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.

Trafficking of immune cells across the blood-brain barrier is modulated by neurofibrillary pathology in tauopathies.

Tauopathies represent a heterogeneous group of neurodegenerative disorders characterized by abnormal deposition of the hyperphosphorylated microtubule-associated protein tau. Chronic neuroinflammation in tauopathies is driven by glial cells that potentially trigger the disruption of the blood-brain barrier (BBB). Pro-inflammatory signaling molecules such as cytokines, chemokines and adhesion molecules produced by glial cells, neurons and endothelial cells, in general, cooperate to determine the integrity of BBB by influencing vascular permeability, enhancing migration of immune cells and altering transport systems. We considered the effect of tau about vascular permeability of peripheral blood cells in vitro and in vivo using primary rat BBB model and transgenic rat model expressing misfolded truncated protein tau. Immunohistochemistry, electron microscopy and transcriptomic analysis were employed to characterize the structural and functional changes in BBB manifested by neurofibrillary pathology in a transgenic model. Our results show that misfolded protein tau ultimately modifies the endothelial properties of BBB, facilitating blood-to-brain cell transmigration. Our results suggest that the increased diapedesis of peripheral cells across the BBB, in response to tau protein, could be mediated by the increased expression of endothelial signaling molecules, namely ICAM-1, VCAM-1, and selectins. We suggest that the compensation of BBB in the diseased brain represents a crucial factor in neurodegeneration of human tauopathies.

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.

miRNAs as biofluid markers for diagnostics of Alzheimer's disease: recent status and perspectives.

After many decades of research in the field of neurodegeneration, we have no effective cure for Alzheimer's disease (AD), a major form of dementia. It is mainly due to the lack of early, reliable and sensitive biomarkers and incomplete understanding of disease mechanisms at molecular level. Several recently employed biomarkers, especially their combinations, can discriminate advanced stages of AD from other forms of dementia or neuropathy. They do not provide much information on molecular mechanisms of disease rather they reflect the amount of key histopathological markers in the diseased brain. This review is focussed on novel class of potentially very promising AD biomarkers: extracellular miRNAs in body liquids, such as cerebrospinal fluid and blood. They have a great potential not only to indicate the presence of AD, but more importantly, to reflect the molecular mechanisms playing a role early at the beginning of the pathogenic pathways consequently leading to AD. We believe this comprehensive review on deregulated miRNAs in AD can be a good source of information for thorough in silico analyses aiming to identification, development and validation of miRNAs as "diseases mechanism engaged" candidate biomarkers. Having such molecules could bring us closer to the goal - successful treatment of AD.

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.

Proteomic Analysis of the Spatio-temporal Based Molecular Kinetics of Acute Spinal Cord Injury Identifies a Time- and Segment-specific Window for Effective Tissue Repair.

Spinal cord injury (SCI) represents a major debilitating health issue with a direct socioeconomic burden on the public and private sectors worldwide. Although several studies have been conducted to identify the molecular progression of injury sequel due from the lesion site, still the exact underlying mechanisms and pathways of injury development have not been fully elucidated. In this work, based on OMICs, 3D matrix-assisted laser desorption ionization (MALDI) imaging, cytokines arrays, confocal imaging we established for the first time that molecular and cellular processes occurring after SCI are altered between the lesion proximity, i.e. rostral and caudal segments nearby the lesion (R1-C1) whereas segments distant from R1-C1, i.e. R2-C2 and R3-C3 levels coexpressed factors implicated in neurogenesis. Delay in T regulators recruitment between R1 and C1 favor discrepancies between the two segments. This is also reinforced by presence of neurites outgrowth inhibitors in C1, absent in R1. Moreover, the presence of immunoglobulins (IgGs) in neurons at the lesion site at 3 days, validated by mass spectrometry, may present additional factor that contributes to limited regeneration. Treatment in vivo with anti-CD20 one hour after SCI did not improve locomotor function and decrease IgG expression. These results open the door of a novel view of the SCI treatment by considering the C1 as the therapeutic target.

Tau-mediated synaptic damage in Alzheimer's disease.

Synapses are the principal sites for chemical communication between neurons and are essential for performing the dynamic functions of the brain. In Alzheimer's disease and related tauopathies, synapses are exposed to disease modified protein tau, which may cause the loss of synaptic contacts that culminate in dementia. In recent decades, structural, transcriptomic and proteomic studies suggest that Alzheimer's disease represents a synaptic disorder. Tau neurofibrillary pathology and synaptic loss correlate well with cognitive impairment in these disorders. Moreover, regional distribution and the load of neurofibrillary lesions parallel the distribution of the synaptic loss. Several transgenic models of tauopathy expressing various forms of tau protein exhibit structural synaptic deficits. The pathological tau proteins cause the dysregulation of synaptic proteome and lead to the functional abnormalities of synaptic transmission. A large body of evidence suggests that tau protein plays a key role in the synaptic impairment of human tauopathies.

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.

A comparative study of PKH67, DiI, and BrdU labeling techniques for tracing rat mesenchymal stem cells.

Mesenchymal stem cells (MSCs) have generated a great deal of promise as a potential source of cells for cell-based therapies. Various labeling techniques have been developed to trace MSC survival, migration, and behavior in vitro or in vivo. In the present study, we labeled MSCs derived from rat bone marrow (rMSCs) with florescent membrane dyes PKH67 and DiI, and with nuclear labeling using 5 µM BrdU and 10 µM BrdU. The cells were then cultured for 6 d or passaged (1-3 passages). The viability of rMSCs, efficacy of fluorescent expression, and transfer of the dyes were assessed. Intense fluorescence in rMSCs was found immediately after membrane labeling (99.3 ± 1.6% PKH67+ and 98.4 ± 1.7% DiI+) or after 2 d when tracing of nuclei was applied (91.2 ± 4.6% 10 µM BrdU+ and 77.6 ± 4.6% 5 µM BrdU+), which remained high for 6 d. Viability of labeled cells was 91 ± 3.8% PKH67+, 90 ± 1.5% DiI+, 91 ± 0.8% 5 µM BrdU+, and 76.9 ± 0.9% 10 µM BrdU+. The number of labeled rMSCs gradually decreased during the passages, with almost no BrdU+ nuclei left at final passage 3. Direct cocultures of labeled rMSCs (PKH67+ or DiI+) with unlabeled rMSCs revealed almost no dye transfer from donor to unlabeled recipient cells. Our results confirm that labeling of rMSCs with PKH67 or DiI represents a non-toxic, highly stable, and efficient method suitable for steady tracing of cells, while BrdU tracing is more appropriate for temporary labeling due to decreasing signal over time.

Disorder of sexual development in a Yorkshire terrier (78, XY; SRY-positive).

A 9-month-old Yorkshire terrier was admitted to the clinic because of abnormal sexual behaviour and clitoral hypertrophy. External examination confirmed standard development of caudal genital organs: vagina, vulva and cervix uteri. Serum profile of gonadotropin hormones 17 ß-estradiol (<10.0 pg.ml(-1)) and testosterone (9.1 ng.ml(-1)) revealed the presence of testicular tissue. A midline laparotomy was performed to detect the cranial parts of the genital system. Gonads resembling testicles, structures indicating epididymis and rudimentary deferent ducts were resected, along with adherent part of the uterus. Cytogenetic analysis showed a male chromosomal complement 78, XY in all metaphases of the studied Yorkshire terrier dog. The chromosomal constitution was confirmed by fluorescence in situ hybridisation (FISH) with whole-chromosome painting probes specific for chromosomes X and Y, as well as by polymerase chain reaction (PCR) amplification of the 271-bp Y-linked fragment of SRY (the sex-determining region on the Y chromosome) gene. Sequencing of the dog's SRY gene coding region did not reveal any mutation. To search for potential mutation in the SOX9 gene (Sry-box containing gene 9), which is considered to be one of the key genes involved in the sex determination process, the PCR fragments of exons 1, 2 and 3 originating from the canine patient were sequenced in order to compare with both male and female healthy control dogs. In the analysed regions of the SOX9 gene, no mutation was found.

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.

Umbilical cord blood cells CD133+/CD133- cultivation in neural proliferation media differentiates towards neural cell lineages.

BACKGROUND AND AIMS: Umbilical cord blood (UCB) has been identified as a good source of hematopoietic and nonhematopoietic stem cells that can be easily isolated. In the present study we investigated the possibility of whether stem cells in mononuclear UCB grown under defined conditions can produce progeny with neural phenotype. METHODS: A combination of antigen-driven magnetic cell sorting (MACs) method and defined culture conditions specific for cells of neural lineages were used for isolation, expansion and differentiation of CD133+/- cells from UCB. Both UCB-derived fractions were expanded by exposure to growth factors (EGF, bFGF). Differentiation was induced by replacing them with fetal bovine serum. Using immunocytochemistry, the cell markers for neural (MAP2, GFAP, RIP) and non-neural lineages (S-100, von Willebrand factor) were detected. RESULTS: The analysis revealed occurrence of fully mature neural and non-neural lineages, which showed qualitative and quantitative differences between population of CD133+ and CD133- cells. The expression levels of MAP2 and RIP in CD133+ were significantly higher than in CD133-, more GFAP positive cells were found in the CD133-. At the same time, S-100 was expressed by 32.47 ± 6.24% of CD133- cells and 29.42 ± 1.32% of CD133- cell expressed a von Willebrand factor antigen. CONCLUSIONS: Our results indicate that stem cells derived from umbilical cord blood are easy to obtain, proliferate and are able to differentiate towards the cells of neural lineages, which represents a promising way for their utilization in cell-based therapies for CNS injuries and diseases.

Identification of NO/sGC signalling in the bulbospinal respiratory pathway after C2-C3 hemisection of the spinal cord in rats.

Guanylyl cyclase (GC) as the effector molecule for nitric oxide (NO) plays a key role in the NO/cGMP signalling cascade. Based on these observations, our study focused on NO/sGC signalization in the bulbospinal respiratory pathway. The distribution of neuronal nitric oxide synthase (nNOS), ß1 subunit of soluble guanylyl cyclase (ß1sGC) and synaptophysin (SYN) was explored in the upper part of the respiratory pathway after C2-C3 hemisection of the spinal cord in male Wistar rats. Unilateral injection of Fluorogold into the phrenic nucleus (PN) at C4 level and survival of animals for 2 days revealed many Fluorogold fluorescent neurons in the ventral respiratory group (VRG) of the medulla, mostly on the contralateral side. Under physiological conditions we noted nNOS-fluorescent terminals of VRG neurons around ß1sGC fluorescent motoneurons in the PN. A strong depletion of nNOS/SYN fluorescent terminals was noted 8 days after hemisection around alpha motoneurons in the PN on the contralateral side. On the side of injury, nNOS/SYN fluorescent puncta were detected around phrenic motoneurons only sporadically. Phrenic alpha motoneurons responded to C2-C3 hemisection by a loss of ß1sGC positivity. The results confirm, that ß1sGC immunoreactive phrenic motoneurons are innervated by nNOS positive terminals coming from the VRG.

The effect of cadmium in combination with zinc and selenium on ovarian structure in Japanese quails.

In this study the effect of cadmium, cadmium+selenium and cadmium+zinc administration on the ovarian structure in Japanese quails was studied. The morphometric analysis of the relative volume of primary follicles detected the highest value in control group with a similar value in the group with administration of cadmium with selenium. Lower relative volume is reported in group with cadmium and zinc administration and the group with simple cadmium administration (P < 0.05). The relative volume of growing follicles was very similar in all studied groups (11.33-15.35%), and the relative volume of stroma was very stable (82.59-86.45%). In the evaluation of the number of follicles undergoing atresia detected significantly higher number of atretic primary follicles as well as atretic growing follicles in the group with cadmium administration and cadmium with selenium administration in comparison with control group. In comparison of normal and atretic follicles we report the most negative effect of single cadmium administration on ovarian structure. Selenium co-administration shows protective effects but only the co-administration with zinc prevent significant cadmium ovarian alterations.

Cobalt-induced alterations in hamster testes in vivo.

The purpose of this study was to assess the effects of cobalt on the testicular structure of adult golden hamsters. Hamsters in group A received cobalt (CoCl2) in single intraperitoneal dose 20 mg/kg, in group B 10 mg/kg and in group C 5 mg CoCl2/kg body weight and were killed fourty eight hours after cobalt administration. Afer a preparation of histological samples the results were compared with control. After a cobalt administration dilatation of blood capillaries in interstitium, undulation of basal membrane and occurrence of empty spaces in seminiferous epithelium was detected. Morphometric analysis showed that in all cobalt-treated groups the relative volume of seminiferous epithelium was significantly decreased. In the relative volume of interstitium a significant increase was found between control group and experimental groups. After cobalt administration we have found linear non-significant decrease. Evaluation of diameter seminiferous tubules found increase of this parameter in the all experimental group in comparison with control. Height of seminiferous epithelium was relatively constant and in all groups but the difference between control and group A was significant (P < 0.05). Analysis of the lumen diameter of seminiferous tubules detected significantly increase mainly group B. Evaluation of the number of cell nuclei per a constant area detected an increase of this parameter in experimental group. Results of this study report a negative effect of cobalt on structure and function of testes.