Hippocampal capillaries in different age groups of chronically ethanol-intoxicated rats.

The influence of chronic ethanol intoxication on the terminal vascularization of particular hippocampal fields and layers was investigated in different age groups of rats. Thirty-six male Wistar rats aged 6 weeks were used in the study. For twelve months 24 of them drank only 25% ethanol — 12 starting at 6-week-age and 12 at 3-month-age. The control group of 12 rats drank only water. As an effect of long-term ethanol exposure on hippocampal capillaries we observed the increase in the terminal vessel diameter and the decrease in microvascular length, surface, and volume densities. These changes varied between different age groups and between particular hippocampal regions. The observed age and regional differentiation of ethanol-related microvascular changes did not correlate well with the damaging effects of alcohol on corresponding neuronal elements, which emphasizes the very complicated pathogenesis of ethanol-induced injuries

The growth and differentiation of aortal smooth muscle cells after calcitriol treatment are associated with microtubule reorganisation - an in vitro study

The smooth muscle cells (SMCs) of the arterial media play a predominant role in functional and structural alterations of the arterial wall. The transition from the “contractile” to the “synthetic” phenotype appears to be an early critical event in the development of atherosclerotic disease. A number of observations suggest that 1,25(OH)2D3 (calcitriol) is of importance in maintaining normal cardiovascular function through its receptors in cardiac myocytes or aortal SMCs. The present study has focused on the microtubular (MT) network reorganisation after exposure to calcitriol. SMCs isolated by enzymatic digestion from the aortal media of neonatal rats were cultured on glass cover slips. 1 μM of 1,25(OH)2D3 was added to the culture medium every second day. The cytoskeletal features of SMCs after calcitriol were visualised by the immunofluorescence staining of α-tubulin. The alterations in α-tubulin expression and the distribution of microtubules related to the activities of the vascular smooth muscle cells, namely adhesion, migration, multilayer formation and cell division, were observed. A spindle shape, decreased cell adhesion, low expression of α-tubulin and a longitudinally arranged microtubular network manifested the high rate of SMC differentiation in the calcitriol-treated culture. A flat stellate morphology, high expression of α-tubulin and a radially distributed three-dimensional microtubular network were observed in the SMCs of the control culture. Destructive changes in the microtubular architecture which altered the cellular shape were evident in SMCs undergoing apoptosis. Cells with apoptotic features were more frequent in calcitriol-exposed culture. In contrast to the regular SMC divisions observed in the control culture, some of the mitotic cells exposed to calcitriol contained broader bipolar, multipolar or disordered spindles. These alterations in the SMCs’ microtubular cytoskeleton after calcitriol treatment were concomitant with changes in cell growth, differentiation and apoptosis, and may suggest a similarity to atherosclerotic plaque formation

A scanning electron microscopic study of phenotypic plasticity and surface structural changes of aortal smooth muscle cells in primary culture

Phenotypic modulation of smooth muscle cells (SMCs) from a contractile to a synthetic state characterised by active proliferation appears to be an early event in the pathogenesis of atherosclerosis. A similar transition occurs when SMCs are established in culture. In this study the phenotypic plasticity and surface structural changes of aortal smooth muscle cells during the transition from the contractile to the synthetic state and during maturation have been structurally assessed by scanning electron microscope (SEM). The experiments were performed on SMCs obtained from aorta of neonatal rats after enzymatic digestion and then cultured on glass cover slips. SEM observations revealed a three-dimensional appearance characteristic for different stages of SMCs. Intensively proliferating cells from monolayer region were large, polygonal in shape with lamellipodia and well spread. Long, uniform in diameter, finger-like microvilli were densely arranged on the surface of these cells. In the thickened region of culture, the cells were rather small, generally spindle-shaped, not well spread, with low density of short, bubble-like microvilli on the surface. Numerous plasma membrane structural alterations in apoptotic cells were observed by SEM: loss of cellular adhesion, smoothing, shrinkage and outpouching of membrane segments have been recognised as markers associated with the cell injury and death. It was concluded that scanning microscopy observations would allow a more complete understanding of SMCs and their changes in culture and atherosclerotic disease

Ultrastructure of the ventromedial hypothalamic nucleus in fasted and refed young and old rats

Many hypothalamic nuclei are involved in the regulation of food intake and energy homeostasis. An ultrastructural investigation of the hypothalamic ventromedial nucleus (VMN), a hypothetical "satiety centre" was performed to explore the morphological basis of altered feeding behaviour of old rats in an experimental model of fasting/refeeding. Young (5 months old, n = 12) and old (24 months old, n = 12) male Wistar rats were fasted for 48 hours, then refed for 24 hours and sampled thereafter. Brain tissue was fixed by perfusion, histological and ultrathin sections were obtained by routine methods. Although food intake was similar in control young and old rats, during refeeding old animals consumed less chow than young ones. The EM analysis of VMN neurones of old control rats revealed, besides typical age-related residual bodies, deep indentations of the nuclear envelope and the presence of long, undulating rough endoplasmic reticulum cisternae in the cell periphery. In both young and old rats fasting for 48 hours led to the expansion of Golgi complexes and increased folds of the nuclear envelope, which is suggestive of enhanced cellular activity of the VMN neurones. These fasting-induced alterations were sustained in the VMN neurones of refed rats in both age groups. The results showed that the VMN neurones of old control rats differ at the ultrastructural level from young ones. However, starvation and subsequent refeeding cause similar alterations in the hypothalamic neurones of "satiety centre" of both young and old rats

The effect of fasting and refeeding on the ultrastructure of the hypothalamic paraventricular nucleus in young and old rats

In order to explore the morphological basis of the altered feeding behaviour of old rats, an ultrastructural investigation of the magnocellular neurons of the hypothalamic paraventricular nucleus (PVN) was performed. Young and old male Wistar rats, 5 and 24 months old, respectively, and with each age group comprising 12 animals, were divided into 3 groups. The rats in Group I were used as controls (normally fed), the rats of Group II were fasted for 48 hours and in Group III the rats were fasted for 48 hours and then refed for 24 hours. The brains were fixed by perfusion and histological and ultrathin sections were obtained by routine methods. Common features of the magnocellular PVN neurons of young and old rats were abundant Golgi complexes and short fragments of RER localised at the cell periphery. In contrast to young rats, the PVN neurons of old animals showed deep indentations of the nuclear envelope and agerelated residual bodies. In both age groups fasting for 48 hours led to the expansion of the Golgi complexes and dilatation of RER cisternae. In contrast to those in fed rats, RER cisternae in the neurons of old fasted animals were situated between the nuclear envelope and the Golgi zone. Prolonged RER cisternae were distributed in the peripheral cytoplasm of refed old rats. Our observations suggest that at the ultrastructural level the process of ageing does not change the responsiveness of magnocellular PVN neurons to fasting-refeeding

Regulatory effects of 1,25-dihydroxyvitamin D3 on vascular smooth muscle cells

Inflammatory response has been recognized as a central feature in the development and progression of atherosclerosis, and VSMCs (Vascular Smooth Muscle Cells) - the main cellular component of media, play an important role in this process. Many reports indicate that the biologically active vitamin D metabolite - 1,25-dihydroxyvitamin D3 (1,25(OH)2D3 = calcitriol), besides its well established role in calcium homeostasis, plays an essential role in the regulation of the inflammation process. The aim of this study was to determine the regulatory effects of calcitriol, applied at two supra-physiological doses (10 nM and 100 nM), in VSMC culture. Secretion of the pro-inflammatory cytokines, IL-6 and TNF-α, was significantly attenuated in calcitriol-treated VSMC culture, but the level of anti-inflammatory TGF-β was generally unchanged. Since in advanced atherosclerosis lesions several cell types, including VSMCs, overproduce the HSP70 chaperone protein, we also checked the effects of calcitriol on its synthesis. The presence of 1,25(OH)2D3 did not affect HSP70 synthesis under physiological conditions but the synthesis of HSP70 in VSMCs exposed to heat shock was significantly inhibited by calcitriol (=100 nM). We observed that 1,25(OH)2D3 induced SOD 1 activity, stimulated the expression of IκB-α, and did not influence the level of NF-κB-p65 in VSMCs. The results of our study suggest that 1,25(OH)2D3 may serve as a natural anti-inflammatory agent and may therefore play a beneficial role in the physiology of VSMC in some contexts of atherosclerosis

Molecular Mechanism of Silver Nanoparticles-Induced Human Osteoblast Cell Death: Protective Effect of Inducible Nitric Oxide Synthase Inhibitor

<div><p>Background</p><p>Silver nanoparticles (AgNPs) show strong antibacterial properties, making them excellent candidates to be used in orthopaedic repair and regeneration. However, there are concerns regarding the cytotoxicity of AgNPs and molecular mechanisms underlying AgNPs-induced bone cells toxicity have not been elucidated. Therefore, the aim of our study was to explore mechanisms of AgNPs-induced osteoblast cell death with particular emphasis on the role of nitric oxide (NO) generated by inducible nitric oxide synthase (iNOS).</p><p>Methods and Result</p><p>Silver nanoparticles used in this study were 18.3±2.6 nm in size, uncoated, spherical, regular shape and their zeta potential was -29.1±2.4 mV as measured by transmission electron microscopy (TEM) and zetasizer. The release of silver (Ag) from AgNPs was measured in cell culture medium by atomic absorption spectroscopy (AAS). The exposure of human osteoblast cells (hFOB 1.19) to AgNPs at concentration of 30 or 60 μg/mL for 24 or 48 hours, respectively resulted in cellular uptake of AgNPs and changes in cell ultrastructure. These changes were associated with apoptosis and necrosis as shown by flow cytometry and lactate dehydrogenase (LDH) assay as well as increased levels of pro-apoptotic Bax and decreased levels of anti-apoptotic Bcl-2 mRNA and protein. Importantly, we have found that AgNPs elevated the levels of nitric oxide (NO) with concomitant upregulation of inducible nitric oxide synthase (iNOS) mRNA and protein. A significant positive correlation was observed between the concentration of AgNPs and iNOS at protein and mRNA level (r = 0.837, r = 0.721, respectively; p<0.001). Finally, preincubation of osteoblast cells with N-iminoethyl-l-lysine (L-NIL), a selective iNOS inhibitor, as well as treating cells with iNOS small interfering RNAs (siRNA) significantly attenuated AgNPs-induced apoptosis and necrosis. Moreover, we have found that AgNPs-induced cells death is not related to Ag dissolution is cell culture medium.</p><p>Conclusion</p><p>These results unambiguously demonstrate that increased expression of iNOS and generation of NO as well as NO-derived reactive species is involved in AgNPs-induced osteoblast cell death. Our findings may help in development of new strategies to protect bone from AgNPs-induced cytotoxicity and increase the safety of orthopaedic tissue repair.</p></div

AgNPs-induced generation of nitrotyrosine in hFOB 1.19 and its inhibition by L-NIL.

<p>(A) A bar graph showing data as measured by ELISA; (B) Representative immunoblots. Data are expressed as means ± SD of 3 independent experiments. *p<0.05; **p<0.01 exposed cells v/s control or as indicated.</p

AgNPs-induced necrosis in hFOB 1.19 cells and protective effect of L-NIL.

<p>Nec-1 failed to protect cells from AgNPs-induced cell death. Data are expressed as means ± SD of 3 independent experiments. *p<0.05; ***p<0.001 exposed cells v/s control or as indicated.</p

Evaluation of cytotoxicity of AgNPs and Ag released in cell medium from 30 and 60 μg/mL AgNPs on hFOB 1.19 cells.

<p>Results are expressed as % LDH release by cells into the culture medium and presented as mean ± standard deviation of 3 independent experiment. ***p<0.001 exposed cells v/s control.</p