Monosodium glutamate (MSG)-obese rats develop glucose intolerance and insulin resistance to peripheral glucose uptake

Different levels of insulin sensitivity have been described in several animal models of obesity as well as in humans. Monosodium glutamate (MSG)-obese mice were considered not to be insulin resistant from data obtained in oral glucose tolerance tests. To reevaluate insulin resistance by the intravenous glucose tolerance test (IVGTT) and by the clamp technique, newborn male Wistar rats (N = 20) were injected 5 times, every other day, with 4 g/kg MSG (N = 10) or saline (control; N = 10) during the first 10 days of age. At 3 months, the IVGTT was performed by injecting glucose (0.75 g/kg) through the jugular vein into freely moving rats. During euglycemic clamping plasma insulin levels were increased by infusing 3 mU . kg-1 . min-1 of regular insulin until a steady-state plateau was achieved. The basal blood glucose concentration did not differ between the two experimental groups. After the glucose load, increased values of glycemia (P<0.001) in MSG-obese rats occurred at minute 4 and from minute 16 to minute 32. These results indicate impaired glucose tolerance. Basal plasma insulin levels were 39.9 ± 4 µU/ml in control and 66.4 ± 5.3 µU/ml in MSG-obese rats. The mean post-glucose area increase of insulin was 111% higher in MSG-obese than in control rats. When insulinemia was clamped at 102 or 133 µU/ml in control and MSG rats, respectively, the corresponding glucose infusion rate necessary to maintain euglycemia was 17.3 ± 0.8 mg . kg-1 . min-1 for control rats while 2.1 ± 0.3 mg . kg-1 . min-1 was sufficient for MSG-obese rats. The 2-h integrated area for total glucose metabolized, in mg . min . dl-1, was 13.7 ± 2.3 vs 3.3 ± 0.5 for control and MSG rats, respectively. These data demonstrate that MSG-obese rats develop insulin resistance to peripheral glucose uptake

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

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.

Facilitation of the main generator source of earthworm muscle contraction by a peripheral neuron

A constant facilitation of responses evoked in the earthworm muscle contraction generator neurons by responses evoked in the neurons of its peripheral nervous system was demonstrated. It is based on the proposal that these two responses are bifurcations of an afferent response evoked by the same peripheral mechanical stimulus but converging again on this central neuron. A single-peaked generator response without facilitation was demonstrated by sectioning the afferent route of the peripheral facilitatory modulatory response, or conditioning response (CR). The multipeaked response could be restored by restimulating the sectioned modulatory neuron with an intracellular substitutive conditioning stimulus (SCS). These multi-peaked responses were proposed to be the result of reverberating the original single peaked unconditioned response (UR) through a parallel (P) neuronal circuit which receives the facilitation of the peripheral modulatory neuron. This peripheral modulatory neuron was named "Peri-Kästchen" (PK) neuron because it has about 20 peripheral processes distributed on the surface of a Kästchen of longitudinal muscle cells on the body wall of this preparation as revealed by the Lucifer Yellow-CH-filling method.

Persistent attenuation and enhancement of the earthworm main muscle contraction generator response induced by repeated stimulation of a peripheral neuron

Responses evoked in the earthworm, Amynthas hawayanus, main muscle contraction generator M-2 (postsynaptic mechanical-stimulus-sensitive) neuron by threshold mechanical stimuli in 2-s intertrial intervals (ITI) were used as the control or unconditioned responses (UR). Their attenuation induced by decreasing these intervals in non-associative conditioning and their enhancement induced by associating the unconditioned stimuli (US) to a train of short (0.1 s) hyperpolarizing electrical substitutive conditioning stimuli (SCS) in the Peri-Kästchen (PK) neuron were measured in four parameters, i.e., peak numbers (N) and amplitude ()averaged from 120 responses, sum of these amplitudes (SAMP) and the highest peak amplitude (V) over a period of 4 min. Persistent attenuation similar to habituation was induced by decreasing the control ITI to 0.5 s and 2.0 s in non-associative conditioning within less than 4 min. Dishabituation was induced by randomly pairing one of these habituated US to an electrical stimulus in the PK neuron. All four parameters of the UR were enhanced by forward (SCS-US), but not backward (US-SCS), association of the US with 25, 100 and 250-Hz trains of SCS with 40-ms interstimulus intervals (ISI) for 4 min and persisted for another 4 min after turning off the SCS. The enhancement of these parameters was proportional to the SCS frequencies in the train. No UR was evoked by the SCS when the US was turned off after 4 min of classical conditioning.

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.

Detection and analysis of apoptosis in peripheral blood cells from breast cancer patients

Apoptosis is a well-known specific process of cell death that normally occurs in physiological situations such as tissue or organ development and involution. During tumor growth there is a balance between proliferation and cell death which involves apoptotic mechanisms. In the present study genomic DNAs from 120 breast tumor biopsies were analyzed by agarose gel electrophoresis and none of them presented the fragmentation pattern characteristic of the apoptosis process. However, 33% of the 105 breast cancer patients clearly showed the apoptotic pattern when DNA from blood cells was analyzed. None of the DNAs from healthy volunteer blood cells showed any trace of apoptosis. Since the breast cancer patients were not receiving chemo- or hormone therapy, the possible relationship between blood cortisol levels and the apoptotic pattern found in patient blood cells was investigated. Using a chemoluminescence immunodetection assay, similar cortisol levels were observed in breast cancer patient sera presenting or not apoptotic blood cells and in healthy volunteer sera. Analysis of the clinical data obtained from 60 of these patients showed that patients bearing tumors of smaller size (under 20 mm) were more susceptible to the apoptotic effect in blood cells. According to the Elston grade, it was observed that 7 of 12 patients with grade III tumors (58%) presented apoptotic peripheral blood cells, in contrast to 10 of 48 patients with grade I and grade II tumors. These observations may reflect the immunosuppression characteristic of some breast cancer patients, which may contribute to tumor growth. Therefore, further studies are necessary to elucidate the factor(s) involved in such massive blood cell death.

Ischemic preconditioning reduces peripheral oxidative damage associated with brain ischemia in rats

Brain ischemia followed by reperfusion causes neuronal death related to oxidative damage. Furthermore, it has been reported that subjects suffering from ischemic cerebrovascular disorders exhibit changes in circulating platelet aggregation, a characteristic that might be important for their clinical outcome. In the present investigation we studied tert-butyl hydroperoxide-initiated plasma chemiluminescence and thiol content as measures of peripheral oxidative damage in naive and preconditioned rats submitted to forebrain ischemia produced by the 4-vessel occlusion method. Rats were submitted to 2 or 10 min of global transient forebrain ischemia followed by 60 min or 1, 2, 5, 10 or 30 days of reperfusion. Preconditioned rats were submitted to a 10-min ischemic episode 1 day after a 2-min ischemic event (2 + 10 min), followed by 60 min or 1 or 2 days of reperfusion. It has been demonstrated that such preconditioning protects against neuronal death in rats and gerbils submitted to a lethal (10 min) ischemic episode. The results show that both 2 and 10 min of ischemia cause an increase of plasma chemiluminescence when compared to control and sham rats. In the 2-min ischemic group, the effect was not present after reperfusion. In the 10-min ischemic group, the increase was present up to 1 day after recirculation and values returned to control levels after 2 days. However, rats preconditioned to ischemia (2 + 10 min) and reperfusion showed no differences in plasma chemiluminescence when compared to controls. We also analyzed plasma thiol content since it has been described that sulfhydryl (SH) groups significantly contribute to the antioxidant capacity of plasma. There was a significant decrease of plasma thiol content after 2, 10 and 2 + 10 min of ischemia followed by reperfusion when compared to controls. We conclude that ischemia may cause, along with brain oxidative damage and cell death, a peripheral oxidative damage that is reduced by the preconditioning phenomenon.

Monitoring human cytomegalovirus viral load in peripheral blood leukocytes of renal transplant recipients by a simple limiting dilution-PCR assay

To assess the clinical relevance of a semi-quantitative measurement of human cytomegalovirus (HCMV) DNA in renal transplant recipients within the typical clinical context of a developing country where virtually 100% of both receptors and donors are seropositive for this virus, we have undertaken HCMV DNA quantification using a simple, semi-quantitative, limiting dilution polymerase chain reaction (PCR). We evaluated this assay prospectively in 52 renal transplant patients from whom a total of 495 serial blood samples were collected. The samples scored HCMV positive by qualitative PCR had the levels of HCMV DNA determined by end-point dilution-PCR. All patients were HCMV DNA positive during the monitoring period and a diagnosis of symptomatic infection was made for 4 of 52 patients. In symptomatic patients the geometric mean of the highest level of HCMV DNAemia was 152,000 copies per 106 leukocytes, while for the asymptomatic group this value was 12,050. Symptomatic patients showed high, protracted HCMV DNA levels, whereas asymptomatic patients demonstrated intermittent low or moderate levels. Using a cut-off value of 100,000 copies per 106 leukocytes, the limiting dilution assay had sensitivity of 100%, specificity of 92%, a positive predictive value of 43% and a negative predictive value of 100% for HCMV disease. In this patient group, there was universal HCMV infection but relatively infrequent symptomatic HCMV disease. The two patient groups were readily distinguished by monitoring with the limiting dilution assay, an extremely simple technology immediately applicable in any clinical laboratory with PCR capability.

Non-neuronal cells are not the limiting factor for the low axonal regeneration in C57BL/6J mice

Peripheral axonal regeneration was investigated in adult male mice of the C57BL/6J (C), BALB/cJ (B) and A/J (A) strains and in their F1 descendants using a predegenerated nerve transplantation model. Four types of transplants were performed: 1) isotransplants between animals of the C, B and A strains; 2) donors of the C strain and recipients of the C x B and C x A breeding; 3) donors of the B strain and recipients of the C x B breeding, and 4) donors of the A strain and recipients of the C x A breeding. Donors had the left sciatic nerve transected and two weeks later a segment of the distal stump was transplanted into the recipient. Four weeks after transplantation the regenerated nerves were used to determine the total number of regenerated myelinated fibers (TMF), diameter of myelinated fibers (FD) and myelin thickness (MT). The highest TMF values were obtained in the groups where C57BL/6J mice were the donors (C to F1 (C x B) = 4658 ± 304; C to F1 (C x A) = 3899 ± 198). Also, A/J grafts led to a significantly higher TMF (A to F1 (C x A) = 3933 ± 565). Additionally, isotransplant experiments showed that when the nerve is previously degenerated, C57BL/6J mice display the largest number of myelinated fibers (C to C = 3136 ± 287; B to B = 2759 ± 170, and A to A = 2835 ± 239). We also observed that when C57BL/6J was the graft donor, FD was the highest and MT did not differ significantly when compared with the other groups. These morphometric results reinforce the idea that Schwann cells and the nerve environment of C57BL/6J provide enough support to the regenerative process. In this respect, the present results support the hypothesis that the non-neuronal cells, mainly Schwann cells, present in the sciatic nerve of C57BL/6J mice are not the main limiting factor responsible for low axonal regeneration.

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.

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.

The early facilitatory effect of a peripheral spatially noninformative prime stimulus depends on target stimulus features

We investigated the dependency of the early facilitatory effect of a prime stimulus (S1) on the physical characteristics of the target stimulus (S2). A go-no go reaction time paradigm was used. The S1 was a gray ring and the S2s were a white vertical line, a white horizontal line, a white cross and a white small ring, all inside a white ring with the same dimensions as the S1. S1 onset-S2 onset asynchrony was 100 ms. The stimuli appeared randomly in any one of the quadrants of a monitor screen. The S2 could occur at the same position as the S1 or at a different one. We observed a strong facilitatory effect when the vertical line or the horizontal line was the go stimulus and no effect when the cross was the go stimulus. These results show that the features of the target stimulus can be decisive for the appearance of the facilitatory effect of a peripheral spatially noninformative prime stimulus.

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.