The beam walking test revealed pronounced behavioral symptoms in the rats that had been exposed to 6-OHDA (F (3.36) = 10.54; p = 0.001). All rodents from the control group successfully got to the end of the walking beam; the time it took them to cover the whole distance was 13.88 ± 1.73 seconds on average. However, only 3 of 11 animals from the 6-OHDA+NaCl group were able to reach the safety platform. Other rats demonstrated freezing behavior and could not finish the test. Therefore, we used an additional scale to assess the neurotic-like behavior of the animals (tab. 2). The rats that had received carnosine or LA did not have pronounced neurological symptoms. The post hoc analysis revealed no significant differences between any of these groups and the controls. At the same time, the differences between these 2 groups and the 6-OHDA+NaCl group were significant (tab. 2).
No significant changes in horizontal activity were observed between the experimental groups in the OF test (F (3.36) = 1.83; p = 0.16). However, paired sample comparison demonstrated significant differences between the control and the 6-OHDA+NaCl groups (7.97 ± 0.42 and 4.21 ± 0.77, respectively; p = 0.001). ANOVA suggested a change in vertical activity (F (3.36) = 3.04; p = 0.04). The post hoc analysis revealed that vertical activity was low only in the 6-OHDA+ NaCl group, in contrast to the animals who had been treated with carnosine or LA (tab. 2).

Quantification of dopaminergic neurons

The number of dopaminergic neurons in SNpc was inferred from the immunohistochemical localization of TH, the marker of dopamine neurons. Staining for TH was indicative of neuronal loss in the midbrain sections of all animals injected with 6-OHDA (F (3.10) = 6.33; p = 0.01) (fig. 1); it also showed that AO had produced no therapeutic effect (F (2.8) = 0.51; p = 0.61). For example, the examined regions of SNpc of the control animals contained an average of 166 ± 48 neurons, whereas all rats exposed to 6-OHDA had only 4 ± 2 neurons (p = 0.01). These findings confirm the validity of our experimental model.

Concentrations of amine neurotransmitters and their metabolites

ANOVA results suggested that the levels of major amine neurotransmitters and their metabolites were changed in the right striatum only, where the neurotoxin had been injected (F (21.81) = 3.29; p = 0.001), in comparison with the left brain hemisphere (F (21.69) = 1.15; p = 0.32). However, paired comparison of the 6-OHDA+NaCl and control groups revealed a significant increase in DOPAC (by 19.0 ± 5.3%) and HVA (by 19.1 ± 3.8%) concentrations in the left-brain hemisphere following the 6-OHDA injection (fig. 2). Carnosine brought DOPAC levels down to the values registered in the control group.
The levels of DA and its major metabolites in the right striatum plummeted by an average of 90% in all animals exposed to the neurotoxin regardless of the therapy they had received (fig. 3). 3-MT concentrations demonstrated a less pronounced decline (by 58%, on average).
Serotonin levels did not change significantly. 5-HIAA, one of serotonin metabolites, was elevated by 38% in the experimental groups, as compared to the controls (fig. 4).
A 5.8-fold (p = 0.007) increase in the striatal DA content was observed in the animals treated with carnosine, relative to the 6-OHDA+NaCl group. The levels of DA metabolites (DOPAC and HVA) were elevated 3.4-fold (p = 0.04) and 8.8-fold (p = 0.01), respectively, following carnosine administration. LA injections caused a 4.7-fold (p = 0.03) and a 7.4-fold (p = 0.04) increase in DOPAC and HVA, respectively (fig. 3). The tested AO induced no changes in 3-MT levels. There was a 23% (p = 0.006) and 36% (p = 0.009) decline in the concentrations of serotonin and its metabolite 5-HIAA, respectively, relative to the 6-OHDA+NaCl group. Carnosine did not have any effect on the levels of serotonin or its metabolites.

DISCUSSION

We attempted to comprehensively assess the effect of carnosine and LA on the performance of rats with induced parkinsonism in the behavioral tests and on the metabolism of DA and serotonin in the striatum. The pathology was induced by a unilateral injection of 6-OHDA into the SN; the first injection of AO was made on day 14 after surgery to prevent their effect on the autoxidation of the toxin and the occurred neuronal loss.
The death of neurons and ipsilateral striatal denervation were confirmed by immunohistochemistry (immunostaining of dopaminergic neurons in SN) and a significant decline in DA and its metabolites in the striatum of the right brain hemisphere, where the toxin had been injected. Regardless of the treatment applied, DA and its metabolites were lower in the animals exposed to 6-OHDA than in the control group. At the same time, carnosine and LA affected DA metabolism and caused an increase in DA metabolites, in comparison with the group of untreated 6-OHDA-exposed animals. However, DA levels were significantly elevated only in the group of animals treated with carnosine. Serotonin and 5-HIAA concentrations were lower in the group treated with LA than in the untreated 6-OHDA-exposed group. Considering that serotonin stimulates neurochemical mechanisms promoting motor dysfunction in PD and that low serotonergic neurotransmission in the basal ganglia has an antiparkinsonian effect [11], the impact of LA on serotonergic innervation may contribute to the improvement of motor function in parkinsonism.
Physiological tests revealed that neurological deficit and behavioral symptoms were significantly attenuated in the animals treated with both tested antioxidants. Four injections of carnosine and LA on days 14 through 20 of the experiment prevented the development of 6-OHDA-induced neurotic symptoms.
Studies involving the use of rat models of toxin-induced parkinsonism have demonstrated neuroprotective effects of both carnosine [12] and lipoic acid [13]. These drugs restored the antioxidant brain status and reduced peroxide oxidation. Besides, lipoic acid improved motor function in the affected animals [13]. However, in the studies cited above the drugs and the toxin were administered simultaneously and the loss of neurons in the SN was not assessed. This complicated interpretation of the results and left open the question about the mechanisms of action of these compounds. Our study provides evidence of symptomatic relief produced by the tested drugs in the late stage of induced parkinsonism.
On the whole, our findings suggest that the tested antioxidants could be used as an adjunctive component to conventional antiparkinsonian therapy. Previously, lipoic acid was proposed for use in combination with levodopa [14], and the combination regimen of carnosine and levodopa was reported to be effective [15].

CONCLUSOINS

Although carnosine and LA did not have a direct neuroprotective effect, they attenuated parkinsonian symptoms due to a compensatory increase in the levels of DA and its metabolites and a simultaneous decrease in serotonin concentrations. Our findings suggest that the tested antioxidants could be used in combination with conventional antiparkinsonian drugs and provide a symptomatic relief.