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.

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.

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.

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.

The vagal afferents discharge and myoelectrical activity in the gastric hyperalgesia model in rats.

A long term exposure of the gastric mucosa to inflammatory factors is suspected to alter the normal stomach motility. The consequence of it is an abnormal sensomotor response to food causing dyspeptic symptoms. Our study aimed to investigate the vagal afferents activity and the gastro-duodenal slow wave response to the mild gastric mucosa inflammation in rats. The gastric mucosal inflammation was induced by addition iodoacetamide to drinking water for 5 days. The gastro-duodenal slow wave, vagal nerve recordings and the gastric mucosa examination were performed on 6th day. The iodoacetamide irritated gastric mucosa presented the minimal inflammatory infiltration with mast cells. The vagal afferent activity was significantly increased after iodoacetamide treatment from 0.3 +/- 0.1 to 1.9 +/- 0.58 Hz, (p<0.05). The gastric slow wave accurate frequencies extracted from the fast Fourier transform spectra accelerated from 0.08 +/- 0.01 to 0.1 +/- 0.02 Hz (p<0.05). The duodenal frequencies remained unchanged (from 0.64 +/- 0.02 to 0.59 +/- 0.1 Hz). These results suggest that mild gastric mucosa irritation sensitizes vagal afferents and alters gastric but not duodenal pacemaker activity which may contribute to dyspeptic sensations.

Dysfunction of the autonomic nervous system activity is responsible for gastric myoelectric disturbances in the irritable bowel syndrome patients.

The pathogenesis of the irritable bowel syndrome (IBS) is still unsolved. Lately most attention has been focused on visceral hypersensitivity related to dysfunction of the autonomic nervous system (ANS). The aim of this study was to evaluate changes in the ANS activity and gastric motility in constipation-predominant IBS patients using the heart rate variability (HRV) and gastric myoelectric activity (EGG) recording. 23 patients (45+/-13 yrs) matching Manning criteria and 30 healthy volunteers (47+/-5 yrs) participated in the study. EGG and HRV in fasted and fed subjects with fasted serum catecholamine levels were measured in both groups. Fasting IBS pts showed gastric dysrrhythmia (29+/-14% vs. 11+/-7%), DP was 128.860 +/- 112.000 vs. 46.000+/- 23.200microV2, DF 2.37+/-0.8 vs. 2.9+/-0.2cpm. Feeding (300 kcal) improved dysrrhythmia to 20+/-13% vs. 8+/-5%, DP decreased to 74.500+/-57.720 vs. 165.600+/-89.000microV(2) and DF increased to 2.53+/-0.7 vs. 3.2+/-0.3cpm. In fasted and fed IBS pts SWC (channels 3-4) was about 60+/-11 vs. 84+/-8% and 68+/-14 vs. 92+/-8% respectively. In IBS pts resting HRV parameters were lower (LF - 650.3 vs. 811.6 ms2; HF - 508.8 vs. 854.6 ms2); with higher LF/HF ratio in IBS patients (1.52 vs. 1.2). The serum fasting level of adrenaline and noradrenaline in IBS pts were higher 1.28+/-0.06 vs. 0.65+/-0.05 nmol/L, and 3.54+/-1.2 vs. 2.89+/- 08 nmol/L, p<0.05 respectively. Increased sympathetic drive in IBS pts reflected by high catecholamine levels and LH/HF ratio is responsible for gastric dysrrhythmias and low DF and coupling. Meal has negligible effect on EGG parameters improvement. The ANS dysfunction observed in IBS patients is most probably responsible for disturbances in gastric myoelectric activity presented as gastric dysrrhythmias resulting in gastric emptying delay and dyspeptic symptoms.

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.

Melatonin and serotonin effects on gastrointestinal motility.

The gastrointestinal tract represents the most important extra pineal source of melatonin. Presence of melatonin (M) suggests that this hormone is somehow involved in digestive pathophysiology. Release of GI melatonin from serotonin-rich enterochromaffin EC cells of the GI mucosa suggest close antagonistic relationship with serotonin (S) and seem to be related to periodicity of food intake. Food deprivation resulted in an increase of tissue and plasma concentrations of M. Its also act as an autocrine and paracrine hormone affecting not only epithelium and immune system but also smooth muscle of the digestive tract. Low doses M improve gastrointestinal transit and affect MMC. M reinforce MMCs cyclic pattern but inhibits spiking bowel activity. Pharmacological doses of M delay gastric emptying via mechanisms that involve CCK2 and 5HT3 receptors. M released in response to lipid infusion exerts a modulatory influence that decreases the inhibitory effects of the ileal brake on gastric emptying. On isolated bowel S induces dose dependent increase in tone and reduction in amplitude of contraction which is affected by M. M reduced the tone but not amplitude or frequency of contraction. M is a promising therapeutic agent for IBS with activities independent of its effects on sleep, anxiety or depression. Since of its unique properties M could be considered for prevention or treatment of colorectal cancer, ulcerative colitis, gastric ulcers and irritable bowel syndrome.

Helicobacter pylori infection in pathogenesis of gastroesophageal reflux disease.

Gastroesophageal reflux disease (GERD) refers to the very common and constantly increasing conditions where reflux of gastric contents into the esophagus leads to development of characteristic symptoms. The esophagus, LES and stomach can be envisioned as single functional unit controlled by neuro-hormonal factors. The abnormalities that contribute to GERD can start in any component of this unit, resulting particularly from disturbances in their control system. It is extremely important to identify factors and mechanisms leading to functional failure of this system so that causative therapy can be effectively applied. The key-role has been attributed to parasympathetic dysfunction, which may adversely affect motor activity of this area by increasing transient LES relaxation number and impairing LES pressure, esophageal acid clearance and motility of the proximal stomach. Recently, numerous investigations have been performed to elucidate the role of Helicobacter pylori (Hp) infection in GERD pathogenesis with the most concern given to its potency to increase gastric acid secretion. However, it appeared that this infection leads to much more complex changes in gastric mucosa including modification of afferent neural signals and specific gastric hormones release. Plasma ghrelin level is low in subjects infected and increases significantly after eradication. Since ghrelin, beside potency to increase gastric secretion has strong prokinetic action on LES functional unit, this phenomenon together with impaired vagal control may contribute to the Hp infection or eradication - related GERD development. Thus, ghrelin and vagal activity could be the missing links that partially explains relationship between GERD and Hp infection.

Peripheral mechanisms of intestinal dysmotility in rats with salsolinol induced experimental Parkinson's disease.

Gastrointestinal dysmotility in Parkinson's disease (PD) has been attributed in part to peripheral neurotoxine action. Our purpose was the evaluation of the salsolinol effect on intramuscular interstitial cells of Cajal (ICC), duodenal myoelectrical activity (DMA) and vagal afferent activity (VAA) in rats with experimental PD. Twenty rats were divided into 2 equal groups. Experimental PD was produced in one group by 3 weeks of the intraperitoneal salsolinol injections (50 mg/kg/day), whereas the 2-nd group served as control. DMA and VAA were recorded in both groups during fasting and stepwise--gastric distension (GD) of 10 ml. Subsequently fragments of duodenum were removed and intramuscular ICC were assessed as c-Kit antigen percentage in the duodenal muscular zone. Analyses of the fasting DMA and VAA recordings didn't reveal differences between the compared groups. During GD increase of DMA dominant frequency (p=0.04) and VAA frequency (p<0.01) was observed in the controls whereas in the salsolinol group both parameters remained unchanged. Image analysis of duodenum revealed decreased c-Kit expression in the salsolinol-injected animals (p=0.05). The results of our study may suggest the direct effect of salsolinol on both ICC and neuronal pathways of gastro-duodenal reflexes.

Peripheral mechanisms of intestinal dysmotility in the morphine tolerant and dependent rats.

Changes of intestinal motility and transit produced by tolerance to and dependence upon morphine have been partly attributed to peripheral mechanisms. We evaluated the effect of chronic peripheral morphine administration and peripheral mu-receptor blockade on vagal afferent activity (VAA) and c-Kit positive intramuscular cells of Cajal (ICCs). Ten rats were subjected to chronic subcutaneous morphine infusion for 72 h with subsequent VAA recording. Potential frequency was evaluated within recordings before and after mu receptor blockade by (D)-Phe -Cys -Tyr -(D)-Trp -Orn -Thr -Phe -Thr (CTOP) i.p. injections. Afterwards the rats were sacrificed and intramuscular c-Kit antigen expression was assessed by image analysis within removed fragments of duodenum and ascending colon. An equal group of rats served as a control for VAA and c-Kit expression. Analysis of VAA revealed similar frequencies of potentials in morphine tolerant / dependent rats before CTOP and in the controls. CTOP increased potential frequency in the morphine group which effect was visible mostly within the first 20 minutes (p=0.01). The morphine infused animals presented also higher c-Kit expression in both the duodenum (p<0.001) and the ascending colon (p<0.001) in comparison to the control group. Results of our study may indicate the involvement of both the intestinal wall and the long vago-vagal reflexes in tolerance to and dependence upon opioids.

Influence of vagal nerve stimulation on food intake and body weight--results of experimental studies.

The paper reviews recent advances in vagal nerve stimulation for the control of food intake and body weight. The vagal nerves are the predominant pathway in the "brain-gut axis" responsible for short term regulation of food intake. Stimulation of afferent vagal traffic attenuates food intake by vagal projections to nucleus tractus solitarius, arcuate nucleus and its convergence's to thalamic center of satiety. A few studies have been published in this field so far. All of them are consistent and show significant decrease in body mass during vagal stimulation. Due to promising results of experimental studies, clinical trials are expected in the near future.

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.

The effects of baclofen on the feeding behaviour and body weight of vagally stimulated rats.

UNLABELLED: It is hypothesised that the GABA(B) receptor agonist baclofen increases or has no effect on food intake, and electrical stimulation of vagal nerves decreases food intake. The aim of this study was to evaluate the effects of baclofen in vagally stimulated rats. MATERIAL AND METHODS: Thirty two Wistar rats were divided into five groups: group A scheduled for microchip implantation for vagal stimulation, group B for sham operation, group C for microchip implantation and baclofen medication, group D for baclofen medication only and group E for gastric motility evaluation under influence of baclofen. The following parameters were then evaluated: food intake and body mass, gastric motility, leptin, insulin, and glucose serum levels. RESULTS: In the comparison of groups B and A, daily food intake and body weight gain decreased by 17% (p<0.05) and by 22% (p<0.05), respectively. Baclofen alone (group D) did not significantly change either food intake nor diurnal body weight compared to the controls, but when used in conjunction with the microchip (group C) it did significantly reduce effect of vagal neuromodulation (p<0.05). Furthermore, a significant decrease in leptin and glucose levels was detected in group C: 677 to 165 pg/ml (p<0.05) and 5,93 to 4,88 mmol/l (p<0.05), respectively. The administration of baclofen stimulated significantly gastric motility and elicited irregular motor migrating complex (327+/-200 against control 255+/-52 cmH2O/s). CONCLUSIONS: These results suggest that microchip vagal neuromodulation through increased vagal afferent activity induces an alteration in the feeding behaviour and decreases nocturnal food intake and body weight. These effects were partially attenuated by baclofen. The data suggests that GABA(B) receptors play an important role in the pathomechanism of attenuation of food intake induced by vagal nerve stimulation.

Disturbances of the parasympathetic branch of the autonomic nervous system in patients with gastroesophageal reflux disease (GERD) estimated by short-term heart rate variability recordings.

Gastro-esophageal reflux disease (GERD) is the result of the acid contents regurgitation back from the stomach into the esophagus. According to the endoscopic findings, GERD can be divided into two main forms: non-erosive (NERD) and erosive reflux esophagitis. The pathogenesis of GERD is associated with the impaired function of the antireflux barrier. Disturbances of the autonomic nervous system (ANS), especially parasympathetic part of the ANS, may be also involved in the pathogenesis of this disease. The aim of our study was to establish the parasympathetic activity in patients with reflux esophagitis and in patients with symptomatic endoscopically negative reflux. Working hypothesis was the question, whether the possible parasympathetic activity disturbances, which are observed in all GERD patients, may be regarded as the primary or secondary to the esophagitis. All the participants (20 pts. with NERD, 20 pts. with reflux esophagitis and 20 healthy controls) underwent esophageal manometry, 24-hour ambulatory pH-monitoring, resting heart rate variability (HRV) recording and the deep breathing (DB) test with the continuous HRV recording. The results of the spectral analysis both of the short-term, resting HRV recordings and DB-evoked revealed the disturbances of the main power spectra components - LF and HF in both groups of patients in comparison with the control group. In our opinion, the observed HRV spectra changes in both groups of patients support the hypothesis that not only is the parasympathetic activity impairment associated with the pathogenesis of GERD but it is also the primary factor contributing to the pathophysiological mechanism of reflux.