Effects of L-arginine on the diaphragm muscle twitches elicited at different frequencies of nerve stimulation

In rats, the nitric oxide (NO)-synthase pathway is present in skeletal muscle, vascular smooth muscle, and motor nerve terminals. Effects of NO were previously studied in rat neuromuscular preparations receiving low (0.2 Hz) or high (200 Hz) frequencies of stimulation. The latter frequency has always induced tetanic fade. However, in these previous studies we did not determine whether NO facilitates or impairs the neuromuscular transmission in preparations indirectly stimulated at frequencies which facilitate neuromuscular transmission. Thus, the present study was carried out to examine the effects of NO in rat neuromuscular preparations indirectly stimulated at 5 and 50 Hz. The amplitude of muscular contraction observed at the end (B) of a 10-s stimulation was taken as the ratio (R) of that obtained at the start (A) (R = B/A). S-nitroso-N-acetylpenicillamine (200 µM), superoxide dismutase (78 U/ml) and L-arginine (4.7 mM), but not D-arginine (4.7-9.4 mM), produced an increase in R (facilitation of neurotransmission) at 5 Hz. However, reduction in the R value (fade of transmission) was observed at 50 Hz. N G-nitro-L-arginine (8.0 mM) antagonized both the facilitatory and inhibitory effects of L-arginine (4.7 mM). The results suggest that NO may modulate the release of acetylcholine by motor nerve terminals.

Uniform decrease of alpha-global field power induced by intermittent photic stimulation of healthy subjects

Nineteen-channel EEGs were recorded from the scalp surface of 30 healthy subjects (16 males and 14 females, mean age: 34 years, SD: 11.7 years) at rest and under trains of intermittent photic stimulation (IPS) at rates of 5, 10 and 20 Hz. Digitalized data were submitted to spectral analysis with fast fourier transformation providing the basis for the computation of global field power (GFP). For quantification, GFP values in the frequency ranges of 5, 10 and 20 Hz at rest were divided by the corresponding data obtained under IPS. All subjects showed a photic driving effect at each rate of stimulation. GFP data were normally distributed, whereas ratios from photic driving effect data showed no uniform behavior due to high interindividual variability. Suppression of alpha-power after IPS with 10 Hz was observed in about 70% of the volunteers. In contrast, ratios of alpha-power were unequivocal in all subjects: IPS at 20 Hz always led to a suppression of alpha-power. Dividing alpha-GFP with 20-Hz IPS by alpha-GFP at rest (R = alpha-GFP IPS/alpha-GFPrest) thus resulted in ratios lower than 1. We conclude that ratios from GFP data with 20-Hz IPS may provide a suitable paradigm for further investigations.

Cholinergic stimulation with pyridostigmine reduces the QTc interval in coronary artery disease

Parasympathetic dysfunction is an independent risk factor in patients with coronary artery disease; thus, cholinergic stimulation is a potential therapeutic measure that may be protective by acting on ventricular repolarization. The purpose of the present study was to determine the effects of pyridostigmine bromide (PYR), a reversible anticholinesterase agent, on the electrocardiographic variables, particularly QTc interval, in patients with stable coronary artery disease. In a randomized double-blind crossover placebo-controlled study, simultaneous 12-lead electrocardiographic tracings were obtained at rest from 10 patients with exercise-induced myocardial ischemia before and 2 h after the oral administration of 45 mg PYR or placebo. PYR increased the RR intervals (pre: 921 ± 27 ms vs post: 1127 ± 37 ms; P<0.01) and, in contrast with placebo, decreased the QTc interval (pre: 401 ± 3 ms vs post: 382 ± 3 ms; P<0.01). No other electrocardiographic variables were modified (PR segment, QT interval, QT and QTc dispersions). Cholinergic stimulation with PYR caused bradycardia and reduced the QTc interval without important side effects in patients with coronary disease. These effects, if confirmed in studies over longer periods of administration, may suggest a cardioprotection by cholinergic stimulation with PYR.

Excitatory amino acid receptor blockade within the caudal pressor area and rostral ventrolateral medulla alters cardiovascular responses to nucleus raphe obscurus stimulation in rats

Pressor responses elicited by stimulation of the nucleus raphe obscurus (NRO) depend on the integrity of the rostral ventrolateral medulla (RVLM). Therefore, to test the participation of excitatory amino acid (EAA) receptors in the cardiovascular responses evoked by NRO stimulation (1 ms, 100 Hz, 40-70 µA, for 10 s), the EAA antagonist kynurenic acid (Kyn) was microinjected at different sites in the ventrolateral medullar surface (2.7 nmol/200 nl) of male Wistar rats (270-320 g, N = 39) and NRO stimulation was repeated. The effects of NRO stimulation were: hypertension (deltaMAP = +43 ± 1 mmHg, P<0.01), bradycardia (deltaHR = -30 ± 7 bpm, P<0.01) and apnea. Bilateral microinjection of Kyn into the RVLM, which did not change baseline parameters, almost abolished the bradycardia induced by NRO stimulation (deltaHR = -61 ± 3 before vs -2 ± 3 bpm after Kyn, P<0.01, N = 7). Unilateral microinjection of Kyn into the CVLM did not change baseline parameters or reduce the pressor response to NRO stimulation (deltaMAP = +46 ± 5 before vs +48 ± 5 mmHg after Kyn, N = 6). Kyn bilaterally microinjected into the caudal pressor area reduced blood pressure and heart rate and almost abolished the pressor response to NRO stimulation (deltaMAP = +46 ± 4 mmHg before vs +4 ± 2 mmHg after Kyn, P<0.01, N = 7). These results indicate that EAA receptors on the medullary ventrolateral surface play a role in the modulation of the cardiovascular responses induced by NRO stimulation, and also suggest that the RVLM participates in the modulation of heart rate responses and that the caudal pressor area modulates the pressor response following NRO stimulation.

Modulation of hormone secretion by functional electrical stimulation of the intact and incompletely dysfunctional dog pancreas

The purpose of the present study was to modulate the secretion of insulin and glucagon in Beagle dogs by stimulation of nerves innervating the intact and partly dysfunctional pancreas. Three 33-electrode spiral cuffs were implanted on the vagus, splanchnic and pancreatic nerves in each of two animals. Partial dysfunction of the pancreas was induced with alloxan. The nerves were stimulated using rectangular, charge-balanced, biphasic, and constant current pulses (200 µs, 1 mA, 20 Hz, with a 100-µs delay between biphasic phases). Blood samples from the femoral artery were drawn before the experiment, at the beginning of stimulation, after 5 min of stimulation, and 5 min after the end of stimulation. Radioimmunoassay data showed that in the intact pancreas stimulation of the vagal nerve increased insulin (+99.2 µU/ml) and glucagon (+18.7 pg/ml) secretion and decreased C-peptide secretion (-0.15 ng/ml). Splanchnic nerve stimulation increased insulin (+1.7 µU/ml), C-peptide (+0.01 ng/ml), and glucagon (+50 pg/ml) secretion, whereas pancreatic nerve stimulation did not cause a marked change in any of the three hormones. In the partly dysfunctional pancreas, vagus nerve stimulation increased insulin (+15.5 µU/ml), glucagon (+11 pg/ml), and C-peptide (+0.03 ng/ml) secretion. Splanchnic nerve stimulation reduced insulin secretion (-2.5 µU/ml) and increased glucagon (+58.7 pg/ml) and C-peptide (+0.39 ng/ml) secretion, and pancreatic nerve stimulation increased insulin (+0.2 µU/ml), glucagon (+5.2 pg/ml), and C-peptide (+0.08 ng/ml) secretion. It was concluded that vagal nerve stimulation can significantly increase insulin secretion for a prolonged period of time in intact and in partly dysfunctional pancreas.

Fuzzy expert system in the prediction of neonatal resuscitation

In view of the importance of anticipating the occurrence of critical situations in medicine, we propose the use of a fuzzy expert system to predict the need for advanced neonatal resuscitation efforts in the delivery room. This system relates the maternal medical, obstetric and neonatal characteristics to the clinical conditions of the newborn, providing a risk measurement of need of advanced neonatal resuscitation measures. It is structured as a fuzzy composition developed on the basis of the subjective perception of danger of nine neonatologists facing 61 antenatal and intrapartum clinical situations which provide a degree of association with the risk of occurrence of perinatal asphyxia. The resulting relational matrix describes the association between clinical factors and risk of perinatal asphyxia. Analyzing the inputs of the presence or absence of all 61 clinical factors, the system returns the rate of risk of perinatal asphyxia as output. A prospectively collected series of 304 cases of perinatal care was analyzed to ascertain system performance. The fuzzy expert system presented a sensitivity of 76.5% and specificity of 94.8% in the identification of the need for advanced neonatal resuscitation measures, considering a cut-off value of 5 on a scale ranging from 0 to 10. The area under the receiver operating characteristic curve was 0.93. The identification of risk situations plays an important role in the planning of health care. These preliminary results encourage us to develop further studies and to refine this model, which is intended to implement an auxiliary system able to help health care staff to make decisions in perinatal care.

Air pollution and neonatal deaths in São Paulo, Brazil

Air pollution has been associated with health effects on different age groups. The present study was designed to assess the impact of daily changes in air pollutants (NO2, SO2, CO, O3, and particle matter (PM10)) on total number of daily neonatal deaths (those that occur between the first and the 28th days of life) in São Paulo, from January 1998 to December 2000, since adverse outcomes such as neonatal deaths associated with air pollution in Brazil have not been evaluated before. Generalized additive Poisson regression models were used and nonparametric smooth functions (loess) were adopted to control long-term trend, temperature, humidity, and short-term trends. A linear term was used for holidays. The association between air pollutants and neonatal deaths showed a short time lag. Interquartile range increases in PM10 (23.3 µg/m³) and SO2 (9.2 µg/m³) were associated with increases of 4% (95% CI, 2-6) and 6% (95% CI, 4-8), respectively. Instead of adopting a two-pollutant model we created an index to represent PM10 and SO2 effects. For an interquartile range increase in the index an increase of 6.3% (95% CI, 6.1-6.5) in neonatal deaths was observed. These results agree with previous studies performed by our group showing the deleterious effects of air pollutants during the perinatal period. The method reported here represents an alternative approach to analyze the relationship between highly correlated pollutants and public health problems, reinforcing the idea of the synergic effects of air pollutants in public health.

Neonatal administration of citalopram delays somatic maturation in rats

We investigated the somatic maturation of neonate rats treated during the suckling period with citalopram, a selective serotonin reuptake inhibitor. Groups with 6 male neonates were randomly assigned to different treatments 24 h after birth. Each litter was suckled by one of the dams until the 21st postnatal day. Body weight, head axis and tail length were measured daily from the 1st to the 21st postnatal day. Time of ear unfolding, auditory conduit opening, incisor eruption, and eye opening was determined. Pups received 5 mg (Cit5), 10 mg (Cit10) or 20 mg/kg (Cit20) citalopram sc, or saline (0.9% NaCl, w/v, sc). Compared to saline, body weight was lower (24.04%, P < 0.01) for Cit10 from the 10th to the 21st day and for Cit20 from the 6th to the 21st day (38.19%, P < 0.01). Tail length was reduced in the Cit20 group (15.48%, P < 0.001) from the 8th to the 21st day. A reduction in mediolateral head axis (10.53%, P < 0.05) was observed from the 11th to the 21st day in Cit10 and from the 6th to the 21st day in Cit20 (13.16%, P < 0.001). A reduction in anteroposterior head axis was also observed in the Cit20 group (5.28%, P < 0.05) from the 13th to the 21stday. Conversely, this axis showed accelerated growth from the 12th to the 21stday in the Cit5 group (13.05%, P < 0.05). Auditory conduit opening was delayed in the Cit5 and Cit20 groups and incisor eruption was delayed in all citalopram groups. These findings show that citalopram injected during suckling to rats induces body alterations and suggest that the activity of the serotoninergic system participates in growth mechanisms.

Umbilical cord blood and neonatal endothelin-1 levels in preterm newborns with and without respiratory distress syndrome

Increased pulmonary vascular resistance in preterm newborn infants with respiratory distress syndrome is suggested, and endothelin-1 plays an important role in pulmonary vascular reactivity in newborns. We determined umbilical cord blood and neonatal (second sample) levels of endothelin-1 in 18 preterm newborns with respiratory distress syndrome who had no clinical or echocardiographic diagnosis of pulmonary hypertension and 22 without respiratory distress syndrome (gestational ages: 31.4 ± 1.6 and 29.3 ± 2.3 weeks, respectively). Umbilical cord blood and a second blood sample taken 18 to 40 h after birth were used for endothelin-1 determination by enzyme immunoassay. Median umbilical cord blood endothelin-1 levels were similar in both groups (control: 10.9 and respiratory distress syndrome: 11.4 pg/mL) and were significantly higher than in the second sample (control: 1.7 pg/mL and respiratory distress syndrome: 3.5 pg/mL, P < 0.001 for both groups). Median endothelin-1 levels in the second sample were significantly higher in children with respiratory distress syndrome than in control infants (P < 0.001). There were significant positive correlations between second sample endothelin-1 and Score for Neonatal Acute Physiology and Perinatal Extension II (r = 0.36, P = 0.02), and duration of mechanical ventilation (r = 0.64, P = 0.02). A slower decline of endothelin-1 from birth to 40 h of life was observed in newborns with respiratory distress syndrome when compared to controls. A significant correlation between neonatal endothelin-1 levels and some illness-severity signs suggests that endothelin-1 plays a role in the natural course of respiratory distress syndrome in preterm newborns.

Distribution of microglial cells in the cerebral hemispheres of embryonic and neonatal chicks

The distribution, morphology and morphometry of microglial cells in the chick cerebral hemispheres from embryonic day 4 (E4) to the first neonatal day (P1) were studied by histochemical labeling with a tomato (Lycopersicon esculentum) lectin. The histochemical analysis revealed lectin-reactive cells in the nervous parenchyma on day E4. Between E4 (5.7 ± 1.35 mm length) and E17 (8.25 ± 1.2 mm length), the lectin-reactive cells were identified as ameboid microglia and observed starting from the subventricular layer, distributed throughout the mantle layer and in the proximity of the blood vessels. After day E13, the lectin-reactive cells exhibited elongated forms with small branched processes, and were considered primitive ramified microglia. Later, between E18 (5.85 ± 1.5 mm cell body length) and P1 (3.25 ± 0.6 mm cell body length), cells with more elongated branched processes were observed, constituting the ramified microglia. Our findings provide additional information on the migration and differentiation of microglial cells, whose ramified form is observed at the end of embryonic development. The present paper focused on the arrangement of microglial cells in developing cerebral hemispheres of embryonic and neonatal chicks, which are little studied in the literature. Details of morphology, morphometry and spatial distribution of microglial cells contributed to the understanding of bird and mammal central nervous system ontogeny. Furthermore, the identification and localization of microglial cells during the normal development could be used as a morphological guide for embryonic brain injury researches.

Electrical field stimulation improves bone mineral density in ovariectomized rats

Osteoporosis and its consequent fractures are a great social and medical problem mainly occurring in post-menopausal women. Effective forms of prevention and treatment of osteoporosis associated with lower costs and the least side effects are needed. Electrical fields are able to stimulate osteogenesis in fractures, but little is known about their action on osteoporotic tissue. The aim of the present study was to determine by bone densitometry the effects of electrical stimulation on ovariectomized female Wistar rats. Thirty rats (220 ± 10 g) were divided into three groups: sham surgery (SHAM), bilateral ovariectomy (OVX) and bilateral ovariectomy + electrical stimulation (OVX + ES). The OVX + ES group was submitted to a 20-min session of a low-intensity pulsed electrical field (1.5 MHz, 30 mW/cm²) starting on the 7th day after surgery, five times a week (total = 55 sessions). Global, spine and limb bone mineral density were measured by dual-energy X-ray absorptiometry (DXA Hologic 4500A) before surgery and at the end of protocol (84 days after surgery). Electrical stimulation improved (P < 0.05) global (0.1522 ± 0.002), spine (0.1502 ± 0.003), and limb (0.1294 ± 0.003 g/cm²) bone mineral density compared to OVX group (0.1447 ± 0.001, 0.1393 ± 0.002, and 0.1212 ± 0.001, respectively). The OVX + ES group also showed significantly higher global bone mineral content (9.547 ± 0.114 g) when compared to both SHAM (8.693 ± 0.165 g) and OVX (8.522 ± 0.207 g) groups (P < 0.05). We have demonstrated that electrical fields stimulate osteogenesis in ovariectomized female rats. Their efficacy in osteoporosis remains to be demonstrated.

Changes in types of muscle fibers induced by transcutaneous electrical stimulation of the diaphragm of rats

The objective of the present study was to assess the effect of transcutaneous electrical diaphragmatic stimulation (TEDS) on different types of diaphragm muscle fibers. Male Wistar rats (8-12 weeks old) were divided into 2 experimental groups (N = 8 in each group): 1) control, 2) animals submitted to TEDS [frequency = 50 Hz; T ON/T OFF (contraction/relaxation time) = 2/2 s; pulse duration = 0.4 ms, intensity = 5 mA with a 1 mA increase every 3 min for 20 min] for 7 days. After completing this treatment period, the I, IIA, IIB, and IID diaphragm muscle fibers were identified using the mATPase technique. Statistical analysis consisted of the normality, homoscedasticity and t-tests (P < 0.05). There was a 19.6% (P < 0.05) reduction in the number of type I fibers and a 49.7% increase (P < 0.05) in type IID fibers in the TEDS group compared with the control group. An important result of the present study was that electrical stimulation with surface electrodes was efficient in altering the distribution of fibers in diaphragm muscle. This therapeutic resource could be used in the treatment of respiratory muscle alterations.

Effects of systemic administration of ciliary neurotrophic factor on Bax and Bcl-2 proteins in the lumbar spinal cord of neonatal rats after sciatic nerve transection

Ciliary neurotrophic factor (CNTF) is a cytokine that plays a neuroprotective role in relation to axotomized motoneurons. We determined the effect of daily subcutaneous doses of CNTF (1.2 µg/g for 5 days; N = 13) or PBS (N = 13) on the levels of mRNA for Bcl-2 and Bax, as well as the expression and inter-association of Bcl-2 and Bax proteins, and the survival of motoneurons in the spinal cord lumbar enlargement of 2-day-old Wistar rats after sciatic nerve transection. Five days after transection, the effects were evaluated on histological and molecular levels using Nissl staining, immunoprecipitation, Western blot analysis, and reverse transcriptase-polymerase chain reaction. The motoneuron survival ratio, defined as the ratio between the number of motoneurons counted on the lesioned side vs those on the unlesioned side, was calculated. This ratio was 0.77 ± 0.02 for CNTF-treated rats vs 0.53 ± 0.02 for the PBS-treated controls (P < 0.001). Treatment with CNTF modified the level of mRNA, with the expression of Bax RNA decreasing 18% (with a consequent decrease in the level of Bax protein), while the expression of Bcl-2 RNA was increased 87%, although the level of Bcl-2 protein was unchanged. The amount of Bcl-2/Bax heterodimer increased 91% over that found in the PBS-treated controls. These data show, for the first time, that the neuroprotective effect of CNTF on neonatal rat axotomized motoneurons is associated with a reduction in free Bax, due to the inhibition of Bax expression, as well as increased Bcl-2/Bax heterodimerization. Thus, the neuroprotective action of the CNTF on axotomized motoneurons can be related to the inhibition of this apoptotic pathway.

Neonatal screening for cystic fibrosis in São Paulo State, Brazil: a pilot study

Cystic fibrosis is one of the most common autosomal recessive hereditary diseases in the Caucasian population, with an incidence of 1:2000 to 1:3500 liveborns. More than 1000 mutations have been described with the most common being F508del. It has a prevalence of 23-55% within the Brazilian population. The lack of population-based studies evaluating the incidence of cystic fibrosis in São Paulo State, Brazil, and an analysis concerning the costs of implantation of a screening program motivated the present study. A total of 60,000 dried blood samples from Guthrie cards obtained from April 2005 to January 2006 for neonatal screening at 4 reference centers in São Paulo State were analyzed. The immunoreactive trypsinogen (IRT)/IRT protocol was used with the cut-off value being 70 ng/mL. A total of 532 children (0.9%) showed IRT >70 ng/mL and a 2nd sample was collected from 418 (80.3%) of these patients. Four affected children were detected at two centers, corresponding to an incidence of 1:8403. The average age at diagnosis was 69 days, and 3 of the children already showed severe symptoms of the disease. The rate of false-positive results was 95.2% and the positive predictive value for the test was 8%. The cost of detecting an affected subject was approximately US$8,000.00 when this cystic fibrosis program was added to an existing neonatal screening program. The present study clearly shows the difficulties involved in cystic fibrosis screening using the IRT/IRT protocol, particularly in a population with no long-term tradition of neonatal screening.

Frequency of 8 CFTR gene mutations in cystic fibrosis patients in Minas Gerais, Brazil, diagnosed by neonatal screening

The nature and frequency of cystic fibrosis mutations in Brazil is not uniform due to the highly varied ethnic composition of the population. The average frequency of the F508del mutation has been reported to be 48.6%. Other common mutations in Brazil are G542X, R1162X, and N1303K. The aim of this study was to analyze the frequency of 8 mutations (F508del, G542X, R1162X, N1303K, W1282X, G85E, 3120+1G>A, and 711+1G>T) in a sample of 111 newborn patients with cystic fibrosis diagnosed by the Cystic Fibrosis Neonatal Screening Program of Minas Gerais State. The mutations were tested by allele-specific oligonucleotide PCR with specially designed primers. An allele frequency of 48.2% was observed for the F508del mutation, and allele frequencies of 5.41, 4.50, 4.05, and 3.60% were found for the R1162X, G542X, 3120+1G>A, and G85E mutations, respectively. The genotypes obtained were in Hardy-Weinberg equilibrium. These data demonstrate that the 8-mutation panel studied here has extensive coverage (68%) for the cystic fibrosis mutations in Minas Gerais. These data improve our knowledge of cystic fibrosis in Brazil, particularly in this region. In addition, this investigation contributed to the establishment of a sensitive and population-specific mutation panel, which can be helpful for molecular diagnosis of cystic fibrosis.