Effects of voltage sensitive dye di-4-ANEPPS on guinea pig and rabbit myocardium.

Voltage-sensitive dyes (VSDs) are used to record transient potential changes in various cardiac preparations. In our laboratory, action potentials have been recorded by optical probe using di-4-ANEPPS. In this study, the effects of two different ways of staining were compared in guinea pig and rabbit isolated hearts perfused according to Langendorff: staining either by coronary perfusion with low dye concentration or with concentrated dye as a bolus into the aorta. Staining with low dye concentration lead to its better persistence in the tissue. Electrogram and coronary flow were monitored continuously. During the staining and washout of the dye, prominent electrophysiological changes occurred such as a decrease in spontaneous heart rate, partial atrioventricular block and changes of ST-T segment, accompanied by a decrease in mean coronary flow. No production of hydroxyl radicals was found by HPLC which excluded significant ischemic damage of the myocardium. Good viability of the stained preparation was supported by unchanged electron microscopy. Since in rabbit hearts the VSD-induced arrhythmogenesis was less pronounced, we conclude that the rabbit myocardium is more resistant to the changes triggered by VSD application. It may be due to different properties of the membrane potassium channels in the cardiomyocytes of these two species.

Comparison of dentine-resin interface in total-etch and self-etching adhesives using electron microscopy.

The important condition for the adhesive bond between dentine and composite filling is the change of dentine property from hydrophilic to hydrophobic. Substances able to accomplish this change, dentinal adhesives, have undergone complex development. Recently, the so-called self-etching adhesives have been formulated. The aim of the present paper was to compare the structural pattern of dentin/resin interface after the application of dentinal adhesives used in total- and self-etching technique. Dentinal adhesives Single bond, Prime bond, Prompt-L-pop Adper, and Xeno III were used. The former two belong to the total etch technique, the latter two to the self-etching technique. The dentin/resin interface was studied in transmission (TEM) and scanning (SEM) electron microscope. In TEM sixteen teeth were investigated, each adhesive was applied in four teeth. Under SEM forty teeth were studied, each adhesive was applied in ten teeth. The obtained results demonstrated that all the dentine adhesives studied were able to penetrate into dentinal tubules and to form a hybrid layer with the demineralized intertubular and peritubular dentine. There was no substantial difference in morphology of the dentine/resin interface between the techniques studied. Our results suggest that the hybrid layer function of all four adhesives might be of the same quality.

The influence of moisture wound healing on the incidence of bacterial infection and histological changes in healthy human skin after treatment of interactive dressings.

In this article the authors discuss the problem faced by physicians when trying to use moisture-retentive dressing in pressure sores (decubitus ulcers). First, they report the results of an in vitro study using a new model of experimental wound (radio-isotopic investigation) that assesses the release of Ringer's solution from interactive dressings continually during fourteen hours. Second, they perform an animal experiment that assesses the incidence of wound infection in defects treated conventionally or using interactive dressings. The defects treated with interactive pads had lower incidence of wound infection, and the process of wound healing was rapid. Finally, the authors discuss their experience in four paraplegic patients with decubitus ulcers where they used moisture-retentive dressing on ulcers and on the surrounding intact skin before surgical procedure to detect the possibility of maceration of healthy skin. Histological evaluation was performed in order to find microscopically changes after moisture healing. The changes of healthy skin were not significant after treatment of moisture-retentive dressings.

[Treatment of deep chondral defects of the knee using autologous chondrocytes cultured on a support--preparation of the cartilage graft]. / Terapie hlubokých chondrálních defektu kolena pomocí autologních kultivovaných chondrocytu na nosici--príprava chondrograftu.

PURPOSE OF THE STUDY: Chondral and osteochondral defects represent an important topic in the knee surgery. The knee traumas are often followed by premature development of osteoarthritis due to limited reparative processes in the cartilage. Today's diagnostic possibilities and progress in arthroscopic techniques promote the early diagnostics and exact classification of osteochondral defects. Authors present therapeutic ways for treatment of cartilage injuries and adjacent part of the subchondral bone. New possible method for treatment of deep chondral defects on the weight-bearing area of the knee is the application of autologous cultivated chondrocytes in Tissucol (Baxter, Austria). MATERIAL AND METHODS: Chondrocyte retrieval and in vitro cultivation is today a commonly known method. The goal of the knee surgery is to find a suitable three-dimensional bearer for chondrocyte culture. After serial laboratory tests we decided to use the tissue fibrin glue Tissucol (Baxter, Austria) as a chondrocyte carrier. Very good cell viability and capability of cell migration and outgrow in Tissucol were documented. Authors describe a new chondrograft type, its characteristics. They focused on quality control and documentation of key parameters necessary for graft use in humans. Following methods for quality control were used: Sterility control Determination of cells number Determination of cell viability by trypan blue staining. Viability between 90-95% was demonstrated. Determination of proliferative activity was tested using growth quality control. Migration and outgrowth of chondrocytes was documented. The morphological characteristics were studied by: Light microscopy (haematoxylin-eosin staining). Electron microscopy (transmission and scanning). A good stage of cellular organelles and production of extracellular matrix with protocollagenic fibrils were found in all cases. Immuno-fluorescence microscopy (using monoclonal antibodies against vimentin). RESULTS: Preclinical testing on pigs and cadaverous was provided following laboratory investigations. Testing on cadaverous demonstrated a chondrograft ability to fill up completely the chondral defect to renovate the anatomic surface of the joint cartilage. Tests on pigs described hyaline-like cartilage. Publication contains detailed description of chondrograft preparation to allow reproducibility of described technique. Chondrograft transplantation consists of following phases: Arthroscopic diagnostic of defect. Arthroscopic sampling of cartilage for in vitro cultivation. Transport of samples to tissue bank. Enzymatic isolation, in vitro cultivation of chondrocytes. Graft formation (combination of fibrin glue Tissucol with chodrocytes), quality control. Transplantation (chondrograft agglutination into defect). DISCUSSION: The goal of the nowadays tissue engineering is to find an optimal three-dimensional carrier for autologous chondrocyte culture. A new type of carrier based on hyaluronic acid--Hyalograft C was presented by Marcani et al in 2001 year. Published results from the first 20 clinical cases using Hyalograft C do not report any method complications. Our chondrograft technique represents an alternative option when compared to method described by Marcani. CONCLUSIONS: Based on the successful preclinical outcomes observed in this study, authors conclude that autologous chondrocyte transplantation in Tissucol offers an important treatment option for large full thickness chondral lesions located on the weight-bearing surfaces of the femur.

Reduced nephrotoxicity of conventional amphotericin B therapy after minimal nephroprotective measures: animal experiments and clinical study.

Amphotericin B (AmB)-treated rats develop severe polyuria, polydypsia, impairment of renal concentrating ability, and morphologic signs of tubular damage. However, renal insufficiency develops quickly only in animals in which water intake is restricted to the median volume drunk by rats of the control group. Therefore, vigorous hydration seems crucial for prevention of AmB-induced nephrotoxicity. In a clinical study, 61 patients with hematologic malignancies receiving AmB therapy were massively hydrated to ensure urine output of > or =4000 mL/day. Urine sodium, potassium, and magnesium were also measured, and all losses were supplemented (potassium as a 7.45% solution via central venous catheter). AmB-treated patients developed signs of renal tubular damage (increased fractional excretion of sodium and potassium) and required large amounts of ion supplementation. The serum ion concentration and creatinine clearance remained stable. No clinically significant renal damage developed to force premature cessation of AmB treatment.

[Safety of long-term administration of conventional amphotericin B in oncology patients]. / Bezpecné dlouhodobé podávání konvencního amfotericinu B u onkologicky nemocných.

In cancer patients with persistent fever and neutropenia, amphotericin B is administered for the early treatment and prevention of the clinically occult invasive fungal infections. The major drawback of the conventional amphotericin B is its nephrotoxicity. We have previously showed that massive potassium, sodium, and magnesium supplementation, which corresponds to the amount lost in kidney during amphotericin B administration, as well as vigorous hydration, can markedly reduce nephrotoxicity of conventional amphotericin B. Clinical courses of four oncological patients treated with conventional amphotericin B suspected or proven fungal infection for a period of 24 days (two patients), 39 days, and 47 days are described. During the long-term amphotericin B administration with nephroprotective measures, no severe renal function decrease was observed in any of our patients. Massive ion supplementation corresponding to the amounts lost in kidney, as well as the vigorous hydration are effective in the prevention of the renal function decrease induced by the long-term conventional amphotericin B therapy.

[Prevention of mycotic infections in oncology patients with regard to the toxicity of antimycotic agents]. / Moznosti profylaxe mykotických infekcí u onkologických nemocných s prihlédnutím k toxicite antimykotických léku.

Profound and prolonged neutropenia following chemotherapy is a major risk factor for systemic fungal infections. It is still unclear how effective the various measures that are currently being used are in preventing serious fungal infection. Although a number of studies have been published on antifungal prophylaxis in neutropenic patients, there are a lot of conflicting data. At the present time, it seems that antifungal prophylaxis is more a matter of faith rather than science. The prophylactic use of fluconazole or itraconazole may reduce the incidence of systemic fungal infections in high-risk patient groups, mainly in patients with haematological malignancies. The antifungals currently used for prophylaxis in immunocompromised patients, and the toxicity of the antifungals are reviewed.

Histochemical and ultrahistochemical localization of heavy metals in calf organs.

Cadmium, zinc, selenium, and copper were administered, singly or in combination, orally or subcutaneously. Experiment I included 32 calves of both sexes; six received Cd (two groups), Zn, Cd, and Zn, and Cd and Se (two groups) and one group was a control. In Experiment II (21 bulls), three were given Cd, Cd, and Cu, and Cd and Zn, respectively, and one group was a control. For light microscopy, in Experiment I the highest amounts of silver granules were present in the samples of liver, small intestine, and vesicular gland of all the exposed groups; in Experiment II the most affected organs were liver, kidney, and small intestine. For electron microscopy, in Experiment I, after administration of Cd and Zn, the highest amounts of granules were seen in the cytoplasm of hepatocytes and cells of the proximal and distal renal tubules and the lowest amounts were found in glandular cells of the pancreas. Administration of Cd and Se resulted in the presence of large numbers of granules in the nuclei and nucleoli of spermatogonies. In Experiment II, ingestion of Cd and Zn in feed led to the appearance of highest amounts of granules in the nucleoli, nuclei, and cytoplasm of cells in testes, kidneys, and pancreas. Following Cd intake, the highest accumulation of granules was observed in the nucleoli of hepatocytes and cells of the proximal and distal renal tubules. Combined Cd and Cu produced the highest number of granules in cells of the proximal and distal renal tubules and in the nucleoli and nuclei of germinal epithelium.

Distribution of exogenous heavy metals in the hepatocytes of calves: a morphometric study.

The objective of this study was to evaluate the presence of heavy metals in the hepatocytes of the animals fed a cadmium-supplemented diet and also receiving zinc and/or selenium in the injection form. The experiment involved four groups of calves (6-8, both sexes) receiving the heavy metals in various combinations for 95 days. Electron micrographs of liver cells were prepared and statistically evaluated using Student's t-test. A modified morphometric apparatus was used for morphometric examination. Exogenous cadmium showed marked accumulation in the hepatocytes. If, however, the cadmium diet was combined with zinc or selenium administration the amount of the reduction product was much lower.

[Use of skin substitutes in treatment of large hypertrophic scars due to burn injury]. / Uzití dermálních náhrad k resení plosných hypertrofických jizev po popálení.

The study concerns a clinical experiment in two patients with a dermal substitute based on atelocollagen and hyaluronic acid, and allogeneic acellular dermis. In both cases two-step grafting was performed. At the first step the dermal substitute was implanted into the wound and it was grafted at the second step with thin dermoepidermal autograft. Large hypertrophic scars after burn injury were treated. In both patients the quality of skin cover was significantly improved.

Determination of intracytoplasmic filaments in the chondrocytes of articular cartilage.

Intracytoplasmic filaments were demonstrated in the chondrocytes of articular cartilage of cattle at 82 days post partum and at 3 and 4 years of age using transmission electron microscopy and immunocytochemistry. Samples of cartilage for electron microscopy were processed in a standard way. Immunocytochemical procedures were performed with semithin sections made from the material prepared for electron microscopy after embedding medium and osmium compounds had been eliminated. Vimentin filaments were demonstrated by means of anti-vimentin monoclonal antibody visualized by peroxidase-labelled immunoglobulin. Both techniques showed that intracytoplasmic filaments were situated around the nucleus and only occasionally at the cell periphery, e.g. in cell projections.

Specific antibody against the Aujeszky's disease virus in the complex cumulus oophorus-oocyte of sows.

Oocytes collected from sows vaccinated and revaccinated with an inactivated vaccine against Aujeszky's disease (AD) were examined for the presence of specific antibodies of the IgG class against AD virus in the complex cumulus oophorus-oocyte by means of immunocytochemical methods. No specific antibody of the IgG class was detected in the cumulus-oocyte complexes collected from sows before vaccination. On the other hand, the specific IgG antibody was found in all immunocytochemically examined complexes cumulus-oocyte collected from revaccinated sows. The specific antibody of the IgG class against AD virus and the porcine IgG were immunocytochemically visualized under both light and electron microscopes as a fine granular product disseminated in oocytes and follicular cells.