Genotoxicity of intraperitoneal injection of lipoamphiphile CdSe/ZnS quantum dots in rats.

The main objective of the present in vivo rat study was to determine the genotoxicity of lipoamphiphile-coated CdSe/ZnS Quantum Dots (QDs), in several organs (brain, liver, kidneys, lungs and testicles). The second objective was to establish the correlations between the QDs genotoxic activity and the oxidative stress, the production of a proinflammatory cytokine (TNF-α), a stress-induced chaperone protein, the phosphorylated heat shock protein 70 (pHsp70), and an increase in the caspase-3 apoptosis factor. Four QDs doses were injected into the peritoneal cavity (5, 5×10(-1), 5×10(-2) and 5×10(-3)µg/kg). DNA lesions in the different organs were measured by the comet assay, and chromosome abnormalities were evaluated by the micronucleus assay on blood reticulocytes (MNRET). Twenty-four hours after the QDs injection, genotoxic effects were observed in the brain and liver and, only for the highest QDs concentration, in testicles. No genotoxic effect was seen in the kidney and lung. The MNRET test revealed a dose-response induction of micronuclei. In parallel, we did neither reveal oxidative stress nor significant variations of TNF-α, pHsp70, and caspase-3. In conclusion, the QDs exerted significant genotoxic effects in the brain and liver, even in the absence of any associated oxidative stress and inflammatory processes.

The mechanisms of the widespread production of phosphorylated HSP25 after fatiguing muscle stimulation.

We previously showed that a widespread heat shock protein (HSP) response to fatigue of a single hindlimb muscle was responsible for a global adaptive response to an acute localized stress. We also demonstrated that the HSP response resulted from the activation of nerve afferents from the stimulated muscle. However, we did not examine the role played by the different muscle afferents or the efferent arm of HSP response. In the present study we measured the changes in phosphorylated HSP25 (pHSP25) levels in resting hindlimb muscles and the diaphragm, kidney and brain in response to a fatiguing stimulation of one tibialis anterior muscle that was repeated in five series of experiments: (1) intact muscle innervation, (2) during the selective procaine block of conduction in group IV muscle afferents, (3) after muscle nerve transection to suppress all the sensory messages, and under pharmacological blockade of the (4) alpha-adrenergic or (5) glutamatergic neurotransmission. The data showed that: (1) the pHSP25 response in hindlimb muscles resulted from the stimulation of both group III and IV muscle afferents while the pHSP25 response in the diaphragm, kidney and brain resulted from the sole activation of the group IV fibres, and (2) the blockade of alpha-adrenergic, but not glutamatergic, neurotransmission suppressed the pHSP25 response in all explored tissues except the brain. The present study highlights the role played by the group III and IV muscle afferents in the fatigue-induced pHSP25 response and shows that the sympathetic nerve supply to the muscles and kidney represents the efferent arm of the pHSP25 activation. However, the pHSP25 changes in the brain cannot be explained by the pathways investigated here.

Changes in stationary upright standing and proprioceptive reflex control of foot muscles after fatiguing static foot inversion.

We searched for the consequences of a maximal static foot inversion sustained until exhaustion on the post-exercise stationary upright standing and the proprioceptive control of the foot muscles. Twelve healthy subjects executed an unilateral maximal static foot inversion during which continuous power spectrum analyses of surface electromyograms of the tibialis anterior (TA), peroneus longus (PL), and gastrocnemius medialis (GM) muscles were performed. Superimposed pulse trains (twitch interpolation) were delivered to the TA muscle to identify "central" or "peripheral" fatigue. Before and after the fatiguing task, we measured (1) the repartition of the plantar and barycentre surfaces with a computerized stationary platform, (2) the peak contractile TA response to electrical stimulation (TA twitch), (3) the tonic vibratory response (TVR) of TA and GM muscles, and (4) the Hoffman reflex. During static exercise, "central" fatigue was diagnosed in 5/12 subjects whereas in the 7 others "peripheral" TA fatigue was deduced from the absence of response to twitch interpolation and the post-exercise decrease in twitch amplitude. The sustained foot inversion was associated with reduced median frequency in TA but not in PL and GM muscles. After static exercise, in all subjects both the mean plantar and rearfoot surfaces increased, indicating a foot eversion, the TVR amplitude decreased in TA but did not vary in GM, and the Hoffman reflex remained unchanged. Whatever was the mechanism of fatigue during the maximal foot inversion task, the facilitating myotatic reflex was constantly altered in foot invertor muscles. This could explain the prevailing action of the antagonistic evertor muscles.

Decreased foot inversion force and increased plantar surface after maximal incremental running exercise.

Formulating the hypothesis that a maximal running exercise could induce fatigue of some foot muscles, we searched for electromyographic (EMG) signs of fatigue in the tibialis anterior (TA), peroneus longus (PL), and gastrocnemius medialis (GM) muscles. We also searched for post-exercise alterations of the stationary upright standing in normal-arched feet subjects. Healthy subjects performed a maximal running exercise. Surface EMGs of the TA, PL, and GM muscles were analysed during maximal dynamic efforts. Before and after the running bout, we measured the evoked compound muscle potential (M-wave) in TA, the maximal force into inversion (MIF), and the repartition of the plantar and barycentre surfaces with a computerised stationary platform. During maximal running exercise, the median frequency of the EMG spectra declined in TA while it remained stable in the PL and GM muscles. After the exercise, MIF decreased, and both the rearfoot plantar surface and the barycentre surface increased. We concluded that a maximal running bout elicits EMG signs of fatigue, though only in the TA muscle. It also elicits post-exercise changes in the foot position during stationary upright standing which indicates a foot eversion. These data solely concern a maximal running test and they can not be extrapolated to walking or running at a low speed.

Fatiguing stimulation of one skeletal muscle triggers heat shock protein activation in several rat organs: the role of muscle innervation.

We hypothesised that activation of muscle afferents by fatigue triggers a widespread activation of heat shock proteins (HSPs) in resting muscles and different organs. In anaesthetised rats, HSP25 and HSP70 levels were determined in both tibialis anterior (TA) and extensor digitorum longus (EDL) muscles and in the diaphragm, kidney and brain by ELISA, which mostly identifies phosphorylated HSP, and western blotting. One TA muscle was electrically stimulated and tissues were sampled 10 or 60 min after the stimulation had ended. The nerve supply to the stimulated TA or its counterpart in the contralateral limb was left intact or suppressed. In control rats, no muscle stimulation was performed and tissues were sampled at the same time points (10 or 60 min). After TA stimulation, ELISA showed an increased HSP25 content in the contralateral TA, EDL and diaphragm at 10 min but not at 60 min, and HSP70 increased in all sampled tissues at 60 min. Western blotting did not show any changes in HSP25 and HSP70 at 10 min, while at 60 min HSP25 increased in all sampled tissues except the brain and HSP70 was elevated in all tissues. Denervation of the contralateral non-stimulated limb suppressed HSP changes in TA and after denervation of the stimulated TA the widespread activation of HSPs in other organs was absent. Our data suggest that fatigue-induced activation of skeletal muscle afferents triggers an early increase in phosphorylated HSP25 in muscles and a delayed elevation of non-phosphorylated HSP25 and HSP70 in skeletal and respiratory muscles, kidney and brain.

Combination of two oxidant stressors suppresses the oxidative stress and enhances the heat shock protein 27 response in healthy humans.

We tested the hypothesis that the combination of 2 oxidant stressors (hyperoxia and fatiguing exercise) might reduce or suppress the oxidative stress. We concomitantly measured the plasma concentration of heat shock proteins (Hsp) that protect the cells against the deleterious effects of reactive oxygen species. Healthy humans breathed pure oxygen under normobaric condition for 50-minute periods during which they stayed at rest or executed maximal static handgrip sustained until exhaustion. They also repeated handgrip bouts in normoxic condition. We performed venous blood measurements of 2 markers of the oxidative stress (thiobarbituric acid reactive substances and reduced ascorbic acid) and Hsp27. Under normoxic condition, the handgrip elicited an oxidative stress and a modest increase in plasma Hsp27 level (+7.1 +/- 5.4 ng/mL). Under hyperoxic condition, (1) at rest, compared with the same time schedule in normoxic condition, we measured an oxidative stress (increased thiobarbituric acid reactive substances and decreased reduced ascorbic acid levels) and the plasma Hsp27 level increased (maximal variation, +12.5 +/- 6.0 ng/mL); and (2) after the handgrip, the oxidative stress rapidly disappeared. The combination of both hyperoxia and handgrip bout doubled the Hsp27 response (maximal variation, +24.8 +/- 9.2 ng/mL). Thus, the combination of 2 hits eliciting an oxidative stress seems to induce an adaptive Hsp27 response that might counterbalance an excessive production of reactive oxygen species.

The changes in neuromuscular excitability with normobaric hyperoxia in humans.

Based on previous observations in hyperbaric hyperoxia, we hypothesized that normobaric hyperoxia, often used during general anaesthesia and resuscitation, might also induce a neuromuscular excitability. In healthy volunteers, we studied the consequences of a 50 min period of pure oxygen breathing on the neuromuscular conduction time (CT), the amplitude of the compound evoked muscle potential (M-wave), the latency and amplitude of the Hoffman reflex (H reflex) and the electromyographic tonic vibratory response (TVR) of the flexor digitorum superficialis muscle to explore the proprioceptive reflex loop. Hyperoxia-induced oxidative stress was measured by the changes in blood markers of lipid peroxidation (thiobarbituric acid reactive substances, TBARS) and antioxidant response (reduced ascorbic acid, RAA). During hyperoxia, the M-wave amplitude increased, both CT and H reflex latency were shortened, and the H reflex amplitude increased. By contrast, TVR significantly decreased. Concomitantly, an oxidative stress was assessed by increased TBARS and decreased RAA levels. This study shows the existence of dual effects of hyperoxia, which facilitates the muscle membrane excitability, nerve conduction and spinal reflexes, but reduces the gain of the proprioceptive reflex loop. The activation of the group IV muscle afferents by hyperoxia and the resulting oxidative stress might explain the TVR depression.

Reactive oxygen species activate the group IV muscle afferents in resting and exercising muscle in rats.

We tested the hypothesis that the reactive oxygen species (ROS) produced at rest and mostly during muscle contraction may stimulate the group IV muscle afferents. In rats, afferent activity was recorded in the peroneal nerve innervating the tibialis anterior muscle. Group IV afferents were identified from measurements of their conduction velocity and response to lactic acid. Comparing the group IV response to an intramuscular injection of buffered isotonic NaCl solution, we searched for the effects of a ROS donor (H2O2) or a ROS inhibitor (superoxide dismutase, SOD) on the baseline afferent activity in resting muscles. We also explored the consequences of a pre-treatment with SOD on the afferent nerve response to H2O2 injection or electrical muscle stimulation (MS). In other animals, we measured the changes in intramuscular level of a marker of oxidative stress (isoprostanes) after each test agent. H2O2 injection markedly activated all recorded group IV afferents. SOD injection lowered the baseline activity of 50 out of 70 afferent units, suppressed the afferent response to H2O2 injection, and delayed and reduced the MS-induced activation of all recorded units. Intramuscular isoprostanes level significantly increased after H2O2 injection or MS, the oxidative stress being absent in muscles pre-treated with SOD. We concluded that ROS influence both the spontaneous and contraction-induced activities of the group IV muscle afferents and are a potent stimulus of muscle metaboreceptors.

Reactive oxygen species and inflammatory mediators enhance muscle spindles mechanosensitivity in rats.

We tested the hypothesis that reactive oxygen species (ROS) and inflammatory mediators affect transduction properties of muscle spindles. In rats, muscle spindles response to high-frequency vibration (HFV) was recorded before and after (1) injection of hydrogen peroxide (H2O2) in control rats and animals pre-treated with diclofenac (anti-inflammatory substance), (2) injection of bradykinin and (3) fatigue induced by muscle stimulation (MS) in control rats and rats receiving diclofenac, superoxide dismutase (SOD) or H2O2. Muscular oxidative stress and inflammation induced by H2O2 or MS were assessed by measurements of isoprostanes and IL-6 levels. In control rats, H2O2, bradykinin and MS significantly enhanced the HFV response. Pre-treatment with SOD abolished the post-MS-enhanced HFV response whereas diclofenac lowered the peak HFV response to MS and H2O2. H2O2 injection and MS elicited significant and similar increases in isoprostanes and IL-6. We report a direct modulation of muscle spindles mechanosensitivity by ROS and inflammatory mediators.

Fatigue-induced changes in tonic vibration response (TVR) in humans: relationships between electromyographic and biochemical events.

Fatigue-induced changes in the proprioceptive reflex loop were explored in humans by using the tonic electromyographic (EMG) response to vibration (TVR) and relating it to lactic acidosis (LA) and oxidative stress. TVR was measured in flexor digitorum superficialis before and after sustained or intermittent handgrip at maximal voluntary contraction (MVC). TVR variations were compared with the changes in EMG power spectrum preceding contractile fatigue, the Hoffman reflex (H-reflex), and plasma concentrations of LA and thiobarbituric acid reactive substances (TBARS). After both sustained and intermittent handgrips, TVR amplitude first declined then increased, independently from the changes in EMG power spectrum and H-reflex. TVR depression and facilitation were respectively concomitant with increases in LA and TBARS. The TVR depression was proportional to the increased LA level. The origin of TVR changes after muscle fatigue is questioned because the relationship between TVR depression and LA accumulation might be temporal, not causal, and changes in muscle stiffness were not explored.