19 research outputs found
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