Engaging multisector stakeholders to identify priorities for global health innovation, change and research: an engagement methodology and application to prosthetics service delivery in Cambodia.

PURPOSE: While innovation is known to catalyse solutions to global sustainable development challenges, lack of engagement from stakeholders during conceptualisation and development may influence the degree of success of implementation. METHODS AND MATERIALS: This paper presents a complete and novel engagement methodology, developed from value led business modelling approaches, for working with multi-sector stakeholders. The methodology can be used to determine barriers and facilitators to clinical practice innovations or translational research, within a country-specific context. The approach has then been applied in the Cambodian prosthetics and orthotics sector to provide a practice-based exemplar application of the framework. RESULTS: This approach seeks to ensure the suitability and sustainability of clinical practice and research programmes being implemented within a complex ecosystem. A theoretical basis, drawn from academic and business innovation sectors, has been consolidated and adapted for practical application to design, direct, and inform initiatives in low resource settings. CONCLUSIONS: The methods presented provide a way to both develop and articulate the mission, vision, and goals of any proposed change, and to effectively communicate these with stakeholders in a way that engages the personal and professional values that exist in their ecosystem. It provides a structured process through which meaningful conversations can happen, and a basis for relationship management with key stakeholders; intrinsic to enable a sustained legacy from research and development.

The engagement from stakeholders during conceptualisation and throughout development can determine the success, or not, of any implementation and scale of innovation.This paper presents a conceptual stakeholder-led engagement methodology, developed from value led business modelling approaches, for determining barriers and facilitators to translational global healthcare research in a country-specific context, in this case the Cambodian prosthetics and orthotics sector.Subsequent research and development work in this area needs to carefully manage and negotiate influencing factors identified through the application of the described methodology, to ensure initiatives (whether research or wider national development work) are sustainable and successful.

Preserving the value of legacy film-based teaching files in pediatric radiology.

Senior pediatric radiologists who have spent a major portion of their careers interpreting conventional film-screen radiographic studies have collected a wealth of hard-copy teaching material that is at risk of becoming obsolete. The teaching value and usefulness of analog film teaching files can be preserved using available hardware and standard software. The final product can be made available in a high-quality digital format to students, trainees and faculty without complicated search-and-retrieval methodology. This paper describes a relatively simple and low-cost procedure to preserve and use this source of wisdom and experience. It also emphasizes the role that such a resource can play as part of a comprehensive educational program.

Evaluation of selected protein biomarkers of renal function in rats with an experimental model of acute cyclophosphamide-induced cystitis treated with N-acetylcysteine.

The administration of cyclophosphamide (CP) is associated with the risk of developing cystitis as well as kidney injury. The aim of the study was to verify the uroprotective effect of N-acetylcysteine (NAC), as well as the evaluation of renal function in the experimental model of acute CP-induced cystitis. Rats from group 1 received intraperitoneally only a single dose of 200 mg/kg b.w. of CP. Individuals from groups 2 and 3 additionally received a single dose of 200 mg/kg b.w. of NAC, respectively, orally (p.o.) and intraperitoneally (i.p.). After the administration of the drugs, animals were subject to individual monitoring in metabolic cages to assess 24-hour diuresis and basic vital signs, and then finally sacrificed for the purpose of collecting blood and organs for histopathological analysis. Classic renal parameters (creatinine, urea, uric acid, electrolytes) as well as new markers reflecting renal function, within the filtration-resorption range - cystatin C (CysC), renal tubular integrity - kidney injury molecule-1 (KIM-1) and the condition of the glomerular filtration barrier (nephrin) were determined in the obtained serum and urine samples. In group 1 histopathological development of cystitis was confirmed with the absence of significant pathomorphological disorders of the kidneys, and the initial results of the parameters determined were obtained. In both groups 2 and 3, a decrease of inflammatory changes in urinary bladder was observed, while there were still no morphological disturbances in kidneys. The administration of NAC in both groups 2 and 3 also resulted in a decrease of concentrations in urine and a reduction in 24-hour excretion with urine of all assessed proteins (CysC, KIM-1 and nephrin). NAC, thus exhibited a uroprotective effect, which was accompanied by a functional nephroprotective effect (more accentuated during intraperitoneal administration of this compound), manifested by the reduction of urinary excretion of proteins indicative of developing renal dysfunction.

Effect of the different doses of acrylamide on acetylocholinoesterase activity, thiol groups, malondialdehyde concentrations in hypothalamus and selected muscles of mice.

Acrylamide is a chemical compound that typically forms in starchy food products during high-temperature cooking, including frying, baking and roasting. Acrylamide is a known lethal neurotoxin. Its discovery in some cooked starchy foods in 2002 prompted concerns about the carcinogenicity of those foods. Little is known about acrylamide's influence on the peripheral nerves. In our research we measured acrylamide's influence on the acetylcholinesterase activity in hypothalamus, heart muscle, skeletal muscles of the thigh and smooth muscle of the small intestine (males, Swiss strain) in relation to the thiol groups and malondialdehyde concentration. Acrylamide was injected intraperitoneally (20 and 40 mg/kg, i.e. 0.52 and 1.04 mg per animal). The hypothalamus and muscles were taken 24, 48, and 192 h after the injection. Acetylcholinesterase activity was significantly lower (P < 0.001 to P < 0.05) in all structures. It was accompanied by the statistically significant (P < 0.001 to P < 0.05) increase in malondialdehyde concentrations in most of the studied structures time periods and ACR doses. -SH groups concentrations were significantly depleted in selected structures (P < 0.001 to P < 0.05). The AChE activity evaluation in mice muscles and hypothalamus was very important because there are many evidences that acrylamide affects directly on the peripheral nerves. Thus, it causes structural damages and physiological changes. The results obtained in the present study provide evidence for the occurrence of oxidative stress after intraperitoneal injection of acrylamide to hypothalamus, heart muscle, skeletal muscles of the thigh and smooth muscle of the small intestine.

Changes in U937 cell viability induced by stress factors - possible role of calmodulin.

Current studies were aimed to elucidate influence of magnetic field (MF) stimulation on cell viability and its effect on expression of calmodulin (CaM) and Hsp70 protein which plays a role of cell stress indicator and is a Ca2+-dependent CaM-binding protein. For the experimental model we have chosen U937 cell line exposed to chemical- and/or physical stress factors. Puromycin (PMC) was used as a chemical apoptosis inducer. Alternating (AC) (6.5rms mT, 35 Hz) magnetic field combined with 6 mT static (DC) component, or pulsed electromagnetic field (45 ± 5)mT, 50 Hz (PEMF) acted as physical stressors. Cell viability was assessed by flow cytometry, and the Western blot analysis was carried out for CaM and Hsp70 levels in cytosolic extracts of U937 cells. Cell viability in samples exposed to MF alone did not differ from sham sample, for both types of MF exposure systems. Simultaneous action of MF and PMC influenced cell viability in type of MF stimulation-dependent manner. In contrast to PEMF + PMC stimulated samples, combination of ACDCMF with PMC enhanced cell death compared to PMC control. The observed changes in cell viability were correlated with changes in level of CaM and Hsp70 proteins. Immunoblots have shown, that cytosolic content of both CaM and Hsp70 proteins was enhanced in PMC-treated sample, and further elevated for ACDCMF + PMC. For PEMF + PMC stimulated samples, level of CaM was reduced compared to PMC-treated sample. The results suggest that the changes in expression of CaM and CaM-dependent proteins might modulate effectiveness of cell death under stimulation with MF and/or cytotoxic agents.

Enterohormonal disturbances in colorectal cancer patients.

Gastrointestinal (GI) hormonal peptides play a role in the development of gastrointestinal malignancies, and their abnormal levels may contribute to dysmotility. The aim of this study was to analyze plasma concentrations of enterohormones (motilin, ghrelin, gastrin and pancreatic polypeptide) and to verify if their abnormal levels may contribute to the severity of dyspeptic symptoms in colorectal cancer patients. The study included 60 patients with colorectal malignancies (22 men and 38 women), among them 30 individuals with colon cancers (group A) and 30 subjects with rectal tumors (group B). Fasting plasma levels of pancreatic polypeptide (PP), motilin, gastrin and ghrelin were determined by means of ELISA. The results were compared with the respective parameters of healthy volunteers. Colon cancer patients presented with significantly lower concentrations of ghrelin than the subjects with rectal tumors and healthy controls (156.8±86.7 vs. 260.2±87.6 vs. 258.4±94.2 pg/ml, p=0.02), as well as with significantly higher levels of PP (265.5±66.3 vs. 154.1±54.6 vs. 148.3±64.3 pg/ml, p=0.005). Also the levels of motilin turned out to be lower in colon cancer patients than in the subjects with rectal malignancies and healthy controls. No statistically significant intergroups differences were found in plasma levels of gastrin (388.2±98.6 vs. 475.6±88.7 vs. 428.2±91.2 pg/ml, p>0.05). Epigastric bloating was the most frequent dyspeptic symptom, reported by 63.3% and 40% of patients with colon and rectal tumors, respectively. Our findings imply that colon cancer patients may present with abnormal plasma levels of enterohormones significantly more often than individuals with rectal malignancies. Dysmotility observed in colon cancer patients may result not only from anticancer surgery, but also from abnormal release of enterohormones, induced either by neoplastic process or by changes within the autonomic nervous system.

Prostaglandin-targeting agents and spectral heart rate variability in experimental partial bladder outlet obstruction in rats.

The purpose of this study was to determine the activity of the autonomic nervous system (ANS), using spectral analysis of the heart rate variability (HRV) in the model of partial bladder outlet obstruction (PBOO) in rats treated with selected non-steroidal anti-inflammatory drugs (NSAID): piroxicam (PRX) or meloxicam (MLX), and following administration of PGF2a prostaglandin analogue (Enzaprost F5). Neither the use of PGF2a analogue nor of MLX, caused significant changes in the HRV spectrum (except for HRV spectrum total power reduction with MLX). The use of PRX caused reduction of the total power and powers of all components of the HRV spectrum (except for VLF). Moreover, increased nLF and reduced nHF were observed. The obtained results suggest that the total prostaglandin synthesis block with a non-selective cyclooxygenase inhibitor (PRX) results in reduced ANS total activity, with decreased parasympathetic activity and a relative sympathetic predominance. The preferential cyclooxygenase-2 block (MLX) caused reduction of the total ANS activity as well, however with no clear disproportion of any part of the ANS. Therefore, prostaglandin synthesis inhibition and associated decrease of parasympathetic activity may constitute an additional and favourable feature of NSAID pharmacodynamics in the treatment of BPH.

Disturbances of autonomic nervous system activity and diminished response to stress in patients with celiac disease.

Celiac disease (CED) is immune-mediated enteropathy caused by gluten intolerance affecting genetically predisposed individuals. CED may exert a number of various symptoms, including extra intestinal manifestations. Neurological symptoms can be the first sign of gluten intolerance. However, affected autonomic nervous system (ANS) activity may be linked to other symptoms. We evaluated the frequency of ANS impairment and resting ANS response to several stimuli in CED patients without neurological manifestations. Twenty five neurologically asymptomatic patients with CED were studied. The medical history was taken and ANS activity was determined. ANS tests included heart rate variability (HRV) at rest and after stimulation (sympathetic - stress, and parasympathetic - deep breathing). The results were compared with those of the control group comprising of 30 healthy asymptomatic volunteers. Both the resting HRV parameters and the HRV indices recorded after deep breathing (parasympathetic stimulation) were significantly lower in patients with CED than in the controls (P<0.05). Also the stress-induced increase in normalized low frequency parameter (LFnu) was significantly lower in the CED group than in the control group (P<0.05). Overall, about 20% of CED patients presented with parasympathetic dominancy but 36% with sympathetic dominancy, and 44% of patients did not show changes in sympathetic-vagal balance of the autonomic nervous system. We conclude that sympathetic-parasympathetic imbalance, in favour of more often sympathetic than parasympathetic overactivity occurs among neurologically asymptomatic CED patients. The ANS impairment observed in the course of CED may result from prolonged intestinal inflammation. Therefore, routine ANS testing might be considered in patients presenting with this condition.

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 of urothelial cells induces apoptosis and diminishes necrosis: new insight to magnetic therapy in urology.

The evidence of electromagnetic therapy (EMT) efficacy in stress and/or urge urinary incontinence, as well as in detrusor overactivity is generally lacking in the literature. The potential EMT action of neuromuscular tissue depolarization has been described. Because there is no data on the influence of pulsating electromagnetic fields (PEMF) on the urothelium, we evaluated the effect of PEMF stimulation on rat urothelial cultured cells (RUCC). In our study 15 Wistar rats were used for RUCC preparation. RUCC were exposed to PEMF (50 Hz, 45±5 mT) three times for 4 hours each with 24-hour intervals. The unexposed RUCC was in the same incubator, but in a distance of 35 cm from the PEMF generator. Annexin V-APC (AnV+) labelled was used to determine the percentage of apoptotic cells and propidium iodide (PI+), as standard flow cytometric viability probe to distinguish necrotic cells from viable ones. The results are presented in percentage values. The flow cytometric analysis was carried out on a FACS calibur flow cytometer using Cell-Quest software. In PEMF-unstimulated RUCC, the percentage of AnV+, PI+, and AnV+PI+ positive cells were 1.24±0.34%, 11.03±1.55%, and 12.43±1.96%, respectively. The percentages of AnV+, PI+, and AnV+PI+ positive cells obtained after PEMF stimulation were 1.45±0.16% (p=0.027), 7.03±1.76% (p<0.001), and 9.48±3.40% (p=0.003), respectively. The PEMF stimulation of RUCC induces apoptosis (increase of AnV+ cells) and inhibits necrosis (decrease of PI+ cells) of urothelial cells. This leads us to the conclusion that a low-frequency pulsating electromagnetic field stimulation induces apoptosis and diminishes necrosis of rat urothelial cells in culture.

Morphological and urodynamic evaluation of urinary bladder wall regeneration: muscles guarantee contraction but not proper function--a rat model research study.

BACKGROUND: Numerous studies are ungoing to develop a substitute for the native urinary bladder wall. The principals of tissue engineering approaches to urinary bladder wall augmentation require a favorable environment for smooth muscle regeneration, which is crucial for bladder function. This study was performed to evaluate bone marrow mesenchymal stem cells (BMSC) seeded on to amniotic membranes fixed to Tachosil sponges as grafts for urinary bladder muscle layer augmentation in a syngenic rat model. MATERIALS AND METHODS: Amniotic membranes seeded with BMSC and covered by Tachosil sponges were implanted as multilayer grafts into nine rats to regenerate the urinary bladder wall. The control group consisted of 12 healthy rats. Urodynamic examinations included contraction, elasticity, compliance, and urinary bladder motor activity. Hematocylin and eosin and Masson's trichrome stains were used to evaluate muscle regeneration; histological data were digitally analyzed with the ImageJ tool. RESULTS: The area of muscle bundles ranged from 5% to 25% or 32% to 41% in control versus reconstructed bladders, respectively. Among nine animals with reconstructed urinary bladders, urodynamic evaluation revealed bladder motor hyperactivity with regular (n = 4) or irregular (n = 1) storage and voiding phases, as well as proper bladder motor activity with a large bladder capacity (n = 1). No bladder contractility was recorded in one case and large stones developed in two animals, which made functional studies impossible. CONCLUSIONS: Regenerated smooth muscle cells created an autonomic cell population that was poorly assimilated to the rest of the urinary bladder wall. The histological presence of a regenerated muscle layer did not guarantee proper urinary bladder function.

Exogenous melatonin abolishes mechanical allodynia but not thermal hyperalgesia in neuropathic pain. The role of the opioid system and benzodiazepine-gabaergic mechanism.

Melatonin (MT) is a neurohormone synthesized and secreted by the pineal gland. MT plays an important role in the regulation of physiological and neuroendocrine functions. The purpose of this study was to assess the overall effect of melatonin on neuropathic pain, the type of melatonin receptor involved, and potential role of the opioid system and GABA(A) receptors. The experiments were conducted by using the animal neuropathic pain model (CCI). The rats with CCI showed the characteristic for the mechanical allodynia and thermal hyperalgesia signs that were calculated by using the von Frey's and Hargreaves' tests. The conducted studies measured the effects of intraperitoneal administration of naloxone (opioid antagonist), prazosin (MT3 antagonist), luzindole (MT1/MT2 receptor antagonist), picrotoxin (GABA(A) antagonist) and flumazenil (benzodiazepine antagonist) on the antinociceptive effects caused by melatonin. Melatonin caused the increase in the pain threshold of the mechanical allodynia and the slight increase in the threshold of the thermal hyperalgesia. The pre-treatment with naloxone completely abolished the antinociceptive effects of melatonin in von Frey's test, but not thermal sensation in the Hargreaves's test. Prazosin did not have any effects, while administration of luzindole significantly suppressed the antinociceptive effect of melatonin. The antiallodynic effect of MT was also abolished by flumazenil and picrotoxin. Melatonin influences the mechanical allodynia but not thermal hyperalgesia via activation of opioid system and benzodiazepine-GABAergic pathway. Antinociceptive effects of melatonin are mostly related to the MT1/MT2 receptors interaction.

Effect of partial and complete blockade of vanilloid (TRPV1-6) and ankyrin (TRPA1) transient receptor potential ion channels on urinary bladder motor activity in an experimental hyperosmolar overactive bladder rat model.

UNLABELLED: The study investigated the mechanisms through which the hyperosmolarity might induce detrusor overactivity (DO). We compared the bladder activity in response to partial and complete blockade of TRPV1-6 and TRPA1 receptors. Experiments were performed on 42 rats. DO was induced by using hyperosmolar saline. All animals were randomly divided into six groups. The measurements represent the average of five bladder micturition cycles. Hyperosmolar saline induced DO. The complete blockade of TRPV1-6 and TRPA1 prevented DO. The partial blockade of TRPV1 didn't prevented DO. In the voiding phase periodical bladder contractions complexes occurred leading to slow urine flow due to bladder distension. Ruthenium red and capsaicin resulted in complete disorganisation of detrusor muscle contractility impairing urine voiding and leading to constantly lasting urine retention in healthy rats. CONCLUSIONS: hyperosmolar-induced DO is mediated by TRPV and TRPA1 channels; the hyperosmolar stimuli of urinary bladder might be transmitted mostly via ruthenium red sensitivity pathway.

Electrical vagus nerve stimulation decreases food consumption and weight gain in rats fed a high-fat diet.

There is growing evidence that vagus nerve stimulation (VNS) has a suppressive effect on both short- and long-term feeding in animal models. We previously showed that long-term VNS (102 days) with low-frequency electrical impulses (0.05 Hz) decreased food intake and body weight in rats. In the present study, we investigated the effect of high frequency (10 Hz) VNS on feeding behavior and appetite in rats fed a high-fat diet; peptide secretion and other parameters were assessed as well. Adult male Wistar rats were each implanted subcutaneously with a microstimulator (MS) and fed a high-fat diet throughout the entire study period (42 days). The left vagus nerve was stimulated by rectangular electrical pulses (10 ms, 200 mV, 10 Hz, 12 h a day) generated by the MS. Body weight and food intake were measured each morning. At the end of the experimental period, animals were euthanized and blood samples were taken. Serum levels of ghrelin, leptin and nesfatin-1 were assessed using radioimmunoassays. Adipose tissue content was evaluated by weighing epididymal fat pads, which were incised at the time of sacrifice. To determine whether VNS activated the food-related areas of the brain, neuronal c-Fos induction in the nuclei of the solitary tract (NTS) was assessed. Chronic vagus nerve stimulation significantly decreased food intake, body weight gain and epididymal fat pad weight in animals that received VNS compared with control animals. Significant neuronal responses in the NTS were observed following VNS. Finally, serum concentrations of ghrelin were increased, while serum levels of leptin were decreased. Although not significant, serum nesfatin-1 levels were also elevated. These results support the theory that VNS leads to reductions in food intake, body weight gain and adipose tissue by increasing brain satiety signals conducted through the vagal afferents. VNS also evoked a feed-related hormonal response, including elevated blood concentrations of nesfatin-1.

Functional, histological structure and mastocytes alterations in rat urinary bladders following acute and [corrected] chronic cyclophosphamide treatment.

Neurogenic inflammation is linked to urinary bladder overactivity development. Cyclophosphamide (CYP) damages all mucosal defence lines of urinary bladder and induces cystitis with overactivity. The aim of this study was to estimate the effect of CYP on rat urinary bladder function, histological structure and mastocytes numbers following acute and chronic CYP treatment. Fourty two female rats were divided into four groups: I (control), II (acute cystitis), III (chronic cystitis), IV (sham group). Acute and chronic cystitis were induced by CYP in single dose and four doses (1(st), 3(rd), 5(th), 7(th) day), respectively. In group I-III the cystometric evaluation was performed. Sections of the bladder were stained with HE and toluidine blue for the detection of mastocytes. The severity of inflammation was examined according to mucosal abrasion, haemorrhage, leukocyte infiltration and oedema. Acute and chronic CYP treatment caused inflammatory macroscopic and microscopic changes (mucosal abrasion, haemorrhage, oedema) and increased infiltration of inflammatory cells in urinary bladder. Acute treatment induced the infiltration of mastocytes within bladder wall contrary to chronic one decrement. Acute treatment caused more severe mucosal abrasion, whereas chronic one revealed more developed haemorrhage changes. Additionally, cystometric evaluation revealed urinary bladder overactivity development in both types of cystitis. Basal pressure and detrusor overactivity index after acute treatment increased considerably in comparison with the increase obtained after chronic one. Our results proved that acute model of CYP-induced cystitis in rats is more credible for further evaluation of neurogenic inflammation response in pathogenesis of overactive bladder as compared to chronic one.

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.

Magnetically induced vagus nerve stimulation and feeding behavior in rats.

Vagus nerve (VN) contribute to the bidirectional communication between the gastrointestinal tract and the central nervous system. Stimulation of the VN by a magnetically-driven solenoid with parameters similar to those during food-induced stomach distension has been thought to mimic short-term signaling of satiety and suppress food intake. In this study, the determination of optimal parameters of vagal neuro-modulation to achieve decreased food intake with a resulting reduction in body mass of rats is explored as therapy to treat obesity. The experimental design consisted of three groups of obese adult male Wistar rats: Group 1: VEMF - with solenoid's electrodes placed on the left VN in the magnetic field exposure (MFE); Group 2: EMF - without solenoid's electrodes on the VN in MFE; Group 3: CON - without solenoid's electrodes on the VN outside the MFE. This study suggests that the rats with solenoid's electrodes placed on the left VN significantly decreased their food intake, weight gain and serum leptin concentrations when compared to that of the CON group. PP levels were found to be higher in the VEMF group when compared to the controls groups. It was found that the most effective parameters of vagal stimulation on eating behavior were 3631, 7861, 14523 A(2) x h/m(2). The magnetic field by unknown mechanisms also influences feeding behavior. This study suggests that vago-vagal reflexes are involved in the feeding homeostasis and that neuromodulation might be an effective method for managing obesity. Further studies are required to confirm these effects in humans.

Urodynamic effects of the bladder C-fiber afferent activity modulation in chronic model of overactive bladder in rats.

Previous studies have suggested that, different types of unmyelinated bladder afferent C-fibres, such as capsaicin-sensitive and capsaicin-resistant mediate the voiding reflex in overactive bladder (OAB). Considering its polymodal features, we explored the urodynamic effect of primary afferent neurons modulation on detrusor activity in normal and OAB rats. Experiments were performed on 48 female rats. OAB was induced by intraperitoneal administration of cyclophosphamide. All the surgical procedures and urodynamic studies were performed under urethane anaesthesia. Cystometry was done after a 1 h recovery period from the surgical procedure. All animals were randomly divided into six groups: control, chronic OAB, chronic OAB after capsaicin or lidocaine instillation, control capsaicin or lidocaine instillation. The measurements represent the average of five bladder micturition cycles. We analyzed: basal, threshold, micturition voiding pressure; intercontraction interval; compliance; functional bladder capacity; motility index; detrusor overactivity index. We used chronic cyclophosphamide OAB model for further investigations. In healthy rats, intravesical instillation of capsaicin caused complete inhibition of detrusor contractility preventing from proper voiding function of the bladder. Contrary, lidocaine has no influence on micturition cycles in intact animals. Also, intravesical instillation of capsaicin and lidocaine reduced the severity of detrusor overactivity of OAB rats leading to improvement of cystometric parameters.

The role of sensory C-fibers in response of vagal afferent stimulation by gastric distension in rats with experimental chronic gastric ulcer.

There is growing evidence that gastric vagal afferent input may contribute to the altered sensations associated with gastrointestinal disorders. The aim of our study was to evaluate gastric vagal afferents (VA) activity in rats with experimental gastric ulcer and ulcer healing. The study was carried out on rats with gastric ulcer (GU), including, a group with perivagal capsaicin pretreatment (CAP), a group with capsaicin administration in gastric ulcer (CAP+GU) animals and control rats. In all rats electrical VA activity was recorded and analysed. In GU rats recordings were carried out in chronic ulcer and ulcer healing. In GU and CAP+GU groups gastric balloon distensions with vagal recording was performed on 3(rd) day after ulcer induction. Usually, experimental GU healed spontaneously within 2 weeks. Three days after acetic acid application when GU fully develop, the frequency of the basal VA activity was almost 3-times higher than in the control intact rats and remained elevayed until 4(th) week after ulcer induction. VA response to gastric distension increased concomitantly with increased balloon volume in both GU and control animals, but it was several times higher in GU rats. Perivagal capsaicin application decreased the frequency of spontaneous VA activity and decreased the response of VA to gastric distension. In CAP+GU, spontaneous activity as well as the response to gastric distension were higher than in CAP rats. Our study shows that GU induced inflammatory changes increase sensitivity of gastric VA. Capsaicin-sensitive vagal afferent fibers may play some role in this phenomenon. Peripheral sensitization of VA persists even when gastric ulcer is completely healed.