Swimming training attenuates oxidative damage and increases enzymatic but not non-enzymatic antioxidant defenses in the rat brain.

Although it is well known that physical training ameliorates brain oxidative function after injuries by enhancing the levels of neurotrophic factors and oxidative status, there is little evidence addressing the influence of exercise training itself on brain oxidative damage and data is conflicting. This study investigated the effect of well-established swimming training protocol on lipid peroxidation and components of antioxidant system in the rat brain. Male Wistar rats were randomized into trained (5 days/week, 8 weeks, 30 min; n=8) and non-trained (n=7) groups. Forty-eight hours after the last session of exercise, animals were euthanized and the brain was collected for oxidative stress analysis. Swimming training decreased thiobarbituric acid reactive substances (TBARS) levels (P<0.05) and increased the activity of the antioxidant enzyme superoxide dismutase (SOD) (P<0.05) with no effect on brain non-enzymatic total antioxidant capacity, estimated by FRAP (ferric-reducing antioxidant power) assay (P>0.05). Moreover, the swimming training promoted metabolic adaptations, such as increased maximal workload capacity (P<0.05) and maintenance of body weight. In this context, the reduced TBARS content and increased SOD antioxidant activity induced by 8 weeks of swimming training are key factors in promoting brain resistance. In conclusion, swimming training attenuated oxidative damage and increased enzymatic antioxidant but not non-enzymatic status in the rat brain.

Swimming training attenuates oxidative damage and increases enzymatic but not non-enzymatic antioxidant defenses in the rat brain

Although it is well known that physical training ameliorates brain oxidative function after injuries by enhancing the levels of neurotrophic factors and oxidative status, there is little evidence addressing the influence of exercise training itself on brain oxidative damage and data is conflicting. This study investigated the effect of well-established swimming training protocol on lipid peroxidation and components of antioxidant system in the rat brain. Male Wistar rats were randomized into trained (5 days/week, 8 weeks, 30 min; n=8) and non-trained (n=7) groups. Forty-eight hours after the last session of exercise, animals were euthanized and the brain was collected for oxidative stress analysis. Swimming training decreased thiobarbituric acid reactive substances (TBARS) levels (P<0.05) and increased the activity of the antioxidant enzyme superoxide dismutase (SOD) (P<0.05) with no effect on brain non-enzymatic total antioxidant capacity, estimated by FRAP (ferric-reducing antioxidant power) assay (P>0.05). Moreover, the swimming training promoted metabolic adaptations, such as increased maximal workload capacity (P<0.05) and maintenance of body weight. In this context, the reduced TBARS content and increased SOD antioxidant activity induced by 8 weeks of swimming training are key factors in promoting brain resistance. In conclusion, swimming training attenuated oxidative damage and increased enzymatic antioxidant but not non-enzymatic status in the rat brain.

Trypan blue exclusion assay by flow cytometry

Dye exclusion tests are used to determine the number of live and dead cells. These assays are based on the principle that intact plasma membranes in live cells exclude specific dyes, whereas dead cells do not. Although widely used, the trypan blue (TB) exclusion assay has limitations. The dye can be incorporated by live cells after a short exposure time, and personal reliability, related to the expertise of the analyst, can affect the results. We propose an alternative assay for evaluating cell viability that combines the TB exclusion test and the high sensitivity of the flow cytometry technique. Previous studies have demonstrated the ability of TB to emit fluorescence when complexed with proteins. According to our results, TB/bovine serum albumin and TB/cytoplasmic protein complexes emit fluorescence at 660 nm, which is detectable by flow cytometry using a 650-nm low-pass band filter. TB at 0.002% (w/v) was defined as the optimum concentration for distinguishing unstained living cells from fluorescent dead cells, and fluorescence emission was stable for 30 min after cell treatment. Although previous studies have shown that TB promotes green fluorescence quenching, TB at 0.002% did not interfere with green fluorescence in human live T-cells stained with anti-CD3/fluorescein isothiocyanate (FITC) monoclonal antibody. We observed a high correlation between the percentage of propidium iodide+CD3/FITC+ and TB+CD3/FITC+ cells, as well as similar double-stained cell profiles in flow cytometry dot-plot graphs. Taken together, the results indicate that a TB exclusion assay by flow cytometry can be employed as an alternative tool for quick and reliable cell viability analysis.

Trypan blue exclusion assay by flow cytometry.

Dye exclusion tests are used to determine the number of live and dead cells. These assays are based on the principle that intact plasma membranes in live cells exclude specific dyes, whereas dead cells do not. Although widely used, the trypan blue (TB) exclusion assay has limitations. The dye can be incorporated by live cells after a short exposure time, and personal reliability, related to the expertise of the analyst, can affect the results. We propose an alternative assay for evaluating cell viability that combines the TB exclusion test and the high sensitivity of the flow cytometry technique. Previous studies have demonstrated the ability of TB to emit fluorescence when complexed with proteins. According to our results, TB/bovine serum albumin and TB/cytoplasmic protein complexes emit fluorescence at 660 nm, which is detectable by flow cytometry using a 650-nm low-pass band filter. TB at 0.002% (w/v) was defined as the optimum concentration for distinguishing unstained living cells from fluorescent dead cells, and fluorescence emission was stable for 30 min after cell treatment. Although previous studies have shown that TB promotes green fluorescence quenching, TB at 0.002% did not interfere with green fluorescence in human live T-cells stained with anti-CD3/fluorescein isothiocyanate (FITC) monoclonal antibody. We observed a high correlation between the percentage of propidium iodide+CD3/FITC+ and TB+CD3/FITC+ cells, as well as similar double-stained cell profiles in flow cytometry dot-plot graphs. Taken together, the results indicate that a TB exclusion assay by flow cytometry can be employed as an alternative tool for quick and reliable cell viability analysis.

Reduced hippocampal GABAergic function in Wistar audiogenic rats

Epilepsy is a neurological disorder associated with excitatory and inhibitory imbalance within the underlying neural network. This study evaluated inhibitory γ-amino-butyric acid (GABA)ergic modulation in the CA1 region of the hippocampus of male Wistar rats and Wistar audiogenic rats (aged 90 ± 3 days), a strain of inbred animals susceptible to audiogenic seizures. Field excitatory postsynaptic potentials and population spike complexes in response to Schaffer collateral fiber stimulation were recorded in hippocampal slices before and during application of picrotoxin (50 µM, 60 min), a GABA A antagonist, and the size of the population spike was quantified by measuring its amplitude and slope. In control audiogenic-resistant Wistar rats (N = 9), picrotoxin significantly increased both the amplitude of the population spike by 51 ± 19 percent and its maximum slope by 73 ± 21 percent. In contrast, in slices from Wistar audiogenic rats (N = 6), picrotoxin caused no statistically significant change in population spike amplitude (33 ± 46 percent) or slope (11 ± 29 percent). Data are reported as means ± SEM. This result indicates a functional reduction of GABAergic neurotransmission in hippocampal slices from Wistar audiogenic rats.

Reduced hippocampal GABAergic function in Wistar audiogenic rats.

Epilepsy is a neurological disorder associated with excitatory and inhibitory imbalance within the underlying neural network. This study evaluated inhibitory γ-amino-butyric acid (GABA)ergic modulation in the CA1 region of the hippocampus of male Wistar rats and Wistar audiogenic rats (aged 90 ± 3 days), a strain of inbred animals susceptible to audiogenic seizures. Field excitatory postsynaptic potentials and population spike complexes in response to Schaffer collateral fiber stimulation were recorded in hippocampal slices before and during application of picrotoxin (50 µM, 60 min), a GABA A antagonist, and the size of the population spike was quantified by measuring its amplitude and slope. In control audiogenic-resistant Wistar rats (N = 9), picrotoxin significantly increased both the amplitude of the population spike by 51 ± 19% and its maximum slope by 73 ± 21%. In contrast, in slices from Wistar audiogenic rats (N = 6), picrotoxin caused no statistically significant change in population spike amplitude (33 ± 46%) or slope (11 ± 29%). Data are reported as means ± SEM. This result indicates a functional reduction of GABAergic neurotransmission in hippocampal slices from Wistar audiogenic rats.

Computerized invasive measurement of time-dependent intraocular pressure.

Several methods have been described to measure intraocular pressure (IOP) in clinical and research situations. However, the measurement of time varying IOP with high accuracy, mainly in situations that alter corneal properties, has not been reported until now. The present report describes a computerized system capable of recording the transitory variability of IOP, which is sufficiently sensitive to reliably measure ocular pulse peak-to-peak values. We also describe its characteristics and discuss its applicability to research and clinical studies. The device consists of a pressure transducer, a signal conditioning unit and an analog-to-digital converter coupled to a video acquisition board. A modified Cairns trabeculectomy was performed in 9 Oryctolagus cuniculus rabbits to obtain changes in IOP decay parameters and to evaluate the utility and sensitivity of the recording system. The device was effective for the study of kinetic parameters of IOP, such as decay pattern and ocular pulse waves due to cardiac and respiratory cycle rhythm. In addition, there was a significant increase of IOP versus time curve derivative when pre- and post-trabeculectomy recordings were compared. The present procedure excludes corneal thickness and error related to individual operator ability. Clinical complications due to saline infusion and pressure overload were not observed during biomicroscopic evaluation. Among the disadvantages of the procedure are the requirement of anesthesia and the use in acute recordings rather than chronic protocols. Finally, the method described may provide a reliable alternative for the study of ocular pressure dynamic alterations in man and may facilitate the investigation of the pathogenesis of glaucoma.

Computerized invasive measurement of time-dependent intraocular pressure

Several methods have been described to measure intraocular pressure (IOP) in clinical and research situations. However, the measurement of time varying IOP with high accuracy, mainly in situations that alter corneal properties, has not been reported until now. The present report describes a computerized system capable of recording the transitory variability of IOP, which is sufficiently sensitive to reliably measure ocular pulse peak-to-peak values. We also describe its characteristics and discuss its applicability to research and clinical studies. The device consists of a pressure transducer, a signal conditioning unit and an analog-to-digital converter coupled to a video acquisition board. A modified Cairns trabeculectomy was performed in 9 Oryctolagus cuniculus rabbits to obtain changes in IOP decay parameters and to evaluate the utility and sensitivity of the recording system. The device was effective for the study of kinetic parameters of IOP, such as decay pattern and ocular pulse waves due to cardiac and respiratory cycle rhythm. In addition, there was a significant increase of IOP versus time curve derivative when pre- and post-trabeculectomy recordings were compared. The present procedure excludes corneal thickness and error related to individual operator ability. Clinical complications due to saline infusion and pressure overload were not observed during biomicroscopic evaluation. Among the disadvantages of the procedure are the requirement of anesthesia and the use in acute recordings rather than chronic protocols. Finally, the method described may provide a reliable alternative for the study of ocular pressure dynamic alterations in man and may facilitate the investigation of the pathogenesis of glaucoma.

Translocation of protein kinase C by halothane in cholinergic cells.

Protein kinase C (PKC) is a signal transducing enzyme that is an important regulator of multiple physiologic processes and a potential molecular target for volatile anaesthetic actions. However, the effects of these agents on PKC activity are not yet fully understood. Volatile anaesthetics increase intracellular calcium concentration ([Ca(2+)](i)) in a variety of cells, thus their effects on PKC activity may be indirect due to [Ca(2+)](i) increase. Alternatively, the anaesthetics could directly stimulate PKC activity. In order to distinguish these two possibilities in intact cells, we used a fully functional green fluorescent protein conjugated PKCbetaII (GFP-PKCbetaII) and confocal microscopy to evaluate the dynamic redistribution of PKC in living SN56 cells, a cholinergic cell line, in response to halothane. Halothane induced PKC translocation in SN56 cells transfected with GFP-PKCbetaII. This effect was not suppressed by dantrolene, a drug that blocks halothane-induced Ca(2+) release from intracellular stores in these cells. These findings indicate that halothane induces PKC translocation in SN56 cells independently of its ability to release calcium from internal stores.

Halothane-induced intracellular calcium release in cholinergic cells.

Low concentrations of halothane and isoflurane can release acetylcholine in an extracellular Ca(2+)-independent manner. In the present study, a cholinergic cell line (SN56) was used to examine whether release of calcium from intracellular stores occurs in the presence of halothane. Changes in intracellular calcium concentration ([Ca(2+)](i)) were measured using fluo-3, a fluorescent calcium-sensitive dye and laser scanning confocal microscopy. Halothane, at sub-anesthetic concentrations (14, 28, 40 and 56 microM), increased [Ca(2+)](i) in SN56 cells. This effect remained even when the cells were perfused with medium lacking extracellular calcium, suggesting the involvement of intracellular Ca(2+) sources. SN56 cells responded to ryanodine by increasing [Ca(2+)](i) and this effect was blocked by dantrolene, an inhibitor of Ca(2+)-release from ryanodine-sensitive stores. The effect of halothane was attenuated after the increase in [Ca(2+)](i) induced by ryanodine and it was suppressed by dantrolene, suggesting the participation of ryanodine-sensitive stores. Using cyclopiazonic acid, a Ca(2+)-ATPase inhibitor, we investigated whether the depletion of intracellular Ca(2+) stores interfered with the effect of halothane. Cyclopiazonic acid significantly decreased the increase in [Ca(2+)](i) induced by the volatile anesthetic. It is suggested that sub-anesthetic concentrations of halothane may increase [Ca(2+)](i) by releasing Ca(2+) from intracellular stores in cholinergic cells.

Effects of a Lasiodora spider venom on Ca2+ and Na+ channels.

The venom of a Brazilian spider, Lasiodora sp (Mygalomorphae, Theraphosidae), was screened for activity against ion channels using Ca2+ imaging and whole-cell patch clamp in GH3 cells. When tetrodotoxin (TTX) was present to block Na+ channels, the venom abolished the Ca2+ oscillations that are normally present in these cells and reduced the basal level of intracellular Ca2+. Under patch clamp, the venom reduced the L-type Ca2+ channel conductance and caused a positive shift in its voltage dependence of activation. In addition to these effects, when applied without TTX, the venom also caused a slow and noisy increase in intracellular Ca2+. The sensitivity of this second effect to TTX suggested an effect on Na+ channels, which was tested using patch clamp. Control Na+ currents inactivated completely as a single exponential. Treatment with the venom did not affect the amplitude of I(Na), but caused it to divide in two slower exponential components plus a sustained component, all of which were suppressed by TTX. The venom also caused a negative shift in the voltage dependence of activation and steady-state inactivation of I(Na). The observed effects of this venom on whole-cell currents explain the changes it causes in intracellular Ca2+ in GH3 cells and demonstrate that the venom of this spider is a source of toxins active against ion channels.

Calcium channels coupled to depolarization-evoked glutamate release in the myenteric plexus of guinea-pig ileum.

Glutamate is the major excitatory neurotransmitter in the CNS. The recent characterization of glutamate as a neurotransmitter in the enteric nervous system opened a new line of investigation concerning the role of glutamate in that system. The present study aimed to further characterize the enteric glutamate release and the calcium channels coupled to it. For this study the myenteric plexus-longitudinal muscle of guinea-pig ileum was stimulated with potassium chloride or with electrical pulses. The released glutamate was detected by spectrofluorimetry. Laser scanning confocal microscopy was used for analysis of immunolabeled enteric tissue for co-localization studies of calcium channels (N- and P/Q-type) and glutamate transporters (EAAC1). Here we report the effects of known Ca(2+)-channel blockers on glutamate release evoked by KCl-depolarization or electrical stimulation in the myenteric plexus. We find that N-type Ca(2+) channels control a major portion of evoked glutamate release from this system, with a very small contribution from L-type Ca(2+) channels. Moreover, alpha(1A)-like (P-type Ca(2+) channel) and alpha(1B)-like (N-type Ca(2+ )channel) immunoreactivity co-localized with glutamate transporters in the myenteric plexus. In addition, KCl-evoked or electrically stimulated glutamate release was sensitive to omega-agatoxin IVA, in a frequency-dependent manner, suggesting that P-type channels are also coupled to the release of glutamate. We, thus, conclude that both N-type and P-type Ca(2+) channels control most of the evoked glutamate release from the enteric nervous system, as also occurs in some parts of the CNS.

Expression of the vesicular acetylcholine transporter, proteins involved in exocytosis, and functional calcium signaling in varicosities and soma of a murine septal cell line.

The expression and localization of the vesicular acetylcholine transporter in a septal cell line, SN56, were investigated. Immunoprecipitation and immunoblot analysis of postnuclear supernatants indicated that this cell line expresses reasonable amounts of the transporter. Immunofluorescence and confocal microscopy experiments showed that the vesicular transporter is present in varicosities and also in the cell body of differentiated cells. Varicosities have the potential to be functional sites of transmitter release because they responded to depolarization with calcium influx through voltage-gated calcium channels and expressed the synaptic proteins synaptotagmin, SV2, synaptophysin, and a subunit of P/Q calcium channels. In the soma of SN56 cells, the transporter immunoreactivity was similar to that for synaptotagmin, and it colocalized with synaptophysin, but it did not colocalize with SV2. Labeling for SV2 appeared prominently in a defined perinuclear structure, whereas the two former proteins were widely distributed in the soma, where several endocytic compartments could be identified with the vital dye FM4-64. These data suggest that distinct synaptic vesicle proteins exist in different subcellular compartments, and consequently they may follow distinct pathways in neurites before reaching sites of transmitter storage and release in SN56 cells.

Alpha- and beta-scorpion toxins evoke glutamate release from rat cortical synaptosomes with different effects on [Na+]i and [Ca2+]i.

Scorpion toxins have long been used as tools in the investigation of neurotransmitter release mechanisms. We have used rat cortical synaptosomes to study the effects of a beta-type scorpion toxin (TiTX-gamma) on the release of glutamate and on the concentrations of free sodium and calcium ions inside the synaptosomes. The effects are compared with those of an alpha-type scorpion toxin (TsTX), on which there have been more studies. TsTX increased overall internal sodium and calcium ion concentrations and glutamate release in an incremental, dose dependent manner. TiTX-gamma similarly evoked glutamate release in an incremental, dose dependent manner. However, TiTX-gamma caused little increase in the overall internal sodium and calcium ion concentrations at low doses that evoked a significant release of glutamate and a maximal increase in these ions at somewhat higher doses. The results suggest that TiTX-gamma preferentially binds sodium channels close to the active zones for glutamate release and indicates that modifications of the activation or inactivation of the Na+-channel can lead to very different changes in the cytosolic concentrations of free Na+and Ca2+, with consequences for neurotransmission. This provides an interesting perspective concerning modulation of neurotransmitter release via pharmacological manipulation of Na+-channel properties, that may lead to a better comprehension of its physiological and pathological roles.