Experimental unilateral intracerebral hemorrhage induces delayed bilateral neurodegeneration of sciatic nerve fibres in rats.

Pathological processes, such as inflammatory effects, oxidative stress, apoptosis and cytotoxicity of blood after an intracerebral hemorrhage (ICH), generally contribute to a secondary injury. One of the secondary ICH consequences in the nervous system may be delayed neurodegeneration of the peripheral nerves. Therefore, the aim of our study was to investigate possible structural changes in the sciatic nerve and changes in the conduction velocity via this nerve at different terms after experimental ICH in male Wistar rats. Intracerebral hemorrhage was provided by direct injection of autologous blood into the capsula interna. On the 10th day after ICH mean conduction velocity in sciatic nerve was 15% smaller compared to the control. On the 30th and 90th days after ICH, highly significant decreases in the conduction velocity by 62% and 60%, respectively in comparison with the control group of animals were observed. The data of morphometric analysis demonstrated significant decreases in the mean diameter and density of myelinated fibres at all examined terms after ICH. A number of the myelin sheaths were swollen and lost their regular laminations. Axoplasmic and myelin degenerations were the most frequent events in these nerve fibres; reductions of the diameter of the axis cylinders were also observed. In the contralateral nerve (related to the hemisphere with ICH), negative changes were greater, while the ipsilateral nerve was also subjected to those. Our data demonstrate that the consequences of unilateral ICH in the capsula interna induce bilateral negative changes in the peripheral nervous system of rats.

C60 Fullerene Reduces 3-Nitropropionic Acid-Induced Oxidative Stress Disorders and Mitochondrial Dysfunction in Rats by Modulation of p53, Bcl-2 and Nrf2 Targeted Proteins.

C60 fullerene as a potent free radical scavenger and antioxidant could be a beneficial means for neurodegenerative disease prevention or cure. The aim of the study was to define the effects of C60 administration on mitochondrial dysfunction and oxidative stress disorders in a 3-nitropropionic acid (3-NPA)-induced rat model of Huntington's disease. Animals received 3-NPA (30 mg/kg i.p.) once a day for 3 consecutive days. C60 was applied at a dose of 0.5 mg/kg of body weight, i.p. daily over 5 days before (C60 pre-treatment) and after 3-NPA exposure (C60 post-treatment). Oxidative stress biomarkers, the activity of respiratory chain enzymes, the level of antioxidant defense, and pro- and antiapoptotic markers were analyzed in the brain and skeletal muscle mitochondria. The nuclear and cytosol Nrf2 protein expression, protein level of MnSOD, γ-glutamate-cysteine ligase (γ-GCLC), and glutathione-S-transferase (GSTP) as Nrf2 targets were evaluated. Our results indicated that C60 can prevent 3-NPA-induced mitochondrial dysfunction through the restoring of mitochondrial complexes' enzyme activity, ROS scavenging, modulating of pro/antioxidant balance and GSH/GSSG ratio, as well as inhibition of mitochondria-dependent apoptosis through the limitation of p53 mitochondrial translocation and increase in Bcl-2 protein expression. C60 improved mitochondrial protection by strengthening the endogenous glutathione system via glutathione biosynthesis by up-regulating Nrf2 nuclear accumulation as well as GCLC and GSTP protein level.

Fatigue-induced Fos immunoreactivity within the lumbar cord and amygdala decreases after С60 fullerene pretreatment.

The fundamental aspects related to the mechanisms of action of C60 fullerene nanoparticles on the level of the central nervous system in different experimental conditions are still unclear. Electrophysiological investigation and immunohistochemical techniques of c-fos expression were combined to determine which neural elements within the lumbar segments and in the central nucleus of the amygdala (CeA) are activated under skeletal muscle fatigue development with prior application of C60 fullerenes (dissolved in dimethyl sulfoxide and in distilled water, FDS). After high-frequency electrical stimulation of the triceps surae muscle, the main fatigue-related increases in the c-Fos expression level were registered ipsilaterally within lamina 1 and 5 of the lumbar segments and within the contralateral capsular part of the CeA. C60 fullerene pretreatment in animals with subsequent electrical stimulation induced a distinct (2-4 times) decrease in the level of Fos immunoreactivity in the observed structures in comparison with only fatigue-induced rats. It can be supposed that FDS, as antioxidant compound, can decrease the concentration of free radicals in fatigued tissue and reduce the transmission intensity of nociceptive information from muscles to the spinal cord and amygdala, thereby changing the level of c-Fos expression within the lumbar segments and CeA.

C60 fullerenes increase the intensity of rotational movements in non­anesthetized hemiparkinsonic rats.

The effect of C60 fullerene aqueous colloid solution (C60FAS) on the intensity of long­lasting (persisting for one hour) rotational movements in non­anesthetized rats was investigated. For this purpose, an experimental hemiparkinsonic animal model was used in the study. Rotational movements in hemiparkinsonic animals were initiated by the intraperitoneal administration of the dopamine receptor agonist apomorphine. It was shown that a preliminary injection of C60FAS (a substance with powerful antioxidant properties) in hemiparkinsonic rats induced distinct changes in animal motor behavior. It was revealed that fullerene­pretreated animals, in comparison with non­pretreated or vehicle­pretreated rats, rotated for 1 h at an approximately identical speed until the end of the experiment, whereas the rotation speed of control rats gradually decreased to 20-30% of the initial value. One can assume that the observed changes in the movement dynamics of the hemiparkinsonic rats after C60FAS pretreatment presumably can be induced by the influence of C60FAS on the dopaminergic system, although the isolated potentiation of the action of apomorphine C60FAS cannot be excluded. Nevertheless, earlier data on the action of C60FAS on muscle dynamics has suggested that C60FAS can activate a protective action of the antioxidant system in response to long­lasting muscular activity and that the antioxidant system in turn may directly decrease fatigue­relate d changes during long­lasting muscular activity.

C60 Fullerene Prevents Restraint Stress-Induced Oxidative Disorders in Rat Tissues: Possible Involvement of the Nrf2/ARE-Antioxidant Pathway.

The effects of C60FAS (50 and 500 µg/kg) supplementation, in a normal physiological state and after restraint stress exposure, on prooxidant/antioxidant balance in rat tissues were explored and compared with the effects of the known exogenous antioxidant N-acetylcysteine. Oxidative stress biomarkers (ROS, O2·-, H2O2, and lipid peroxidation) and indices of antioxidant status (MnSOD, catalase, GPx, GST, γ-GCL, GR activities, and GSH level) were measured in the brain and the heart. In addition, protein expression of Nrf2 in the nuclear and cytosol fractions as well as the protein level of antiradical enzyme MnSOD and GSH-related enzymes γ-GCLC, GPx, and GSTP as downstream targets of Nrf2 was evaluated by western blot analysis. Under a stress condition, C60FAS attenuates ROS generation and O2·- and H2O2 releases and thus decreases lipid peroxidation as well as increases rat tissue antioxidant capacity. We have shown that C60FAS supplementation has dose-dependent and tissue-specific effects. C60FAS strengthened the antiradical defense through the upregulation of MnSOD in brain cells and maintained MnSOD protein content at the control level in the myocardium. Moreover, C60FAS enhanced the GSH level and the activity/protein expression of GSH-related enzymes. Correlation of these changes with Nrf2 protein content suggests that under stress exposure, along with other mechanisms, the Nrf2/ARE-antioxidant pathway may be involved in regulation of glutathione homeostasis. In our study, in an in vivo model, when C60FAS (50 and 500 µg/kg) was applied alone, no significant changes in Nrf2 protein expression as well as in activity/protein levels of MnSOD and GSH-related enzymes in both tissues types were observed. All these facts allow us to assume that in the in vivo model, C60FAS affects on the brain and heart endogenous antioxidative statuses only during the oxidative stress condition.

Fos immunoreactivity in the intermediolateral nucleus induced by tendon vibration of the m. triceps surae in rats pretreated with a nitric oxide blocker or precursor.

We investigated neuronal activation of the rat intermediolateral (IML) nucleus of the thoracolumbar spinal cord, initiated by Achilles tendon vibration, after intramuscular (m. triceps surae) administration of 7­nitroindazole (7­NI) or L­arginine (LA). The spindle afferent response to vibratory stimuli induced a distinct bilateral increase in the activation of c­Fos immunoreactivity in the spinal neurons in three groups of rats (tendon­vibrated, tendon­vibrated + 7­NI and tendon­vibrated + LA). The T5/T13 segments in tendon­vibrated +7­NI animals showed the highest increase of Fos­immunoreactive neurons. This increase was two times higher than that in tendon only­vibrated control rats and endon­vibrated + LA animals. The highest mean number of labelled neurons were observed in the IML nucleus and in layers 4 and 7 of the T5-L3 segments in tendon­vibrated and tendon­vibrated + 7­NI animals, and in the IML nucleus and layer 4 in tendon­vibrated + LA rats. The highest mean number of activated neurons was found ipsilaterally in the IML nucleus of the T5/T13 segment. These results indicate that decreased nitric oxide release after injection of 7­NI was accompanied by a potentiation of the early c­fos gene expression induced by muscle proprioceptive activity within the thoracolumbar region of the rat spinal cord. Thus, enhanced c­Fos immunoreactivity in the IML nucleus indicated that the sympathetic nervous system can exert a direct influence on the muscle spindles.

C60 Fullerenes Diminish Muscle Fatigue in Rats Comparable to N-acetylcysteine or ß-Alanine.

The aim of this study is to detect the effects of C60 fullerenes, which possess pronounced antioxidant properties, in comparison with the actions of the known exogenous antioxidants N-acetylcysteine (NAC) and ß-Alanine in terms of exercise tolerance and contractile property changes of the m. triceps surae (TS) during development of the muscle fatigue in rats. The electrical stimulation of the TS muscle during four 30 min series in control rats led to total reduction of the muscle contraction force. Furthermore, the effects of prior intraperitoneal (i.p.) or oral C60FAS application and preliminary i.p. injection of NAC or ß-Alanine on muscle contraction force under fatigue development conditions is studied. In contrast to control rats, animals with C60FAS, NAC, or ß-Alanine administration could maintain a constant level of muscle effort over five stimulation series. The accumulation of secondary products and changes in antioxidant levels in the muscle tissues were also determined after the fatigue tests. The increased levels of lactic acid, thiobarbituric acid reactive substances and H2O2 after stimulation were statistically significant with respect to intact muscles. In the working muscle, there was a significant (p < 0.05) increase in the activity of endogenous antioxidants: reduced glutathione, catalase, glutathione peroxidase, and superoxide dismutase. Treated animal groups showed a decrease in endogenous antioxidant activity relative to the fatigue-induced animals (P < 0.05). Oral C60FAS administration clearly demonstrated an action on skeletal muscle fatigue development similar to the effects of i.p. injections of the exogenous antioxidants NAC or ß-Alanine. This creates opportunities to oral use of C60FAS as a potential therapeutic agent. Due to the membranotropic activity of C60 fullerenes, non-toxic C60FAS has a more pronounced effect on the prooxidant-antioxidant homeostasis of muscle tissues in rats.

C60 fullerene as promising therapeutic agent for correcting and preventing skeletal muscle fatigue.

BACKGROUND: Bioactive soluble carbon nanostructures, such as the C60 fullerene can bond with up to six electrons, thus serving by a powerful scavenger of reactive oxygen species similarly to many natural antioxidants, widely used to decrease the muscle fatigue effects. The aim of the study is to define action of the pristine C60 fullerene aqueous colloid solution (C60FAS), on the post-fatigue recovering of m. triceps surae in anaesthetized rats. RESULTS: During fatigue development, we observed decrease in the muscle effort level before C60FAS administration. After the application of C60FAS, a slower effort decrease, followed by the prolonged retention of a certain level, was recorded. An analysis of the metabolic process changes accompanying muscle fatigue showed an increase in the oxidative stress markers H 2 O 2 (hydrogen peroxide) and TBARS (thiobarbituric acid reactive substances) in relation to the intact muscles. After C60FAS administration, the TBARS content and H 2 O 2 level were decreased. The endogenous antioxidant system demonstrated a similar effect because the GSH (reduced glutathione) in the muscles and the CAT (catalase) enzyme activity were increased during fatigue. CONCLUSIONS: C60FAS leads to reduction in the recovery time of the muscle contraction force and to increase in the time of active muscle functioning before appearance of steady fatigue effects. Therefore, it is possible that C60FAS affects the prooxidant-antioxidant muscle tissue homeostasis, subsequently increasing muscle endurance.

NADPH-diaphorase reactivity and Fos-immunoreactivity within the ventral horn of the lumbar spinal cord of cats submitted to acute muscle inflammation induced by injection of carrageenan.

The NADPH-diaphorase activity and Fos-immunoreactivity within the ventral horn of the lumbar spinal cord were studied in cats with acute unilateral myositis following injection of carrageenan into the m.m. gastrocnemius-soleus. In carrageenan-injected cats maximum in the mean number of intensely stained NADPH-diaphorase reactive (NADPH-dr) neurons was found in lamina VII (+100%) and VIII (+33%) of the contralateral ventral horn of the L6/L7 segments as compared with control animals. The maximumal level of Fos-immunoreactivity was registered in the same laminae with ipsilateral predominance (39.3±4.6 and 7.6±0.9 cells), in comparison with the contralateral side (13.6±0.8 and 5.5±0.6 cells, respectively; P<0.05). We also visualized low-intensely stained and double labelled (Fos immunoreactive+low-intensely stained NADPH-dr) multipolar and fusiform Renshaw-like cells (RLCs) within the ventral horn on both sides of the L6/L7 segments in carrageenan-injected cats. We visualized the double labelled (Fos-ir+NADPH-dr) multipolar and fusiform Renshaw-like cells (RLCs) within the ventral horn on both sides of the L6/L7 segments in carrageenan-injected cats. A significant difference in the mean number of RLCs was recorded between the ipsi- and contralateral sides in the lamina VII (13.6±2.5 vs. 4.9±0.7 cells, respectively). We suppose that activation of inhibitory RLCs in ipsilateral lamina VII could be directed on attenuation of activation of motoneurons during muscle pain development. Our study showed that a significant contralateral increase in the number of NADPH-dr cells is accompanied by an ipsilateral increase in c-Fos expression in lamina VII. These data may suggest that NADPH-dr neurons of the contralateral ventral horn through commissural connections also involved in the maintenance of the neuronal activity associated with acute muscle inflammation. It is also hypothesized, that during acute myositis, plastic changes in the ventral horn activate the processes of disinhibition due to an increase in the number of NADPH-d-reactive neurons in the spinal gray matter.

7-Nitroindazole enhances c-Fos expression in spinal neurons in rats realizing operant movements.

The expression of c-Fos and NADPH-diaphorase reactivity (NADPH-dr) in the cervical spinal cord was studied in adult male Wistar rats that realized operant reflexes after inhibition of neuronal nitric oxide synthase. Fos-immunoreactive neurons were visualized immunohistochemically in the C6/C7 spinal segments in the control, realized operant movements animals, and/or 7-nitroindazole (7-NI) injected rats. The mean numbers of immunoreactive interneurons and motoneurons (per section) were significantly greater in the Nucleus proprius (+240%) and motor nuclei (+600%) in rats of the 7-NI-pretreated and operant reflex realized group than in the isolated operant reflex realized group. Our study showed intensive staining of NADPH-dr axon terminals on the somata and initial parts of dendrites of motoneurons in experimental rats when the disodium salt of malic acid was added to the staining solution. Suppression of NO release is associated with potentiation of neuronal activation induced by descending supraspinal and proprioceptive signaling within the spinal cord.

Dynorphin B induces lateral asymmetric changes in feline spinal cord reflexes.

The effects of dynorphin B (an agonist of κ-opioid receptors) and naloxone (an antagonist of opioid receptors) on the field potentials (FPs) evoked in the lumbar spinal cord of spinalized cats were examined following successive stimulation of pairs of identical peripheral nerves on both sides of the body. The FPs were recorded bilaterally using microelectrodes from symmetrical sites of the gray matter between the L6 and L7 segments of the spinal cord transected at level of Th11. Significant changes (up to 75%) were registered in the areas of the initial positive components of the FPs evoked by sequential stimulation of the nn. gastrocnemius-soleus, flexor digitorum longus, and tibialis at both hind limbs; a difference between the effects of various nerves was not observed. Two-Way ANOVA analysis showed that two factors, the injection type and recording side, as well as a combination of these factors, strongly influenced the amplitudes of the FPs. Statistically significant side- and injection-dependent differences were registered in the majority of the tests. Both the directions of the changes in the FPs and their relative amplitudes were not strongly connected with a definite side of the spinal cord in different animals. Therefore, it is possible to postulate that the κ-opioid receptors are distributed inhomogeneously over the neuronal populations transmitting the peripheral afferent signals from different hind limbs, thus indicating a possible presence of the lateral asymmetry effects.