The potential adverse effect of 2.45 GHz microwave radiation on the testes of prenatally exposed peripubertal male rats.

In utero development of organs is easily influenced by many environmental factors. The aim of this study was to elucidate the effect of microwave radiation (MR) at a frequency of 2.45 GHz and a specific absorption rate of 1.73 W/kg on intrauterine development of testis. Pregnant albino rats were exposed to whole-body MR for 2 hours per day throughout the pregnancy. Male offspring (n=12, age 35 days) were not exposed to MR after birth. The study revealed that MR applied in utero induced apparent structural changes in the testes, such as irregular shape of seminiferous tubules, significant decrease in the diameter of seminiferous tubules (p<0.05) and in the height of the germinal epithelium (p<0.01), disorganisation of germ cells, desquamations of immature germ cells, formation of giant multinucleated cells, and significant (p<0.01) expansion of the interstitium. At the level of transmission electron microscopy, there were observed basement membrane irregularities in seminiferous tubules, vacuolation of the cytoplasm and adversely affected organelles in Sertoli cells, germ cells, Leydig cells, peritubular and endothelial cells. The tight junctions between adjacent Sertoli cells were often incomplete, and necrotizing germ cells were more numerous in experimental animals compared to controls. Enhanced necrotizations of germ cells proved by a Fluoro Jade C method, and declined germ cells proliferation confirmed by proliferating cell nuclear antigen analysis, were detected in MR exposed animals. Our results revealed that the prenatal exposure to MR had an adverse effect on the postnatal testicular development in rats.

Dietary flaxseed's protective effects on body tissues of mice after oral exposure to xylene.

Xylene is a common pollutant in the environment that enters the body of animals and humans in various ways, but most often through the respiratory tract and adversely affects their overall health. However, xylene effects after oral exposure have not been sufficiently studied. This study aimed to investigate the effects of xylene exposure on the mouse organism and to identify possible beneficial effects of flaxseed on such exposure. Eighty mice were divided into four groups: control group C (basal diet + no xylene exposure), group X (oral exposure by 400 mg/kg/day xylene), group F (10% flaxseed supplementation of basal diet), and group XF (10% dietary flaxseed + oral exposure by xylene). Experimental trial took 14 days. Clinical examination, spectroscopic analysis of tissue aminotransferases, total lactate dehydrogenase (TLDH), and acetylcholinesterase (AchE) activities, electrophoretic analysis of LDH isoenzymes, western blot and immunohistochemical analysis of apoptosis as well as routine histology of the kidneys and jejunum, and transmission electron microscopy of the liver were performed. Marked restlessness in group X and high weight losses in mice of all groups were recorded during the experiment. Xylene promoted apoptosis (caspase-3 expression) without causing marked structural changes in the liver and jejunum, although renal cortex structure was affected adversely. In the brain, liver, and kidney of mice, xylene increased levels of liver transaminases, LDH, and decreased AchE activities, reflecting cell membrane damage. Flaxseed feeding improved animal behaviour, leakage of enzymes and prevented selected tissue toxic damage induced by xylene by protecting cell membrane integrity and fluidity and by suppressing apoptosis. These results point at the protective effect of flaxseed consumption on mice.

PHB/CHIT Scaffold as a Promising Biopolymer in the Treatment of Osteochondral Defects-An Experimental Animal Study.

Biopolymer composites allow the creation of an optimal environment for the regeneration of chondral and osteochondral defects of articular cartilage, where natural regeneration potential is limited. In this experimental study, we used the sheep animal model for the creation of knee cartilage defects. In the medial part of the trochlea and on the medial condyle of the femur, we created artificial defects (6 × 3 mm2) with microfractures. In four experimental sheep, both defects were subsequently filled with the porous acellular polyhydroxybutyrate/chitosan (PHB/CHIT)-based implant. Two sheep had untreated defects. We evaluated the quality of the newly formed tissue in the femoral trochlea defect site using imaging (X-ray, Computer Tomography (CT), Magnetic Resonance Imaging (MRI)), macroscopic, and histological methods. Macroscopically, the surface of the treated regenerate corresponded to the niveau of the surrounding cartilage. X-ray examination 6 months after the implantation confirmed the restoration of the contour in the subchondral calcified layer and the advanced rate of bone tissue integration. The CT scan revealed a low regenerative potential in the bone zone of the defect compared to the cartilage zone. The percentage change in cartilage density at the defect site was not significantly different to the reference area (0.06-6.4%). MRI examination revealed that the healing osteochondral defect was comparable to the intact cartilage signal on the surface of the defect. Hyaline-like cartilage was observed in most of the treated animals, except for one, where the defect was repaired with fibrocartilage. Thus, the acellular, chitosan-based biomaterial is a promising biopolymer composite for the treatment of chondral and osteochondral defects of traumatic character. It has potential for further clinical testing in the orthopedic field, primarily with the combination of supporting factors.

Dimethyl Sulfoxide: Morphological, Histological, and Molecular View on Developing Chicken Liver.

Dimethyl sulfoxide (DMSO) is widely used as a solvent for small hydrophobic drug molecules. However, the safe volume allowing to avoid its embryotoxic effect has been poorly studied. In this study, we documented the effects of dimethyl sulfoxide (DMSO) in the developing chicken embryo at morphological, histological, and molecular levels. We focused on the developing chicken liver as the main organ involved in the process of detoxification. In our study, 100% DMSO was administered subgerminally onto the eggshell membrane (membrana papyracea) at various volumes (5, 10, 15, 20, 25, 30, 35, and 50 µL) on 4th embryonic day (ED). We focused on histopathological alterations of the liver structure, and noticed the overall impact of DMSO on developing chicken embryos (embryotoxicity, malformation). At the molecular level, we studied cytochrome P450 complex (CYP) isoform's activities in relation to changes of CYP1A5, CYP3A37, and CYP3A80 gene expression. Total embryotoxicity after application of different doses of DMSO on ED 4, and the embryo lethality increased with increasing DMSO amounts. Overall mortality after DMSO administration ranged below 33%. Mortality was increased with higher amounts of DMSO, mainly from 20 µL. The highest mortality was observed for the highest dose of DMSO over 35 µL. The results also showed a decrease in body weight with increased application volumes of DMSO. At the histological level, we observed mainly the presence of lipid droplets and dilated bile canaliculi and sinusoids in samples over the administration of 25 µL of DMSO. While these findings were not statistically significant, DMSO treatment caused a significant different up-regulation of mRNA expression in all studied genes. For CYP1A5, CYP3A37, and CYP3A80 DMSO volumes needed were 15 µL, 10 µL, and 20 µL, respectively. A significant down-regulation of all studied CYP isoform was detected after application of a DMSO dose of 5 µL. Regarding the morphological results, we can assume that the highest safe dose of DMSO without affecting chicken embryo development and its liver is up to 10 µL. This conclusion is corroborated with the presence of number of malformations and body weight reduction, which correlates with histological findings. Moreover, the gene expression results showed that even the lowest administered DMSO volume could affect hepatocytes at the molecular level causing down-regulation of cytochrome P450 complex (CYP1A5, CYP3A37, CYP3A80).

Potential influence of prenatal 2.45 GHz radiofrequency electromagnetic field exposure on Wistar albino rat testis.

An ever-increasing use of wireless devices over the last decades has forced scientists to clarify their impact on living systems. Since prenatal development is highly sensitive to numerous noxious agents, including radiation, we focused on the assessment of potential adverse effects of microwave radiation (MR) on testicular development. Pregnant Wistar albino rats (3 months old, weighing 282±8 g) were exposed to pulsed MR at a frequency of 2.45 GHz, mean power density of 2.8 mW/cm², and a specific absorption rate of 1.82 W/kg for 2 hours/day throughout pregnancy. Male offspring were no longer exposed to MR following birth. Samples of biological material were collected after reaching adulthood (75 days). In utero MR exposure caused degenerative changes in the testicular parenchyma of adult rats. The shape of the seminiferous tubules was irregular, germ cells were degenerated and often desquamated. The diameters of the seminiferous tubules and the height of the germinal epithelium were significantly decreased (both at ∗∗p<0.01), while the interstitial space was significantly increased (∗∗p<0.01) when compared to the controls. In the group of rats prenatally exposed to MR, the somatic and germ cells were rich in vacuoles and their organelles were often altered. Necrotizing cells were more frequent and empty spaces between Sertoli cells and germ cells were observed. The Leydig cells contained more lipid droplets. An increased Fluoro Jade - C and superoxide dismutase 2 positivity was detected in the rats exposed to MR. Our results confirmed adverse effects of MR on testicular development.

Characterization of Properties, In Vitro and In Vivo Evaluation of Calcium Phosphate/Amino Acid Cements for Treatment of Osteochondral Defects.

Novel calcium phosphate cements containing a mixture of four amino acids, glycine, proline, hydroxyproline and either lysine or arginine (CAL, CAK) were characterized and used for treatment of artificial osteochondral defects in knee. It was hypothesized that an enhanced concentration of extracellular collagen amino acids (in complex mixture), in connection with bone cement in defect sites, would support the healing of osteochondral defects with successful formation of hyaline cartilage and subchondral bone. Calcium phosphate cement mixtures were prepared by in situ reaction in a planetary ball mill at aseptic conditions and characterized. It was verified that about 30-60% of amino acids remained adsorbed on hydroxyapatite particles in cements and the addition of amino acids caused around 60% reduction in compressive strength and refinement of hydroxyapatite particles in their microstructure. The significant over-expression of osteogenic genes after the culture of osteoblasts was demonstrated in the cement extracts containing lysine and compared with other cements. The cement pastes were inserted into artificial osteochondral defects in the medial femoral condyle of pigs and, after 3 months post-surgery, tissues were analyzed macroscopically, histologically, immunohistochemically using MRI and X-ray methods. Analysis clearly showed the excellent healing process of artificial osteochondral defects in pigs after treatment with CAL and CAK cements without any inflammation, as well as formation of subchondral bone and hyaline cartilage morphologically and structurally identical to the original tissues. Good integration of the hyaline neocartilage with the surrounding tissue, as well as perfect interconnection between the neocartilage and new subchondral bone tissue, was demonstrated. Tissues were stable after 12 months' healing.

The effect of 2.45 GHz non-ionizing radiation on the structure and ultrastructure of the testis in juvenile rats.

BACKGROUND: Nowadays, mobile devices that emit non-ionizing electromagnetic radiation (EMR) are predominantly used by juveniles and pubescents. The aim of the present study was to evaluate the effect of whole body pulsed EMR on the juvenile Wistar albino rat testis at a frequency of 2.45 GHz and mean power density of 2.8 mW/cm². METHODS: The investigated animals (n=24) were divided into two control and two EMR groups (5 and 6 week old rats; 6 rats per group). Both EMR groups were irradiated continually for 3 weeks (2h/day) from postnatal days 14 and 21, respectively. RESULTS: EMR caused an irregular shape of seminiferous tubules with desquamated immature germ cells in the lumen, a large number of empty spaces along the seminiferous epithelium and dilated and congested blood vessels in the interstitial tissue of the testis. The cytoplasm of Sertoli cells showed strong vacuolization and damaged organelles, with the cytoplasm full of different heterophagic and lipid vacuoles or the cytoplasm of spermatocytes with swollen mitochondria in both irradiated groups. A significant increase in the total tubular area of seminiferous tubules was observed in both EMR groups compared with controls (P<0.001). A significant increase in the TUNEL-positive apoptotic nuclei (P<0.01) was accompanied by a significant rise in both Cu-Zn-SOD (P<0.01) and Mn-SOD (P<0.001) positive cells in the 6 week old experimental rats compared to control animals. CONCLUSION: Our results confirmed a harmful effect of non-ionizing radiation on the structure and ultrastructure of the juvenile rat testis.

In vitro and in vivo study of microporous ceramics using MC3T3 cells, CAM assay and a pig animal model.

Bone tissue engineering combines biomaterials with biologically active factors and cells to hold promise for reconstructing craniofacial defects. In this study the biological activity of biphasic hydroxyapatite ceramics (HA; a bone substitute that is a mixture of hydroxyapatite and ß-tricalcium phosphate in fixed ratios) was characterized (1) in vitro by assessing the growth of MC3T3 mouse osteoblast lineage cells, (2) in ovo by using the chick chorioallantoic membrane (CAM) assay and (3) in an in vivo pig animal model. Biocompatibility, bioactivity, bone formation and biomaterial degradation were detected microscopically and by radiology and histology. HA ceramics alone demonstrated great biocompatibility on the CAM as well as bioactivity by increased proliferation and alkaline phosphatase secretion of mouse osteoblasts. The in vivo implantation of HA ceramics with bone marrow mesenchymal stem cells (MMSCs) showed de novo intramembranous bone healing of critical-size bone defects in the right lateral side of pig mandibular bodies after 3 and 9 weeks post-implantation. Compared with the HA ceramics without MMSCs, the progress of bone formation was slower with less-developed features. This article highlights the clinical use of microporous biphasic HA ceramics despite the unusually shaped elongated micropores with a high length/width aspect ratio (up to 20) and absence of preferable macropores (>100 µm) in bone regenerative medicine.

Synbiotics suppress the release of lactate dehydrogenase, promote non-specific immunity and integrity of jejunum mucosa in piglets.

The aim of our experiment was to study how synbiotics are able to deal with the problems of post-weaning piglets. Lactobacillus plantarum - Biocenol(TM) LP96 (CCM 7512), Lactobacillus fermentum - Biocenol(TM) LF99 (CCM 7514) and flaxseed (rich in n-3 polyunsaturated fatty acids) were administered to 36 conventional piglets from a problematic breed with confirmed presence of enterotoxigenic Escherichia coli and Coronavirus. The experimental piglets were supplied with probiotic cheeses and crushed flax-seed in the period starting 10 days before weaning and lasting up to 14 days post-weaning. Piglets in the control group were supplied only control cheese. The impact of such additives on the release of lactate dehydrogenase (LDH; spectroscopic and electrophoretic assay), alteration of immunity (index of metabolic activity), jejunum histology (light microscopy), and health of conventional piglets from a problematic breed (monitoring of hematology, consistency and moisture of feces and body temperature) were examined. We found significant decrease in LDH leakage in the blood serum and tissue extracts, indicating better cell membrane integrity in the individual organs of animals. Probiotics and flaxseed applied together seem to be a good source of nutrients to improve the immune status and the integrity of jejunum mucosa during infection. © 2015 Japanese Society of Animal Science.

Blood supply to the thoracolumbar spinal cord in the laboratory mouse using corrosion and dissection techniques.

Mice are used frequently as experimental models in the study of ischemic spinal cord injury. The aim of the present study was to describe the arterial blood supply to the thoracolumbar spinal cord in the mouse. The study was carried out on 20 adult mice using the corrosion and dissection technique. Dorsal intercostal arteries were found as branches of the thoracic aorta: as 7 pairs in 80% of cases, as 8 pairs in 15% of cases and as 9 pairs in 5% of cases. The paired lumbar arteries arising from the abdominal aorta were present as 5 pairs in all cases. Along the entire thoracic and lumbar spinal regions, we observed left-sided branches entering the ventral spinal artery in 64.2% and right-sided branches in 35.8% of cases. Along the entire thoracic and lumbar spinal regions, the branches entering the dorsal spinal arteries were left-sided in 60.8% of cases and right-sided in 39.2% of cases. We found some variations in the site of origin of the artery of Adamkiewicz and in the number of dorsal spinal arteries. Documenting the anatomical variations in spinal cord blood supply in the laboratory mouse will aid the planning of future experimental studies and in determining the clinical relevance of such studies.

Ultrastructural changes in the rabbit liver induced by carbamate insecticide bendiocarb.

Carbamates (CB) are used as insecticides and some of them have been registered as human drugs. The mechanism of CB poisoning involves reversible inhibition of acetylcholine esterase. In the present study, we investigated changes in liver ultrastructure in rabbits (Oryctolagus cuniculus) which were administered bendiocarb for 3, 10, 20, and 30 days. Rabbits in all experimental groups received capsules of bendiocarb (96% Bendiocarb, Bayer, Germany) per os daily at a dose of 5 mg/kg of body weight, and after day 11 received the same dose every 48 h. The observed changes were only moderate, focal, and the effect on the liver was not uniform. On the third day of the experiment, injured hepatocytes had dilated bile capillaries with reduced microvilli. There were no visible alterations in the intercellular contacts. Nuclei of these cells were irregular in shape. Many hepatocytes showed considerable increase in the number of peroxisomes. On day 10 of the experiment, the number of peroxisomes was reduced. Other changes, such as dilated rough endoplasmic reticulum and proliferation of smooth endoplasmic reticulum were observed on day 20. The number of lipid droplets in hepatocytes gradually increased. Usually they were present in low numbers, but on day 30 of the experiment their number increased significantly. They coalesced and formed a single lipid droplet which changed the shape of the nuclei. The results presented in this study indicate that both short and long-term administration of bendiocarb affects the liver ultrastructure. At the same time we also observed rapid onset of regeneration of the damaged tissue through activation of hepatocytes and oval cells.

Structural and ultrastructural study of rat testes influenced by electromagnetic radiation.

This study was conducted to investigate the influence of whole-body electromagnetic radiation (EMR) on testicular parenchyma of Wistar rats. Sexually mature rats were subjected to pulsed electromagnetic field at frequency of 2.45 GHz and mean power density 2.8 mW/cm(2) by 3-h daily applications for 3 wk. Tissue samples were obtained 3 h after the last irradiation and processed by histological techniques for light and transmission electron microscopy. Testes showed apparent degenerative changes of seminiferous epithelium. The seminiferous tubules were mostly irregular in shape, and seminiferous epithelium contained a number of empty spaces of different size. Subsequently, groups of sloughed epithelial cells were often found inside the lumina of tubules. Except for relatively unchanged Sertoli cells, some locations of basal compartment of seminiferous epithelium contained shriveled Sertoli cells with dark cytoplasm. These areas showed degenerative features including necrotizing and shriveled spermatogonia surrounded by empty irregular spaces, and undulating basement membrane. The intertubular spaces were enlarged but interstitial Leydig cells did not show any marked morphological changes. Evidence demonstrates the adverse effects of EMR on testicular parenchyma in rats.

Structural and ultrastructural study of the rabbit testes exposed to carbamate insecticide.

The present study was conducted to investigate the impact of carbamate insecticide - bendiocarb on the testicular structure of adult rabbits. Bendiocarb was perorally administered daily for 10 and 30 days, at a dose 5 mg/kg of body weight. After the histological sampling the tissues were investigated and compared with control. After the bendiocarb administration the absolute and relative testicular weight decreased significantly (P < 0.001) in both time periods. The testicular parenchyma showed structural changes such the sloughing of developing sex cells, occurrence of vacuoles within Sertoli cells and inside various spermatogenic cells. The interstitial Leydig cells were smaller than their control counterpart and possessed shrivelled nuclei and strongly vacuolar dark cytoplasm. The rate of changes was directly proportional on duration of the experiment. The ultrastructural examination proved presence of various cellular defects across the germinal epithelium as well as within the interstitial Leydig cells in both experimental periods. Morphometric analysis manifested decrease in diameters of seminiferous tubules, increase of the diameters of the tubular lumina due to reduction of height of the seminiferous epithelium. Results of this study show distinct negative effects of bendiocarb on structure of rabbit testes.

Alterations in the rabbit lymphoid tissue after bendiocarb administration.

Various pesticides have immuno-suppressive effects, and thus the organisms become responsive to viral, bacterial and parasitic diseases and neoplasm. The aim of the study was to observe the structure of the small intestine (height of enterocytes and crypts), mucosal lymphoid tissue (Payer's patches, lymphocytes in lamina propria) and a lymph node after administration of bendiocarb (2,2-dimethyl-1,3-benzodioxol-4-yl-methylcarbamate) on days 3, 10, 20, 30 and 60 of the experiment. The height of the observed enterocytes showed an increasing tendency. On days 20, 30 and 60 we also observed an increase in diameter of crypts located in intestinal epithelium. The number of cells in lamina propria mucosae was significantly reduced on days 20 and 30 after administration of bendiocarb. Observations of the lymph node showed that on days 10 and 20 there was a significant increase in relative volume of medulla at the expense of the relative volume of the cortex and a decrease in the number of lymphocytes. However, we recorded an increase in diameter of lymphocytes. The intracellular parasite Toxoplasma gondii (T. gondii) belongs to the most common pathogenic parasites in the world and it can cause serious health complications in pregnant and immunodeficient individuals. DNA isolation, standard polymerase chain reaction (PCR) and visualization in a 2.5 % agarose gel, the presence of DNA T. gondii was detected in no examined rabbit brain samples. Using real time PCR T. gondii DNA was detected and quantified in the three rabbit brain samples (10 %).

The another toxic effect of carbamate insecticides.

The activities of the antioxidant and detoxifying enzymes, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSHPx), glutathione reductase (GR), glutathione-S-transferase (GST), and the content of thiobarbituric acid reactive substances (TBARS) were determined in the liver and kidney of rabbits after exposure to bendiocarb. In the liver, the activities of SOD, CAT and GR were not affected by bendiocarb. The induction or inhibition of isoenzymes of SOD (mainly MnSOD) were observed in the experimental groups. The activities of GSHPx-cum and GSHPx-H2O2 significantly decreased on the days 3 and 10 of the experiment. The activity of GST significantly increased on the day 9 of the experiment. In the kidney, the activity of SOD was significantly increased and the new MnSOD isoenzymes were detected. The activities of CAT and GSHPx-H2O2 were significantly decreased in the experimental groups. The activity of GR significantly increased on days 3 and 10, and the activity of GST was significantly increased on days 3, 10, and 30. Exposure of rabbit to bendiocarb did not affect the content of TBARS in the kidney. In the liver, the content of TBARS was significantly increased in the experimental groups as compared to the control. Our results showed that the response of organs to bendiocarb is different and may depend on the specific organ damage and their protective abilities. The alterations in the activities of the antioxidant defence system, increased TBARS values, and changes in the SOD isoenzyme pattern showed that the toxic effect of bendiocarb is not only in the acetylcholine esterase inhibition, but also in ROS production.

Antioxidant and detoxifying enzymes in the liver of rats after subchronic inhalation of the mixture of cyclic hydrocarbons.

The activity of antioxidant and detoxifying enzymes, superoxide dismutase (SOD), glutathione peroxidase (GSHPx), glutathione-S-transferase (GST), the SOD isoenzyme patterns and the contents of thiobarbituric acid reactive substances (TBARS), were determined in the livers of male and female rats after subchronic inhalation of mixtures of benzene, cyclohexanone and cyclohexane. Except for decreased GSHPx (with substrate cumene hydroperoxide) and GST activities in female rats, no differences in the activities of antioxidant and detoxifying enzymes and TBARS content occurred. Between the activities of GSHPx and GST was observed an indirect relationship. The activities of GSHPx-cum and GST were influenced by sex.