{"paper_id":"1a197a81-9b2a-4989-bdf7-ea055e7849c8","body_text":"The animals were treated in accordance with the European Communities Council Directives (86/609/ECC) and the Hungarian Act for the Protection of Animals in Research (Article 32 of Act XXVIII). All experiments involving animal subjects were carried out with the approval of the Hungarian Ethical Committee for Animal Research (registration number: IV/198/2013).\nSprague-Dawley rats (Charles-River Laboratories, Budapest, Hungary) were housed at 22 ± 3°C and a relative humidity of 30%-70%, under a 12 h light/12 h dark cycle. Standard rodent pellet food (Charles-River Laboratories, Budapest, Hungary) and tap water were provided  ad libitum .\nSexually mature female Sprague-Dawley rats (body mass: 140–160 g, 50–60 days old) were mated in the early morning hours. Copulation was confirmed by the presence of a copulation plug or spermatozoa in the vaginal smear. The day of copulation was considered to be the first day of pregnancy.\nFemale, full term pregnant (Day 21 and Day 22 of pregnancy) Sprague-Dawley rats were anaesthetized intraperitoneally (i.p.) with a combination of ketamine (36 mg/kg) and xylazine solution (4 mg/kg). The jugular vein was cannulated for later intravenous (i.v.) drug administration.\nIn the first group of rats (n = 8), a laparotomy was performed and the total gastrointestinal tract was resected from the abdomen under deep anesthesia. A bipolar thread electrode pair (SEN-15-1; MDE GmbH, Walldorf, Germany) was inserted into the myometrium (the distance between the two electrodes was 8 mm), while a bipolar disk electrode pair (SEN-15-2; MDE GmbH, Walldorf, Germany) was placed subcutaneously above the uterus (the distance between the two electrodes was 20 mm). An implantable strain gauge (SEN-04-FSG2; MDE GmbH, Walldorf, Germany) was sutured onto the surface of the left uterine horn, along the long axis of the muscle fibers, in order to detect the mechanical contractions ( Figure 1 ). So as to cover the incision, the surfaces of the abdominal wall were constricted and the abdominal skin was replaced after the positioning of the sensors. The animals were then placed immediately onto a heatable operating table (EXP-D-TC/MA-02; MDE GmbH, Walldorf, Germany) in order to maintain the body temperature (set to 37°C). The basal activity was detected for 60 minutes. The electric signals were recorded and analyzed by an online computer and amplifier system by the S.P.E.L. Advanced ISOSYS Data Acquisition System (MDE GmbH, Walldorf, Germany). Electromyographic (EMG) signals were amplified by using a custom-made amplifier. All analogue signals were filtered with a first-order bandpass Bessel-type filter with a frequency of 0-30 cycles per minute (cpm) and were converted to digital signals at a sample rate of 2 Hz.\nRepresentative picture of the positioning of the electrodes and strain gauge (SG) for recording the myometrial myoelectric and mechanical signals in a rat with a resected gastrointestinal (GI) tract. The thread electrode pair and the SG were positioned on the uterus, while the disk electrode pair was positioned on the abdomen under deep anesthesia.\nDuring the research the animals were under deep anesthesia. When the experiments were completed, we sacrificed the rats with i.v. administered high dose of ketamine (150 mg/kg).\nThe recorded signals were analyzed by fast Fourier transformation (FFT). The frequency of the electric activity was characterized in cpm, and the magnitude of the activity was described as maximum of power spectrum density (PsD max ). When more than one peak was found in the spectrum, only the highest peak was considered during the evaluation. The mechanical contractions were evaluated by area under the curve (AUC) analysis of the primary contractility curves. Before the pharmacological studies, both the mechanical (strain gauge) and electric (thread and disk electrodes) signals were recorded for 30 minutes (n = 8).\nIn the second group of anaesthetized rats (n = 18) with intact gastrointestinal tract, a bipolar disk electrode was placed under the abdominal skin, 1 cm right from the midline of the laparotomy, and 2 strain gauges were sutured one by one onto the surface of the uterus and cecum ( Figure 2 ). The abdominal incision surfaces were closed by surgical staples after the placement of the sensors. Both the mechanical (strain gauges) and electric signals (disk electrode) were recorded for 30 minutes before the administration of the investigated drugs. The myoelectric signals were recorded with the above-mentioned equipment. The analysis and filtering (0-3 cpm) of the signals were carried out as it was described in our previous study ( 9 ).\nThe positioning of the disk electrode and strain gauges (uterus, cecum) for recording of the uterine and gastrointestinal (GI) myoelectric and mechanical signals in a non-GI tract-resected rat under deep anesthesia.\nTwo doses of oxytocin (1 μg/kg) were administered after recording the basal activity, 15 minutes apart. After 30 minutes, a dose of terbutaline (50 μg/kg) was injected i.v. to rats with resected gastrointestinal tract (n = 8 on Day 21 of pregnancy) and rats with intact gastrointestinal tract (n = 10 on Day 22 of pregnancy). One dose of neostigmine (20 μg/kg) and one dose of atropine (300 μg/kg) were administered intravenously 30 minutes apart to rats (n = 8) with intact gastrointestinal tract on Day 22 of pregnancy. Following the administration of each drug, the AUC and FFT were evaluated at 30-minute intervals. The effects were expressed as percentages of the spontaneous activity.\nThe AUC, cpm, and PsD max  values were determined and compared using unpaired  t -test. The  P  values of unpaired  t -tests indicating statistically significant differences are shown in respective figures. Statistical analyses were performed using the statistical program Prism 5.0. (GraphPad Software, La Jolla, CA, USA), and the level of statistical significance was set at  P  < 0.05.\n\nPrimary EMG curves from rats with resected gastrointestinal tract represent characteristic myoelectric signals recorded by thread or disk electrodes. When the primary EMG curves were transformed by FFT, the resulting spectra had sharp and dominant peaks (PsD max ) at low cpm values, and terbutaline reduced the PsD max  of the spectra ( Figure 3 ).\nMyoelectric and mechanical signals of the pregnant uterus ( A ), detected with different electrodes and with the strain gauge (SG). Fast Fourier transformation (FFT) analysis reveals the tissue specific spectra of the myometrium ( B ). Each spectrum has the characteristic frequency expressed in cycles per minute (cpm), determined by the highest peak in the spectrum.\nThe characteristic cpm value for the uterus was found between 1-2.5 cpm measured by thread or disk electrodes ( Figure 4A ). The PsD max  representing the maximum intensity of the signals was higher with the thread electrodes in the cpm range 1-2.5 ( Figure 4B ).\nCycles per minute (cpm) values were measured with thread and disk electrodes ( A ). The intensity of the signals is expressed as the maximum of the power spectrum density (PsD max ), which corresponds to the highest peak in the Fast Fourier transformation spectrum. The signals of the thread electrodes were usually higher than those of the disk electrodes ( B ).\nIn pharmacological experiments oxytocin increased, while terbutaline decreased both the electrical and mechanical signals of the uterus in the rats with resected gastrointestinal tract ( Figure 5 ). The FFT analysis of signals detected by the thread and disk electrodes revealed significant changes in the PsD max  values ( P  = 0.0014, and  P  < 0.001; respectively), while the AUC analysis demonstrated similar changes ( P  < 0.001) in the mechanical contractions. The extents of stimulation or inhibition were similar in both mechanical and electrical changes. A statistically significant positive correlation was found between the changes in the PsD max  and AUC values measured by thread ( Figure 6A ) or disk electrode ( Figure 6B ) in comparison with SG signals ( P  = 0.0008 and  P  < 0.0001, respectively).\nChanges in uterine activity after oxytocin and terbutaline treatments in rats with resected gastrointestinal track. The intensity of the electric signals was expressed as the maximum of the power spectrum density (PsD max ), while the mechanical contraction was evaluated by area under curve (AUC) analysis. Oxytocin increased, while terbutaline reduced the electric and mechanical activities of the smooth muscles relative to the basic activity (100%) ( P  < 0.05*;  P  < 0.01**;  P  < 0.001***).\nCorrelations between myoelectric and mechanical responses induced by oxytocin and terbutaline treatments in GI tract-resected animals. The myoelectric response recorded by thread ( A ) or disk electrode ( B ) is expressed as the maximum of the power spectrum density (PsD max ), while the mechanical response is expressed as the area under the curve (AUC) of the recorded smooth muscle contractions compared to the basic activity.\nThe myoelectric and contractility responses of cecum and pregnant myometrium have been recorded for different drugs in parallel. We have administered neostigmine and atropine, which mainly act on the GI tract, and oxytocin and terbutaline, which have an effect on the myometrium. Neostigmine and atropine treatment caused significant changes in the myoelectric signal of the cecum ( Figure 7A ), while oxytocin and terbutaline had an effect on both the electrical and mechanical signals of the uterus only ( Figure 7B ).\nChanges in gastrointestinal (GI) and myometrial smooth muscle activities after neostigmine and atropine ( A ), or oxytocin and terbutaline ( B ) treatments in rats. with intact GI tract. The intensity of the electric signals was expressed as the maximum of the power spectrum density (PsD max ), while the mechanical contraction was evaluated by area under curve (AUC) analysis ( P  < 0.05*;  P  < 0.01**;  P  < 0.001***).\n\nThe myoelectric activity of pregnant rat myometrium is measurable from the abdominal surface and well-characterized by cpm value. The intensity of contractions can be expressed and followed by the PsD max  values.\nThe currently used electromyography methods can be distinguished into two main techniques: recording the propagation of the electric waves with a multi-electrode array ( 10 ) and investigation of the myoelectric activity with serosal or cutaneous electrode pairs  in vivo  ( 11 ).\nIn the first series of our experiments, the whole gastrointestinal tract was resected for the pure detection of the myoelectric signals from the pregnant uterus. Two pairs of electrodes measured the myoelectric signals: one was inserted into the right horn of the uterus, while the other was laid subcutaneously, above the uterus. The most intensive signals were gained by the organ-inserted electrodes, while the same type of slow wave was detected at the subcutaneous site with a weaker but still well-detectable intensity. This proves that the myometrial myoelectric signals are detectable from the abdominal subcutaneous area, therefore it is possible to get valuable signals of uterine activity from the abdominal surface. The myoelectric activity was measured in parallel with the mechanical contractions by the applied strain gauge. This triple detection along with the gastrointestinal resection and digital filtration provided a clear record of the uterine myoelectric waves in comparison with the contractions.\nThe frequencies of the electric signals of the smooth muscles, such as pregnant uterus, are significantly lower as compared with brain, cardiac or skeletal muscle electric signals. FFT analysis reveals that the frequency ranges of electroencephalograms, electrocardiograms and skeletal muscle electromyograms are 5-50 Hz, 3-20 Hz, and 10-20 Hz, respectively ( 12 , 13 ). The slow waves of smooth muscle electric activity are usually characterized with a 60-fold lower cpm value of Hz. We found that pregnant uterine smooth muscle frequency is between 1-3 cpm (0.017-0.05 Hz), and therefore the higher frequencies must be cut with the digital filters to filter out the non-uterine myoelectric signals.\nThe PsD max  values usually express the intensity of myoelectric signals. We observed a very good correlation between the changes in mechanical contractile response and the changes in electric PsD max  values. This means that the changes in PsD max  values recorded even on the abdominal wall perfectly reflect the real changes in the contractions of the uterine smooth muscle. The oxytocin-induced increase and the terbutaline-induced decrease were clearly detectable via the observation of the changes in the PsD max  intensity of the myoelectric spectra from pregnant uterus.\nIn our previous study, we found that the myoelectric spectrum of the cecum can be detected below 3 cpm ( 9 ). It means that the myoelectric signals of the pregnant uterus and cecum overlap. Therefore, we performed a second series of experiments on pregnant animals with intact gastrointestinal tract to distinguish between the myoelectric signals from these two types of smooth muscles. The mechanical contractions of both organs were detected by strain gauges applied on the surface of the cecum and pregnant myometrium. It is known that the cecum and pregnant myometrium have different responses to drugs. Cecum is much more sensitive to neostigmine (contraction) and atropine (relaxation), while the pregnant uterus has a very intensive response to oxytocin (contraction) and terbutaline (relaxation) ( 9 , 14 , 15 ). We found that drugs acting on myometrial activity altered the PsD max  with a good correlation to myometrial mechanical contractions, but they did not affect cecal activity. The drugs acting on the cecum elicited similar action on cecal activity without influencing the myometrial response. It means that we can follow the uterine contractions with the evaluation of PsD max  values when the uterine activity is predominant. During pregnancy (especially toward the end of pregnancy) the mass of the uterus is significantly increased and its activity is predominant over the cecal contractions. Our method seems to be proper for the detection of the uterine activity in that late-pregnancy period.\nThe limitation of our study is that we have not yet tested our method in non-anaesthetized rats and, therefore, we have no information about its usefulness in wakeful subjects. Another limitation is that we tested this method only in full-term pregnant rats. Nevertheless, we think that we have successfully developed a new, preclinical method to investigate the pregnant myometrial activity,  in vivo . Additionally, this method is already suitable for pharmacological investigations for drugs acting on uterine contractions. It also has the potential for the investigation of pathophysiological processes during parturition, even in conscious animals. The method might support the development of new, non-invasive clinical equipment for obstetricians to detect early signs of premature contractions or predict initiation of labor.","source_license":"CC-BY-4.0","license_restricted":false}