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Open access

Hrvoje Brzica, Davorka Breljak, Marija Ljubojević, Daniela Balen, Vedran Micek, Naohiko Anzai and Ivan Sabolić

Optimal Methods of Antigen Retrieval for Organic Anion Transporters in Cryosections of the Rat Kidney

To localise antigens by immunocytochemistry (IC), the samples of tissues or cells are usually denatured by fixation, and either frozen and cryosectioned, or embedded in paraffin before sectioning. p-Formaldehyde (PFA; formalin) is a common fixative, which preserves antigenicity of proteins, but damages the tissue/cell morphology and "masks" the antibody binding sites (epitopes). In order to "unmask" epitopes, some kind of antigen retrieval (AR) is used. The aim of this study was: a) to find an optimal AR method in cryosections of in vivo PFA-fixed kidneys for organic anion transporters (Oat) that reside in the basolateral (Oat1, Oat3) and brush-border membrane (Oat2, Oat5) of the rat renal proximal tubules, and b) using optimal method, to compare IC staining of Oats in kidneys that had been PFA-fixed in vivo or in vitro. IC staining in untreated cryosections was compared with that following detergent treatment or microwave heating in citrate buffer of pH 3, pH 6, or pH 8, with or without alcohol pre-treatment. The preferred AR method for Oat1, Oat2, and Oat5 was heating of cryosections at pH 6, and for Oat3 heating at pH 3, without alcohol pre-treatment. Compared with tissue fixed in vivo, tissue fixed in vitro exhibited damaged tubule morphology, similar staining intensity of Oat1 and Oat3, and higher staining intensity of Oat2 and Oat5. We conclude that for optimal IC presentation, each Oat in the rat kidney has to be treated individually, with different fixation and AR approach.

Open access

Ana Bogović, Jana Nižetić, Nada Galić, Davor Želježić, Vedran Micek and Marin Mladinić

The Effects of Hyaluronic Acid, Calcium Hydroxide, and Dentin Adhesive on Rat Odontoblasts and Fibroblasts

The aim of this study was to investigate the effects and efficiency of pulp capping preparations based on hyaluronic acid, calcium hydroxide, and dentin adhesive on the pulp tissue of Sprague-Dawley rats. The rats were killed and extracted teeth sectioned transversely through the pulp. The slices were placed in a RPMI 1640 cell culture medium supplemented with 10 % foetal calf serum. During 14 days of cultivation cultures were treated with preparations that contained hyaluronic acid (Gengigel Prof®), and calcium hydroxide (ApexCal®), or with dentin adhesive (Excite®). Cellularity and viability of fibroblasts and odontoblasts was analysed using a haemocytometer. Hyaluronic acid proved most efficient and the least toxic for direct pulp capping. Even though calcium hydroxide and dentin adhesive demonstrated a higher degree of cytotoxicity, their effects were still acceptable in terms of biocompatibility.

Open access

Mirta Milić, Suzana Žunec, Vedran Micek, Vilena Kašuba, Anja Mikolić, Blanka Tariba Lovaković, Tanja Živković Semren, Ivan Pavičić, Ana Marija Marjanović Čermak, Alica Pizent, Ana Lucić Vrdoljak, Rafael Valencia-Quintana, Juana Sánchez-Alarcón and Davor Želježić


In this 28 day-study, we evaluated the effects of herbicide glyphosate administered by gavage to Wistar rats at daily doses equivalent to 0.1 of the acceptable operator exposure level (AOEL), 0.5 of the consumer acceptable daily intake (ADI), 1.75 (corresponding to the chronic population-adjusted dose, cPAD), and 10 mg kg−1 body weight (bw) (corresponding to 100 times the AOEL). At the end of each treatment, the body and liver weights were measured and compared with their baseline values. DNA damage in leukocytes and liver tissue was estimated with the alkaline comet assay. Oxidative stress was evaluated using a battery of endpoints to establish lipid peroxidation via thiobarbituric reactive substances (TBARS) level, level of reactive oxygen species (ROS), glutathione (GSH) level, and the activity of glutathione peroxidase (GSH-Px). Total cholinesterase activity and the activities of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) were also measured. The exposed animals gained less weight than control. Treatment resulted in significantly higher primary DNA damage in the liver cells and leukocytes. Glyphosate exposure significantly lowered TBARS in the liver of the AOEL, ADI, and cPAD groups, and in plasma in the AOEL and cPAD group. AChE was inhibited with all treatments, but the AOEL and ADI groups significantly differed from control. Total ChE and plasma/liver ROS/GSH levels did not significantly differ from control, except for the 35 % decrease in ChE in the AOEL and ADI groups and a significant drop in liver GSH in the cPAD and 100xAOEL groups. AOEL and ADI blood GSH-Px activity dropped significantly, but in the liver it significantly increased in the ADI, cPAD, and 100xAOEL groups vs. control. All these findings show that even exposure to low glyphosate levels can have serious adverse effects and points to a need to change the approach to risk assessment of low-level chronic/sub-chronic glyphosate exposure, where oxidative stress is not necessarily related to the genetic damage and AChE inhibition.