Pulsed electromagnetic field affects intrinsic and endoplasmatic reticulum apoptosis induction pathways in MonoMac6 cell line culture.

Current studies were aimed to elucidate influence of pulsed electromagnetic field stimulation on cell viability and apoptosis induction pathways. For the experimental model we have chosen monocytic cell line MonoMac6 and several apoptosis inducers with different mechanism of death induction like puromycin, colchicine, cyclophosphamide, minocycline and hydrogen peroxide. MonoMac6 cell line was grown at density 1x10(5) cells/well in 96-well culture plates. To induce cell death cell cultures were treated with different apoptosis inducers like puromycin, colchicine, cyclophosphamide, minocycline, hydrogen peroxide and at the same time with pulsed electromagnetic field 50 Hz, 45±5 mT (PEMF) for 4 hour per each stimulation, three times, in 24 hours intervals. Afterwards, cells were harvested for flow cytometry analysis of cell viability measured by annexin V-APC labeled and propidium iodide staining. Expression of apoptosis related genes was evaluated by semi quantitative reverse transcription (RT)-PCR assay. NuPAGE Novex Western blot analysis was carried out for apoptosis inducing factor (AIF) abundance in cytosolic and nuclear extracts of MonoMac6 cells. Puromycin, colchicine and minocycline activated cells and simultaneously treated with PEMF have shown out diminished percentage of annexinV positive (AnV+) cells comparing to controls without PEMF stimulation. MonaMac6 cells puromycin/colchicyne and PEMF treated were to a higher extent double stained (AnV+,PI+), which means increased late apoptotic as well as necrotic (PI+) cells, than non-stimulated controls. On the other hand, minocycline activated cells prior to PEMF treatment showed diminished amount of apoptotic and necrotic (annexin V, annexin V and propidium iodide, propidium iodide positive staining) cells. The opposite effect of PEMF on the percentage of annexin V positively stained cells has been achieved after treatment of MonoMac6 culture with cyclophoshamide and hydrogen peroxide. PEMF enhanced early phase of apoptosis induced by both apoptosis inducing agents. The analysis of expression of the apoptosis related genes in MonoMac6 cultures treated with puromycin and exposed to PEMF performed in reverse transcription of polymerase chain reaction (PCR) assay has shown changes in mRNA of genes engaged in intrinsic apoptotic pathway and pathway with AIF abundance. The most influenced was expression of gene belonging to pro-apoptotic family of Bcl-2 and AIF agent. Examination of immunoblots developed with anti-AIF antibody showed that cytosol content of AIF protein was diminished after puromycin and PEMF treatment of MonoMac6 cells. The obtained results indicate that PEMF affects induction of apoptosis in MonoMac6 cells stimulated to death with inducing agents to a different extent. Main finding of the current results is that, PEMF stimulation of MonoMac6 cells simultaneously treated with puromycin caused changes in the Bcl-family genes expression as well as in caspase independent pathway of apoptosis inducing factor (AIF).

Pulsating electromagnetic field stimulation prevents cell death of puromycin treated U937 cell line.

Aim of study was to verify whether pulsating electromagnetic field (PEMF) can affect cancer cells proliferation and death. U937 human lymphoid cell line at densities starting from 1 x 10(6) cells/ml to 0.0625 x 10(6) cells/ml, were exposed to a pulsating magnetic field 50 Hz, 45+/-5 mT three times for 3 h per each stimulation with 24 h intervals. Proliferation has been studied by counting number of cells stimulated and non-stimulated by PEMF during four days of cultivation. Viability of cells was analyzed by APC labeled Annexin V and 7-AAD (7-amino-actinomycin D) dye binding and flow cytometry. Growing densities of cells increase cell death in cultures of U937 cells. PEMF exposition decreased amount of cells only in higher densities. Measurement of Annexin V binding and 7-AAD dye incorporation has shown that density-induced cell death corresponds with decrease of proliferation activity. PEMF potentiated density-induced death both apoptosis and necrosis. The strongest influence of PEMF has been found for 1 x 10(6)cells/ml and 0.5 x 10(6) cells/ml density. To eliminate density effect on cell death, for further studies density 0.25 x 10(6) cells/ml was chosen. Puromycin, a telomerase inhibitor, was used as a cell death inducer at concentration 100 microg/ml. Combined interaction of three doses of puromycin and three fold PEMF interaction resulted in a reduced of apoptosis by 24,7% and necrosis by 13%. PEMF protects U937 cells against puromycin- induced cell death. PEMF effects on the human lymphoid cell line depends upon cell density. Increased density induced cells death and on the other hand prevented cells death induced by puromycin.

Physiological and morphological effects of long-term vagal stimulation in diet induced obesity in rats.

Some previous studies have shown suppressive effect of the vagal nerve stimulation (VNS) on long - term feeding regulation in rats. We assessed body weight, interstitial cells of Cajal (ICC), myenteric plexus neurons, mast cells in the stomach, duodenum and colon and c-Fos expression in nodose vagal ganglia in the rats with VNS. Male Wistar rats were implanted with microchip (MC) and kept during the whole study (100 days) on high calorie diet. Left vagal nerve was stimulated by electrical pulses (10ms, 200mV, 0.05Hz) generated by MC. After finishing the experiments tissue samples (stomach, duodenum, colon and nodosal vagal ganglia) were taken. Mast cells were toluidine blue stained and counted in mucosa, muscularis externa and serosa. For immunostaining, antibodies for ICC (CD117), myenteric plexus neurons (PGP9.5) and c-Fos were used. Positive cells were assessed by image analysis. Chronic microchip vagal stimulation significantly decreased epididymal fat pad weight, meal size with effect on decreased weight gain in VNS rat. VNS significantly increased mast cells number in all examined parts of the gastrointestinal wall, mainly in the muscularis. There were no significant differences in ICC and myenteric plexus neurons between VNS and control. Expression of c-Fos in nodosal ganglia was higher in VNS group. The effects observed during long-term VNS concern predominantly mast cells. These data support the theory that VNS can increase vagal afferent satiety signals leading to reduced food intake and body weight gain and mast cells are involved in this process.

Magnetic field anti-inflammatory effects in Crohn's disease depends upon viability and cytokine profile of the immune competent cells.

AIM: We investigated effects of pulsating electromagnetic field (PEMF-50 Hz, 45 +/- 5 mT) on viability and cytokine production by human peripheral blood mononuclear cells (PBMC) from healthy donors and from Crohn's disease patients (CD). METHODS: The study was performed after activation of cells with phytohaemaglutinin (PHA) and lipopolisaccharide (LPS). Exposure of PBMC cultures to PEMF from both CD patients and from healthy donors decreased cell's viability of about 10% and 5% (p>0.05) respectively. PEMF influence was most effective after threefold application. Susceptibility of PBMCs to magnetic field exposure differs among the stimulated (PHA, LPS) and not stimulated (NS) cells. Mitogen activated cells during cell division are most susceptible to induction of the cell death as a result of magnetic interaction, contrary PEMF exposure has minimal effect on non-diving PBMCs from CD patients and from controls. Decreased viability of the Crohn derived cells upon magnetic stimulation was accompanied by altered cytokines profile. Exposed and stimulated PBMCs from Crohn patients decreased IFN-gamma proinflammatory and increased IL-10 anti-inflammatory cytokine production. The electromagnetically induced cell death could be an important step for non-invasive PEMF treatment in chronic inflammatory diseases.

Effect of long-term vagal stimulation on food intake and body weight during diet induced obesity in rats.

Regulation of food intake and body weight is accomplished by several mechanisms. CNS receives information from periphery and modifies food intake mainly by vagal nerves that provide the major neuroanatomical link between gastrointestinal sites stimulated during food intake and CNS sites that control feeding behavior and metabolism. Gastric mechanoreceptors and jejunal chemoreceptors activated by food or vagal nerve stimulation (VNS), which mimic the physiological input, suppress feeding within short-term regulation. Our research was aimed on determination the role of electrical VNS in long-term control of food intake and body weight in diet induced obesity fed rats. Food intake, body weight and epididymal fat pad were assessed in male Wistar rats divided into three groups (controls vs. VNS). Rats were implanted with microchip and kept during the whole study (100 days) on diet induced obesity. Vagal nerve was stimulated by electrical rectangular pulses duration 10 ms, amplitude 200 mV, frequency 0.05 Hz generated by microchip. In control group surgery produced no significant changes in meal size and body weight gain as compared to intact group. In contrast, significantly decreased epididymal fat pad weight, decreased meal size with effect on decreased weight gain was observed in VNS rats. Data support theory that VNS can increase vagal afferent signal conduct to CNS and mimics the satiety signals leading to reduce food intake and body weight gain.

Loss of interstitial cells of Cajal after pulsating electromagnetic field (PEMF) in gastrointestinal tract of the rats.

Exposure to the magnetic field has remarkably increased lately due to fast urbanization and widely available magnetic field in diagnosis and treatment. However, biological effects of the magnetic field are not well recognized. The myoelectric activity recorded from the gastrointestinal and urinary systems is generated by specialized electrically active cells called interstitial cells of Cajal (ICCs). Thus it seems rational that ICC have significant vulnerability to physical factors like an electromagnetic field. The aim of this study was to evaluate the influence of pulsating electromagnetic field (PEMF) (frequency 10 kHz, 30ms, 300 muT burst, with frequency 1Hz) on ICCs density in the rat gastrointestinal tract. Rats were divided into two groups (n=32). The first group was exposed to PEMF continuously for 1, 2, 3, and 4 weeks (n = 16), and the second group (n=16) served as a control. Tissue samples of the rat stomach, duodenum and proximal colon were fixed and paraffin embedded. The tangential sections of 5 microm thickness were stained immunohistochemically with anti-c-Kit (sc-168) antibody and visualized finally by DAB as chromogen (brown end product). C-Kit positive branched ICC-like cells were detected under the light microscope, distinguished from the c-kit-negative non-branched smooth muscle cells and from the c-kit positive but non-branched mast cells and quantitatively analyzed by MultiScan computer program. Apoptosis detection was performed with rabbit anti-Bax polyclonal antibody (Calbiochem, Germany) and LSAB 2 visualization system. The surface of c-Kit immunopositive cells decreased after exposure to PEMF in each part of the gastrointestinal tract. Reduced density of ICCs was related to exposure time. The most sensitive to PEMF were ICCs in the fundus of the stomach and in the duodenum, less sensitive were ICCs in the colon and pacemaker areas of the stomach. No marked changes in ICC density in the pyloric part of the stomach were observed. We demonstrate that the PEMF induced apoptosis dependent decrease in ICC expression.

Effects of continuous microchip (MC) vagal neuromodulation on gastrointestinal function in rats.

Afferent fibers from gastrointestinal tract outnumber efferents ten times in vagal nerves. Modifying the afferent input makes possible to change discharge of vagal efferents affecting gastrointestinal functions in process known as neuromodulation (NM). Lately it has been used in the treatment of pain and hyperactive neurogenic bladder in urology. MC induced NM may therefore provide a concurrent to pharmacology tool, in treatment of gastrointestinal disorders. The aim of this study was to investigate the effects of long term neuromodulation procedure with use of MC on gastric motility, secretion and weight control in conscious rats. Experiments were performed on 30 Wistar male rats (250-350 g) divided in two groups: sham operated and microsurgically implanted with MC on left vagal nerve below diaphragm. Following stimulation parameters were used: frequency of 0.5-30 Hz, amplitude of 0.55 V, impulse duration of 10 ms in monophasic fashion. In both groups food intake and body weight were measured through the period of 2 weeks after recovery period. Then gastric fistula was implanted in gastric antrum and fasted gastric motility recorded with use of PowerLab system (Australia). Gastric emptying and secretion were also tested with use of phenol red and automatic titration methods. On the daily basis glucose level with standard test and leptin after MC implantation were measured. Recording of vagal activity in fasted rats showed burst of action potentials about 5 +/- 2.5 in period of 5000 sec, each burst with spike frequency up to 35 Hz. Food (5 ml of Intralipid--intragastrically) almost doubled amount of bursts to 12 +/- 5 in period of 5000 sec with increase in frequency at spike up to 50 Hz. MC induced vagal activity showed continuous spike activity similar to fed pattern. MC induced NM decreases daily food intake by 6% (33.6 +/- 4.8 vs control 35.5 +/- 4.8 g, p < 0.01). Body weight gain in rats before MC implantation decreased by 20% within 2 weeks after recovery (34.8 +/- 9.08 vs control 23.56 +/- 4.15 g). Fasting control glucose level also decreased of 5.5% (93.15 +/- 9.3 vs control 98.5 +/- 11.2 mg%, p < 0.05). Frequency of gastric contractions did not change significantly in MC versus control but amplitude of contractions increased of about 66.7% (2.0 +/- 0.8 vs 1.17 +/- 0.52) at the dominant frequency 0.08 Hz range and about 71.5% (1.17 +/- 0.35 vs 0.68 +/- 0.47, p < 0.05) at the frequency 0.12 Hz. in FFT analysis PowerLab (chart v = 4.01). BAO decreased by 29.25% without H+ concentration changes (0.2 +/- 0.14 vs 0.14 +/- 0.12 mmol/30 min, p < 0.05) but MAO did not change in MC rats (0.37 +/- 0.25 vs 0.42 +/- 0.28 mmol/30 min, p 0.05). Gastric emptying of isotonic solution increased by 10% (90.46 +/- 5.34 vs 80.39 +/- 9.95) percent of marker passing to duodenum/5 min, p < 0.0001). Our results suggest that MC induced NM affect brain-gut axis via influencing metabolic and gastric function and decreases body weight.