours after the lesion has been made. Later the cells in the flattened edge of the moving border also show various types of protrusion which rest on the substratum. Mitotic activity in the surface epithelium and crypts in the surrounding region only increases after closure of the lesion, which usually takes place within 16--24 h.     

A novel form of epithelial wound healing of the embryonic epidermis

The embryonic epidermis of amniotes is a two-cell layer sheet with a periderm positioned superficial to the basal cell layer which, itself, attaches apically to the basal surface of the periderm and basally to the basal lamina. The presence of the periderm is essential to maintain the basal layer as a two-dimensional monolayer. Wounds to the epidermis that remove selectively just the periderm are healed by a stacking of the basal layer cells that constitute the wound bed. Basal cell stacking involves the desertion of the basal lamina by many of the cells so as to increase their contact area with other basal layer cells. This suggests that a preferential adhesion to the planar basal lamina is not important for the monolayered organization of the basal layer but, instead, association with inner surface of the planar periderm is the principal process that maintains the basal layer as a monolayer. The conversion of the basal layer from monolayer to multilayer during wound healing diminishes its planar area, resulting in movement of the wound borders toward the center of the wound. This is a novel scenario for wound healing.     

Bacteriology of human bite wound infections

This study was designed to define the bacteriology of infected soft-tissue wounds from human bites, and to compare this with the bacteriology of infected animal bites in humans as determined in previous studies. The specimens were collected from 57 patients presenting to emergency rooms at 12 locations around the country. Three hundred and eighty organisms were isolated (224 aerobes and 156 anaerobes), for an average of 6.6 per specimen. The most prevalent anaerobes recovered were Prevotella spp. (34%), while streptococci comprised 44% of all aerobic organisms, over half of which were in the "Streptococcus milleri" group, particularly S. anginosus. The study demonstrated that the pathogens in human bite infections differ considerably from those present in animal bites.     

Embryonic wound healing: A primer for engineering novel therapies for tissue repair

Scar is the default tissue repair used by the body in response to most injuries–a response that occurs in wounds ranging in seriousness from minor skin cuts to complete severance of the spinal cord. By contrast, before the third trimester of pregnancy embryonic mammals tend to heal without scarring due to a variety of mechanisms and factors that are uniquely in operation during development in utero. The goal of tissue engineering is to develop safe and clinically effective biological substitutes that restore, maintain, or improve tissue function in patients. This review provides a comparative overview of wound healing during development and maturation and seeks to provide a perspective on just how much the embryo may be able teach us in the engineering of new therapies for tissue repair. Birth Defects Research (Part C) 96:258–270, 2012. © 2012 Wiley Periodicals, Inc.     

Experimental Haemonchus contortus infections in guinea pigs

Approximately 40% of exsheathed Haemonchus contortus larvae administered to guinea pigs established in the stomach and developed into fourth stage larvae. Most worms were then lost between 5 and 7 days after infection and the guinea pigs were resistant to a second infection. Haemorrhage, oedema and infiltration with inflammatory cells, especially eosinophils, developed in the stomach wall of infected guinea pigs and reactive hyperplastic changes occurred in the gastric lymph node. H. contortus infection of guinea pigs has some potential as a model for study of the pathology, immunology and chemotherapy of gastric nematodiasis.     

Verbesina crocata: A pharmacognostic study for the treatment of wound healing

Ethnopharmacological relevanceVerbesina crocata (Cav.) Less. (Arnica or Capitaneja) is an endemic plant from Mexico restricted to the western part of the country. The aerial parts are used in traditional medicine for the treatment of wounds and burns. The objective of this investigation was to carry out a pharmacognostic study of V. crocata and establish markers that allow for the recognition of the characteristics of the plant and validate its traditional use. The study includes anatomical and chemical characteristics of the plant as well as evaluations of its antioxidant capacity and wound healing ability in a murine model.Materials and methodsAn anatomical study of V. crocata was performed on the middle part of the leaf and stem. A methanolic extract of this species (VcME) was obtained by methanolic maceration of the aerial parts. Subsequently, a partition of the VcME was made to obtain a hexanic fraction (VcH). The phytochemical preliminary screening and characterization by high-performance liquid chromatography/mass spectrometry (HPLC-ESI/MS) of the VcME and VcH were performed. The antioxidant activity and total phenolic content were quantified. The wound healing capacity of the methanolic extract was determined in CD-1 mice by the healing rate, the tensiometric method, and histological analysis.ResultsThe anatomical study of V. crocata showed the presence of two types of secretory structures and their position on the leaves. In addition, the characteristics of the middle vein and trichomes are potentially useful for recognition of the species. Chemical compounds detected by HPLC-ESI/MS reveal the presence of sitosterol glycoside and catechin derivatives as principal constituents of V. crocata. The VcME showed low antioxidant capacity and total phenolic. V. crocata had a similar healing effect to Recoveron® in the tensiometric method, but the rate of healing was higher. According to the histological analysis, the treatment of V. crocata promoted the remodelling phase 15 days after the incisional wound.ConclusionThis is the first pharmacognostic study of this species that covers the plant anatomy, chemical content and biological properties related to its traditional use. V. crocata favours wound healing according to physical and histological evaluations. In addition, the characteristics of the middle vein, trichomes and catechin glycosides are potentially useful for the recognition of this species.