Characterization of nerve-induced relaxation of gastrointestinal sphincteric smooth muscle in a South American opossum (Didelphis albiventris)

The presence of inhibitory nonadrenergic noncholinergic (NANC) intrinsic innervation of the circular muscle of the gastrointestinal sphincters of the South American (SA) opossum was investigated in vitro. Isolated circular muscle strips from the esophagogastric and ileocolonic junctions but not from the gastroduodenal (pylorus) region developed spontaneous tension. Tetrodotoxin (TTX, 1 µM) augmented the spontaneous tension only in the ileocolonic junction strips. Electrical field stimulation of esophagogastric and ileocolonic junction strips caused frequency-dependent responses consisting of a relaxation at lower frequencies (<1 Hz) and a biphasic response or contraction at higher frequencies. In the strips from the pyloric region electrical field stimulation abolished the spontaneous activity at lower frequencies and induced contractions at higher frequencies. The responses elicited by electrical field stimulation in the three sphincters were abolished by TTX (1 µM). Electrical field-induced contractions were reduced while relaxations were enhanced by atropine (1 µM). In the presence of atropine (1 µM) and guanethidine (3 µM), electrical field stimulation, nicotine and ATP induced frequency- or concentration-dependent relaxations of the three sphincters that were abolished by TTX (1 µM). Isoproterenol and sodium nitroprusside caused concentration-dependent relaxations which were TTX-resistant. These findings indicate that the sphincteric circular muscle of the SA opossum gastrointestinal tract is relaxed by the activation of intrinsic NANC nerves and therefore can be used as a model for the study of the mechanisms involved in these responses

Glass pipette-carbon fiber microelectrodes for evoked potential recordings

Current methods for recording field potentials with tungsten electrodes make it virtually impossible to use the same recording electrode also as a lesioning electrode, for example for histological confirmation of the recorded site, because the lesioning procedure usually wears off the tungsten tip. Therefore, the electrode would have to be replaced after each lesioning procedure, which is a very high cost solution to the problem. We present here a low cost, easy to make, high quality glass pipette-carbon fiber microelectrode that shows resistive, signal/noise and electrochemical coupling advantages over tungsten electrodes. Also, currently used carbon fiber microelectrodes often show problems with electrical continuity, especially regarding electrochemical applications using a carbon-powder/resin mixture, with consequent low performance, besides the inconvenience of handling such a mixture. We propose here a new method for manufacturing glass pipette-carbon fiber microelectrodes with several advantages when recording intracerebral field potentials

Identification of the main generator source of longitudinal muscle contraction in the earthworm ventral nerve cord

The main generator source of a longitudinal muscle contraction was identified as an M (mechanical-stimulus-sensitive) circuit composed of a presynaptic M-1 neuron and a postsynaptic M-2 neuron in the ventral nerve cord of the earthworm, Amynthas hawayanus, by simultaneous intracellular response recording and Lucifer Yellow-CH injection with two microelectrodes. Five-peaked responses were evoked in both neurons by a mechanical, but not by an electrical, stimulus to the mechanoreceptor in the shaft of a seta at the opposite side of an epidermis-muscle-nerve-cord preparation. This response was correlated to 84% of the amplitude, 73% of the rising rate and 81% of the duration of a longitudinal muscle contraction recorded by a mechano-electrical transducer after eliminating the other possible generator sources by partitioning the epidermis-muscle piece of this preparation. The pre- and postsynaptic relationship between these two neurons was determined by alternately stimulating and recording with two microelectrodes. Images of the Lucifer Yellow-CH-filled M-1 and M-2 neurons showed that both of them are composed of bundles of longitudinal processes situated on the side of the nerve cord opposite to stimulation. The M-1 neuron has an afferent process (A1) in the first nerve at the stimulated side of this preparation and the M-2 neuron has two efferent processes (E1 and E3) in the first and third nerves at the recording side where their effector muscle cell was identified by a third microelectrode.

Effects of sciatic-conditioned medium on neonatal rat retinal cells in vitro

Schwann cells produce and release trophic factors that induce the regeneration and survival of neurons following lesions in the peripheral nerves. In the present study we examined the in vitro ability of developing rat retinal cells to respond to factors released from fragments of sciatic nerve. Treatment of neonatal rat retinal cells with sciatic-conditioned medium (SCM) for 48 h induced an increase of 92.5 ± 8.8% (N = 7 for each group) in the amount of total protein. SCM increased cell adhesion, neuronal survival and glial cell proliferation as evaluated by morphological criteria. This effect was completely blocked by 2.5 µM chelerythrine chloride, an inhibitor of protein kinase C (PKC). These data indicate that PKC activation is involved in the effect of SCM on retinal cells and demonstrate that fragments of sciatic nerve release trophic factors having a remarkable effect on neonatal rat retinal cells in culture.

Use of fluorescent probes to follow membrane traffic in nerve terminals

Optical tracers in conjunction with fluorescence microscopy have become widely used to follow the movement of synaptic vesicles in nerve terminals. The present review discusses the use of these optical methods to understand the regulation of exocytosis and endocytosis of synaptic vesicles. The maintenance of neurotransmission depends on the constant recycling of synaptic vesicles and important insights have been gained by visualization of vesicles with the vital dye FM1-43. A number of questions related to the control of recycling of synaptic vesicles by prolonged stimulation and the role of calcium to control membrane internalization are now being addressed. It is expected that optical monitoring of presynaptic activity coupled to appropriate genetic models will contribute to the understanding of membrane traffic in synaptic terminals.

Axotomy increases NADPH-diaphorase activity in the dorsal root ganglia and lumbar spinal cord of the turtle Trachemys dorbigni

Seven days after transection of the sciatic nerve NADPH-diaphorase activity increased in the small and medium neurons of the dorsal root ganglia of the turtle. However, this increase was observed only in medium neurons for up to 90 days. At this time a bilateral increase of NADPH-diaphorase staining was observed in all areas and neuronal types of the dorsal horn, and in positive motoneurons in the lumbar spinal cord, ipsilateral to the lesion. A similar increase was also demonstrable in spinal glial and endothelial cells. These findings are discussed in relation to the role of nitric oxide in hyperalgesia and neuronal regeneration or degeneration.

Collagen arrangement in hepatic granuloma in mice infected with Schistosoma mansoni: dependence on fiber radiation centers

The collagen structure of isolated and in situ liver granuloma from Swiss Webster mice infected with Schistosoma mansoni was sequentially and three-dimensionally analyzed during different times of infection (early acute, acute, transitional acute-chronic, and chronic phases) by laser scanning confocal microscopy and electron scanning variable vacuum microscopy. The initial granuloma structure is characterized by vascular collagen residues and by anchorage points (or fiber radiation centers), from where collagenous fibers are angularly shed and self-assembled. During the exudative-productive stage, the self-assembly of these fibers minimizes energy and mass through continuous tension and focal compression. The curvature or angles between collagen fibers probably depends on the fibroblastic or myofibroblastic organization of stress fibers. Gradually, the loose unstable lattice of the exudative-productive stage transforms into a highly packed and stable architecture as a result of progressive compactness. The three-dimensional architecture of granulomas provides increased tissue integrity, efficient distribution of soluble compounds and a haptotactic background to the cells.

A comparative study of aging of the elastic fiber system of the diaphragm and the rectus abdominis muscles in rats

In the present study the age-related changes of the striated muscle elastic fiber system were investigated in the diaphragm and rectus abdominis muscles of 1-, 4-, 8- and 18-month-old rats. The activation patterns of these muscles differ in that the diaphragm is regularly mobilized tens of times every minute during the entire life of the animal whereas the rectus abdominis, although mobilized in respiration, is much less and more irregularly activated. The elastic fibers were stained by the Verhoeff technique for mature elastic fibers. Weigert stain was used to stain mature and elaunin elastic fibers, and Weigert-oxone to stain mature, elaunin and oxytalan elastic fibers. The density of mature and elaunin elastic fibers showed a progressive increase with age, whereas the amount of oxytalan elastic fibers decreased in both diaphragm and rectus abdominis muscles and their muscular fascias. These age-related quantitative and structural changes of the elastic fiber system may reduce the viscoelastic properties of the diaphragm and rectus abdominis muscles, which may compromise the transmission of tensile muscle strength to the tendons and may affect maximum total strength.

Early myelin breakdown following sural nerve crush: a freeze-fracture study

In this study we describe the early changes of the myelin sheath following surgical nerve crush. We used the freeze-fracture technique to better evaluate myelin alterations during an early stage of Wallerian degeneration. Rat sural nerves were experimentally crushed and animals were sacrificed by transcardiac perfusion 30 h after surgery. Segments of the nerves were processed for routine transmission electron microscopy and freeze-fracture techniques. Our results show that 30 h after the lesion there was asynchrony in the pattern of Wallerian degeneration, with different nerve fibers exhibiting variable degrees of axon disruption. This was observed by both techniques. Careful examination of several replicas revealed early changes in myelin membranes represented by vacuolization and splitting of consecutive lamellae, rearrangement of intramembranous particles and disappearance of paranodal transverse bands associated or not with retraction of paranodal myelin terminal loops from the axolemma. These alterations are compatible with a direct injury to the myelin sheath following nerve crush. The results are discussed in terms of a similar mechanism underlying both axon and myelin breakdown.

Angiotensin-converting enzyme inhibition by lisinopril enhances liver regeneration in rats

Bradykinin has been reported to act as a growth factor for fibroblasts, mesangial cells and keratinocytes. Recently, we reported that bradykinin augments liver regeneration after partial hepatectomy in rats. Angiotensin-converting enzyme (ACE) is also a powerful bradykinin-degrading enzyme. We have investigated the effect of ACE inhibition by lisinopril on liver regeneration after partial hepatectomy. Adult male Wistar rats underwent 70% partial hepatectomy (PH). The animals received lisinopril at a dose of 1 mg kg body weight-1 day-1, or saline solution, intraperitoneally, for 5 days before hepatectomy, and daily after surgery. Four to six animals from the lisinopril and saline groups were sacrificed at 12, 24, 36, 48, 72, and 120 h after PH. Liver regeneration was evaluated by immunohistochemical staining for proliferating cell nuclear antigen using the PC-10 monoclonal antibody. The value for the lisinopril-treated group was three-fold above the corresponding control at 12 h after PH (P<0.001), remaining elevated at approximately two-fold above control values at 24, 36, 48 (P<0.001), and at 72 h (P<0.01) after PH, but values did not reach statistical difference at 120 h after PH. Plasma ACE activity measured by radioenzymatic assay was significantly higher in the saline group than in the lisinopril-treated group (P<0.001), with 81% ACE inhibition. The present study shows that plasma ACE inhibition enhances liver regeneration after PH in rats. Since it was reported that bradykinin also augments liver regeneration after PH, this may explain the liver growth stimulating effect of ACE inhibitors.

Muscle sympathetic nerve activity and hemodynamic alterations in middle-aged obese women

To study the relationship between the sympathetic nerve activity and hemodynamic alterations in obesity, we simultaneously measured muscle sympathetic nerve activity (MSNA), blood pressure, and forearm blood flow (FBF) in obese and lean individuals. Fifteen normotensive obese women (BMI = 32.5 ± 0.5 kg/m²) and 11 age-matched normotensive lean women (BMI = 22.7 ± 1.0 kg/m²) were studied. MSNA was evaluated directly from the peroneal nerve by microneurography, FBF was measured by venous occlusion plethysmography, and blood pressure was measured noninvasively by an autonomic blood pressure cuff. MSNA was significantly increased in obese women when compared with lean control women. Forearm vascular resistance and blood pressure were significantly higher in obese women than in lean women. FBF was significantly lower in obese women. BMI was directly and significantly correlated with MSNA, blood pressure, and forearm vascular resistance levels, but inversely and significantly correlated with FBF levels. Obesity increases sympathetic nerve activity and muscle vascular resistance, and reduces muscle blood flow. These alterations, taken together, may explain the higher blood pressure levels in obese women when compared with lean age-matched women.

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.

Dietary fiber intake, stool frequency and colonic transit time in chronic functional constipation in children

The objective of the present study was to evaluate associations between fiber intake, colonic transit time and stool frequency. Thirty-eight patients aged 4 to 14 years were submitted to alimentary evaluation and to measurement of colonic transit time. The median fiber intake of the total sample was age + 10.3 g/day. Only 18.4% of the subjects presented a daily dietary fiber intake below the levels recommended by the American Health Foundation. In this group, the median left colonic transit time was shorter than in the group with higher dietary fiber intake (11 vs 17 h, P = 0.067). The correlation between stool frequency and colonic transit time was negative and weak for left colon (r = -0.3, P = 0.04), and negative and moderate for rectosigmoid and total colon (r = -0.5, P<0.001 and r = -0.5, P<0.001, respectively). The stool frequency was lower in the group with slow transit time (0.8 vs 2.3 per week, P = 0.014). In conclusion, most patients with chronic functional constipation had adequate dietary fiber intake. The negative correlation between stool frequency and colonic transit time increased progressively from proximal segments to distal segments of the colon. Patients with normal and prolonged colonic transit time differ in terms of stool frequency.

Effects of terpineol on the compound action potential of the rat sciatic nerve

Terpineol, a volatile terpenoid alcohol of low toxicity, is widely used in the perfumery industry. It is an important chemical constituent of the essential oil of many plants with widespread applications in folk medicine and in aromatherapy. The effects of terpineol on the compound action potential (CAP) of rat sciatic nerve were studied. Terpineol induced a dose-dependent blockade of the CAP. At 100 µM, terpineol had no demonstrable effect. At 300 µM terpineol, peak-to-peak amplitude and conduction velocity of CAP were significantly reduced at the end of 180-min exposure of the nerve to the drug, from 3.28 ± 0.22 mV and 33.5 ± 7.05 m/s, respectively, to 1.91 ± 0.51 mV and 26.2 ± 4.55 m/s. At 600 µM, terpineol significantly reduced peak-to-peak amplitude and conduction velocity from 2.97 ± 0.55 mV and 32.8 ± 3.91 m/s to 0.24 ± 0.23 mV and 2.72 ± 2.72 m/s, respectively (N = 5). All these effects developed slowly and were reversible upon 180-min washout.

Regenerated rat skeletal muscle after periodic contusions

In the present study we evaluated the morphological aspect and changes in the area and incidence of muscle fiber types of long-term regenerated rat tibialis anterior (TA) muscle previously submitted to periodic contusions. Animals received eight consecutive traumas: one trauma per week, for eight weeks, and were evaluated one (N = 8) and four (N = 9) months after the last contusion. Serial cross-sections were evaluated by toluidine blue staining, acid phosphatase and myosin ATPase reactions. The weight of injured muscles was decreased compared to the contralateral intact one (one month: 0.77 ± 0.15 vs 0.91 ± 0.09 g, P = 0.03; four months: 0.79 ± 0.14 vs 1.02 ± 0.07 g, P = 0.0007, respectively) and showed abundant presence of split fibers and fibers with centralized nuclei, mainly in the deep portion. Damaged muscles presented a higher incidence of undifferentiated fibers when compared to the intact one (one month: 3.4 ± 2.1 vs 0.5 ± 0.3%, P = 0.006; four months: 2.3 ± 1.6 vs 0.3 ± 0.3%, P = 0.007, respectively). Injured TA evaluated one month later showed a decreased area of muscle fibers when compared to the intact one (P = 0.003). Thus, we conclude that: a) muscle fibers were damaged mainly in the deep portion, probably because they were compressed against the tibia; b) periodic contusions in the TA muscle did not change the percentage of type I and II muscle fibers; c) periodically injured TA muscles took four months to reach a muscle fiber area similar to that of the intact muscle.