Anti-inflammatory and antioxidative effects of elderberry diet in the rat model of seizure: a behavioral and histological investigation on the hippocampus.

The present study was designed to evaluate whether elderberry (EB) effectively reduces inflammation and oxidative stress in hippocampal cells to modify seizure damage. Seizure was induced in rats by the injection of pentylenetetrazol (PTZ). In the Seizure + EB group, EB powder was added to the rats' routine diet for eight consecutive weeks. The study included several behavioral tests, immunohistopathology, Voronoi tessellation (to estimate the spatial distribution of cells in the hippocampus), and Sholl analysis. The results in the Seizure + EB group showed an improvement in the behavioral aspects of the study, a reduction in astrogliosis, astrocyte process length, number of branches, and intersections distal to the soma in the hippocampus of rats compared to controls. Further analysis showed that EB diet increased nuclear factor-like 2 expression and decreased caspase-3 expression in the hippocampus in the Seizure + EB group. In addition, EB protected hippocampal pyramidal neurons from PTZ toxicity and improved the spatial distribution of hippocampal neurons in the pyramidal layer and dentate gyrus. The results of the present study suggest that EB can be considered a potent modifier of astrocyte reactivation and inflammatory responses.

Long-Term Effects of Hippocampal Low-Frequency Stimulation on Pro-Inflammatory Factors and Astrocytes Activity in Kindled Rats.

OBJECTIVE: Epilepsy is accompanied by inflammation, and the anti-inflammatory agents may have anti-seizure effects. In this investigation, the effect of deep brain stimulation, as a potential therapeutic approach in epileptic patients, was investigated on seizure-induced inflammatory factors. MATERIALS AND METHODS: In the present experimental study, rats were kindled by chronic administration of pentylenetetrazol (PTZ; 34 mg/Kg). The animals were divided into intact, sham, low-frequency deep brain stimulation (LFS), kindled, and kindled +LFS groups. In kindled+LFS and LFS groups, animals received four trains of intra-hippocampal low-frequency deep brain stimulation (LFS) at 20 minutes, 6, 24, and 30 hours after the last PTZ injection. Each train of LFS contained 200 pulses at 1 Hz, 200 µA, and 0.1 ms pulse width. One week after the last PTZ injection, the Y-maze test was run, and then the rats' brains were removed, and hippocampal samples were extracted for molecular assessments. The gene expression of two pro-inflammatory factors [interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α)], and glial fibrillary acidic protein (GFAP) immunoreactivity (as a biological marker of astrocytes reactivation) were evaluated. RESULTS: Obtained results showed a significant increase in the expression of of interleukin-6 (IL-6), tumor necrosis factor (TNF)-α, and GFAP at one-week post kindling seizures. The application of LFS had a long-lasting effect and restored all of the measured changes toward normal values. These effects were gone along with the LFS improving the effect on working memory in kindled animals. CONCLUSION: The anti-inflammatory action of LFS may have a role in its long-lasting improving effects on seizure-induced cognitive disorders.

Ultrastructural maturation of human bone marrow mesenchymal stem cells-derived cardiomyocytes under alternative induction of 5-azacytidine.

Adult cardiomyocytes lack the ability to proliferate and are unable to repair damaged heart tissue, therefore differentiation of stem cells to cardiomyocytes represents an exceptional opportunity to study cardiomyocytes in vitro and potentially provides a valuable source for replacing damaged tissue. However, characteristic maturity of the in vitro differentiated cardiomyocytes and methods to achieve it are yet to be optimized. In this study, differentiation of human bone marrow-mesenchymal stem cells (hBM-MSCs) into cardiomyocytes is accomplished and the process investigated ultrastructurally. The hBM-MSCs were alternatively treated with 5 µM of 5-azacytidine (5-aza) for 8 weeks resulting in differentiation to cardiomyocytes. Expressions of cardiomyocyte-specific genes [cardiac α-actinin, cardiac ß-myosin heavy chain (MHC) and connexin-43] and proteins (cardiac α-actinin, cardiac troponin and connexin-43) were confirmed in a time-dependent manner from the first to the fifth weeks post-induction. Ultrastructural maturation of hBM-MSCs-derived cardiomyocyte (MSCs-CM) corresponded with increase in number and organization of myofilaments in cells over time. Starting from week five, organized myofibrils along with developing sarcomeres were detectable. Later on, MSCs-CM were characterized by the presence of sarcoplasmic reticulum, T-tubules and diads as cardiomyocytes connected to each other by intercalated disc-like structures. Here, we showed the potential of hBM-MSCs as a source for the production of cardiomyocytes and confirmed mature ultrastructural characteristics of these cells using our alternative incubation method.

Effect of low-frequency stimulation on kindling induced changes in rat dentate gyrus: an ultrastructural study.

It has been shown that low-frequency stimulation (LFS) can induce anticonvulsant effects. In this study, the effect of different LFS frequencies on kindling induced behavioral and ultrastructural changes was investigated. For induction of kindled seizures in rats, stimulating and recording electrodes were implanted in perforant path and dentate gyrus, respectively. Animals were stimulated in a rapid kindling manner. Different groups of animals received LFS at different frequencies (0.5, 1 and 5 Hz) following kindling stimulations and their effects on kindling rate were determined using behavioral and ultrastructural studies. Kindling stimulations were applied for 7 days. Then, the animals were sacrificed and their dentate gyrus was sampled for ultrastructural studies under electron microscopy. All three used LFS frequencies (0.5, 1 and 5 Hz) had a significant inhibitory effect on kindling rate and decreased afterdischarge duration and the number of stimulations to achieve stage 4 and 5 seizures significantly. In addition, application of LFS prevented the increase in the post-synaptic density and induction of concave synaptic vesicles following kindling. There was no significant change between anticonvulsant effects of LFS at different frequencies. Obtained results show that LFS application can prevent the neuronal hyperexcitability by preventing the ultrastructural changes during kindling and this may be one of the mechanisms of LFS anticonvulsant effects.

The antiepileptogenic effect of electrical stimulation at different low frequencies is accompanied with change in adenosine receptors gene expression in rats.

PURPOSE: Previous studies have shown that the anticonvulsant effects of low-frequency stimulation (LFS) can be affected by activation of adenosine receptors. In the present study, the effect of LFS at different frequencies on kindling rate and adenosine receptors gene expression was investigated. METHODS: Animals were kindled by perforant path stimulation in a rapid kindling manner. LFS (0.5, 1, and 5 Hz) was applied after termination of each kindling stimulation. Seizure severity was measured according to behavioral and electrophysiologic parameters. At the end of the experiments, adenosine A(1) and A(2A) receptor gene expression were measured. RESULTS: The inhibitory effect of LFS on kindling acquisition was observed at all frequencies. In addition, the inhibitory action of LFS on enhancement of field excitatory postsynaptic potential slope and population spike amplitude during kindling acquisition was not affected by the LFS frequency. However, the effects of LFS on paired-pulse recordings were greater at frequency of 5 Hz. Application of LFS during kindling acquisition also prevented the kindling induced decrease in the A(1) receptor gene expression and attenuated the level of A(2A) receptor gene expression in the dentate gyrus. These effects were also greater at the frequency of 5 Hz. DISCUSSION: According to these data, it may be suggested that the antiepileptogenic effects of LFS, developed through inhibition of synaptic transmission in the dentate gyrus, is mediated somehow through preventing the decrease of A(1) receptor and through attenuating the A(2A) receptor gene expression. These effects might be dependent on the frequency of LFS.

Ultrastructural comparison of developing mouse embryonic stem cell- and in vivo-derived cardiomyocytes.

Embryonic stem cells (ESCs) are expected to become a powerful tool for future regenerative medicine and developmental biology due to their capacity for self-renewal and pluripotency. The present study involves characterization and particularly, the ultrastructure of ESC-derived cardiomyocytes (ESC-CMs). Spontaneously differentiated murine (C57BL/6) ESC-CMs were cultured for 21 days. At different stages, growth characteristics of the CMs were assessed by immunocytochemistry, RT-PCR, transmission electron microscopy, and by addition of chronotropic drugs. EB-derived spontaneously beating cells expressed markers characteristic of CMs including alpha-actinin, desmin, troponin I, sarcomeric myosin heavy chain (MHC), pan-cadherin, connexin 43, cardiac alpha-MHC, cardiac beta-MHC, atrial natriuretic factor (ANF), and myosin light chain isoform-2V (MLC-2V) and responded to drugs in a maturation- and dose-dependent manner. At the ultrasructural level, maturation proceeded with increasing time in culture. In 7+21 days CMs, all sarcomeric components, such as Z-discs, A-, I- and H-bands as well as M-lines, T-tubules, intercalated discs, and the sarcoplasmic reticulum were present. Our data suggest that ESCs can differentiate into functional mature CMs in vitro. Furthermore, ESC-CMs may provide an ideal model for the study of cardiomyocytic development and may be useful for cell therapy of various cardiac diseases.