Evaluation of Ouabain's Tissue Distribution in C57/Black Mice Following Intraperitoneal Injection, Using Chromatography and Mass Spectrometry.

Cardiotonic steroids (CTSs), such as digoxin, are used for heart failure treatment. However, digoxin permeates the brain-blood barrier (BBB), affecting central nervous system (CNS) functions. Finding a CTS that does not pass through the BBB would increase CTSs' applicability in the clinic and decrease the risk of side effects on the CNS. This study aimed to investigate the tissue distribution of the CTS ouabain following intraperitoneal injection and whether ouabain passes through the BBB. After intraperitoneal injection (1.25 mg/kg), ouabain concentrations were measured at 5 min, 15 min, 30 min, 1 h, 3 h, 6 h, and 24 h using HPLC-MS in brain, heart, liver, and kidney tissues and blood plasma in C57/black mice. Ouabain was undetectable in the brain tissue. Plasma: Cmax = 882.88 ± 21.82 ng/g; Tmax = 0.08 ± 0.01 h; T1/2 = 0.15 ± 0.02 h; MRT = 0.26 ± 0.01. Cardiac tissue: Cmax = 145.24 ± 44.03 ng/g (undetectable at 60 min); Tmax = 0.08 ± 0.02 h; T1/2 = 0.23 ± 0.09 h; MRT = 0.38 ± 0.14 h. Kidney tissue: Cmax = 1072.3 ± 260.8 ng/g; Tmax = 0.35 ± 0.19 h; T1/2 = 1.32 ± 0.76 h; MRT = 1.41 ± 0.71 h. Liver tissue: Cmax = 2558.0 ± 382.4 ng/g; Tmax = 0.35 ± 0.13 h; T1/2 = 1.24 ± 0.7 h; MRT = 0.98 ± 0.33 h. Unlike digoxin, ouabain does not cross the BBB and is eliminated quicker from all the analyzed tissues, giving it a potential advantage over digoxin in systemic administration. However, the inability of ouabain to pass though the BBB necessitates intracerebral administration when used to investigate its effects on the CNS.

An assessment of the transport mechanism and intraneuronal stability of L-carnosine.

L-Carnosine (ß-alanyl-L-histidine) is a well-known antioxidant and neuroprotector in various models on animals and cell cultures. However, while there is a plethora of data demonstrating its efficiency as a neuroprotector, there is a distinct lack of data regarding the mechanism of its take up by neurons. According to literature, cultures of rat astrocytes, SKPT cells and rat choroid plexus epithelial cells take up carnosine via the H+-coupled PEPT2 membrane transporter. We've assessed the effectiveness and mechanism of carnosine transport, and its stability in primary rat cortical culture neurons. We demonstrated that neurons take up carnosine via active transport with Km = 119 µM and a maximum velocity of 0.289 nmol/mg (prot)/min. Passive transport speed constituted 0.21∙10-4 nmol/mg (prot)/min (with 119 µM concentration in the medium)-significantly less than active transport speed. However, carnosine concentrations over 12.5 mM led to passive transport speed becoming greater than active transport speed. Using PEPT2 inhibitor zofenopril, we demonstrated that PEPT2-dependent transport is one of the main modes of carnosine take up by neurons. Our experiments demonstrated that incubation with carnosine does not affect PEPT2 amount present in culture. At the same time, after removing carnosine from the medium, its elimination speed by culture cells reached 0.035 nmol/mg (prot)/min, which led to a decrease in carnosine quantity to control levels in culture within 1 h. Thus, carnosine is taken up by neurons with an effectiveness comparable to that of other PEPT2 substrates, but its elimination rate suggests that for effective use as a neuroprotector it's necessary to either maintain a high concentration in brain tissue, or increase the effectiveness of glial cell synthesis of endogenous carnosine and its shuttling into neurons, or use more stable chemical modifications of carnosine.

Neuroprotective Efficacy of a Nanomicellar Complex of Carnosine and Lipoic Acid in a Rat Model of Rotenone-Induced Parkinson's Disease.

Oxidative stress, accompanied by mitochondrial dysfunction, is a key mechanism involved in the pathogenesis of Parkinson's disease (PD). Both carnosine and lipoic acid are potent antioxidants, the applicability of which in therapy is hindered by their limited bioavailability. This study aimed to evaluate the neuroprotective properties of a nanomicellar complex of carnosine and lipoic acid (CLA) in a rotenone-induced rat model of PD. Parkinsonism was induced via the administration of 2 mg/kg rotenone over the course of 18 days. Two doses of intraperitoneal CLA (25 mg/kg and 50 mg/kg) were administered alongside rotenone to assess its neuroprotective effect. At 25 mg/kg CLA decreased muscle rigidity and partially restored locomotor activity in animals that received rotenone. Furthermore, it caused an overall increase in brain tissue antioxidant activity, accompanied by a 19% increase in neuron density in the substantia nigra and increased dopamine levels in the striatum relative to animals that only received rotenone. Based on the acquired results, it may be concluded that CLA have neuroprotective properties and could potentially be beneficial in PD treatment when used in conjunction with the base therapy.

Na+,K+-ATPase and Cardiotonic Steroids in Models of Dopaminergic System Pathologies.

In recent years, enough evidence has accumulated to assert that cardiotonic steroids, Na+,K+-ATPase ligands, play an integral role in the physiological and pathophysiological processes in the body. However, little is known about the function of these compounds in the central nervous system. Endogenous cardiotonic steroids are involved in the pathogenesis of affective disorders, including depression and bipolar disorder, which are linked to dopaminergic system dysfunction. Animal models have shown that the cardiotonic steroid ouabain induces mania-like behavior through dopamine-dependent intracellular signaling pathways. In addition, mutations in the alpha subunit of Na+,K+-ATPase lead to the development of neurological pathologies. Evidence from animal models confirms the neurological consequences of mutations in the Na+,K+-ATPase alpha subunit. This review is dedicated to discussing the role of cardiotonic steroids and Na+,K+-ATPase in dopaminergic system pathologies-both the evidence supporting their involvement and potential pathways along which they may exert their effects are evaluated. Since there is an association between affective disorders accompanied by functional alterations in the dopaminergic system and neurological disorders such as Parkinson's disease, we extend our discussion to the role of Na+,K+-ATPase and cardiotonic steroids in neurodegenerative diseases as well.

Time Course of Neurobehavioral Disruptions and Regional Brain Metabolism Changes in the Rotenone Mice Model of Parkinson's Disease.

Parkinson's disease (PD) is characterized by slow progression with a long prodromal stage and the gradual evolution of both neuropsychological symptoms and subtle motor changes, preceding motor dysfunction. Thus, in order for animal models of PD to be valid, they should reproduce these characteristics of the disease. One of such models, in which neuropathology is induced by chronic injections of low doses of mitochondrial toxin rotenone, is well established in rats. However, data on this model adapted to mice remain controversial. We have designed the study to describe the timecourse of motor and non-motor symptoms during chronic subcutaneous administration of rotenone (4 mg/kg daily for 35 days) in C57BL/6 mice. We characterize the underlying neuropathological processes (dopaminergic neuron degeneration, regional brain metabolism, monoamine neurotransmitter and lipid peroxidation changes) at different timepoints: 1 day, 2 weeks and 5 weeks of daily rotenone exposure. Based on the behavioral data, we can describe three stages of pathology: cognitive changes from week 2 of rotenone exposure, subtle motor changes in week 3-4 and motor dysfunction starting roughly from week 4. Neuropathological changes in this model include a general decrease in COX activity in different areas of the brain (acute effect of rotenone) and a more specific decrease in midbrain (chronic effect), followed by significant neurodegeneration in SNpc but not VTA by the 5th week of rotenone exposure. However, we were unable to find changes in the level of monoamine neurotransmitters neither in the striatum nor in the cortex, nor in the level of lipid peroxidation in the brainstem. Thus, the gradual progression of pathology in this model is linked with metabolic changes, rather than with oxidative stress or tonic neurotransmitter release levels. Overall, this study supports the idea that a low-dose rotenone mouse model can also reproduce different stages of PD as well as rats.

Neurokinin-1 receptor antagonist rolapitant suppresses anxiety and alcohol intake produced by repeated withdrawal episodes.

Neurokinin-1 receptor (NK1r) antagonists have been shown to suppress operant self-administration of alcohol, voluntary alcohol consumption and stress-induced reinstatement of alcohol-seeking behaviour. Considering the long half-life and anxiolytic-like properties of NK1r antagonist rolapitant, we expected that it may be an effective option for reducing anxiety and alcohol motivation during early withdrawal. Voluntary alcohol intake (two-bottles paradigm) was recorded in male Wistar rats during the three periods: 24 days (basal level), 6-day period when rats received 5 mg·kg-1 rolapitant or vehicle and 12-h period after repeated withdrawal episodes (alcohol cessation for 36 h). We found that upon intraperitoneal (i.p.) administration, rolapitant rapidly penetrated into specific rat brain regions - amygdala, hypothalamus and neocortex - implicated in the control of anxiety and reward. Rolapitant did not affect basal voluntary alcohol intake, but significantly suppressed anxiety-like behaviour and alcohol consumption following withdrawal episodes. Our findings suggest that rolapitant should be further investigated as a novel treatment option for relapse prevention in alcohol-dependent patients.

Neurokinin-1 Receptor Antagonist Reverses Functional CNS Alteration Caused by Combined γ-rays and Carbon Nuclei Irradiation.

BACKGROUND: Ionizing Radiation (IR) is one of the major limiting factors for human deep-space missions. Preventing IR-induced cognitive alterations in astronauts is a critical success factor. It has been shown that cognitive alterations in rodents can be inferred by alterations of a psycho- emotional balance, primarily an anxiogenic effect of IR. In our recent work, we hypothesized that the neurokinin-1 (NK1) receptor might be instrumental for such alterations. OBJECTIVE: The NK1 receptor antagonist rolapitant and the classic anxiolytic diazepam (as a comparison drug) were selected to test this hypothesis on Wistar rats. METHODS: Pharmacological substances were administered through intragastric probes. We used a battery of tests for a comprehensive ethological analysis. High-performance liquid chromatography was applied to quantify monoamines content. An analysis of mRNA expression was performed by real-time PCR. Protein content was studied by the Western blotting technique. RESULTS: Our salient finding includes no substantial changes in anxiety, locomotor activity and cognitive abilities of treated rats under irradiation. No differences were found in the content of monoamines. We discovered a synchronous effect on mRNA expression and protein content of 5- HT2a and 5-HT4 receptors in the prefrontal cortex, as well as decreased content of serotonin transporter and increased content of tryptophan hydroxylase in the hypothalamus of irradiated rats. Rolapitant affected the protein amount of a number of serotonin receptors in the amygdala of irradiated rats. CONCLUSION: Rolapitant may be the first atypical radioprotector, providing symptomatic treatment of CNS functional disorders in astronauts caused by IR.

Expression Analysis of Genes Involved in Transport Processes in Mice with MPTP-Induced Model of Parkinson's Disease.

Processes of intracellular and extracellular transport play one of the most important roles in the functioning of cells. Changes to transport mechanisms in a neuron can lead to the disruption of many cellular processes and even to cell death. It was shown that disruption of the processes of vesicular, axonal, and synaptic transport can lead to a number of diseases of the central nervous system, including Parkinson's disease (PD). Here, we studied changes in the expression of genes whose protein products are involved in the transport processes (Snca, Drd2, Rab5a, Anxa2, and Nsf) in the brain tissues and peripheral blood of mice with MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-induced models of PD. We detected changes in the expressions of Drd2, Anxa2, and Nsf at the earliest modeling stages. Additionally, we have identified conspicuous changes in the expression level of Anxa2 in the striatum and substantia nigra of mice with MPTP-induced models of PD in its early stages. These data clearly suggest the involvement of protein products in these genes in the earliest stages of the pathogenesis of PD.

The dynamics of nigrostriatal system damage and neurobehavioral changes in the rotenone rat model of Parkinson's disease.

Subcutaneous administration of rotenone to rats is currently a widely used method of reproducing Parkinson's disease (PD) symptoms, due to its convenience and effectiveness. Despite this, its influence on the temporal dynamics of parkinsonism development has yet to be investigated. The present study characterizes behavioral and neurochemical disruptancies underlying the dynamics of parkinsonism development in rats, induced by chronic subcutaneous administration of 2 mg/kg rotenone over the course of 18 days. In this article, the presence of two stages of pathology development in the model in question - the premotor and motor disability stages - are illustrated through a complex assessment of animal behavior, the development of an original neurological symptoms scale, and the establishment of the dynamics of histological and neurochemical changes in the brain. The premotor stage was observed up to 3 days of rotenone administration, and was characterized by a decrease in the motivational component of behavior, shown both in the food-getting task and in the "sucrose preference" test. A 30 % decrease in the number of cells in the substantia nigra pars compacta by the 3rd day of rotenone administration was also shown during the premotor stage. No changes in the metabolism of dopamine and other monoamine mediators were observed at this time. At the same time, acute administration of rotenone caused an increase in the GSH / GSSG ratio by 69 %. The motor stage developed after a decrease in the number of cells in the SNpc by more than 30 %, and was characterized by changes in the dopaminergic system, leading up to a 71 % reduction in dopamine levels in the striatum. It was shown that starting from 4 to 6 days of rotenone injection, experimental group animals begin to develop motor symptoms of Parkinson's disease, including bradykinesia, rigidity and postural instability. The development of motor impairment in all rats of this group was accompanied by significantly reduced activity of the antioxidant system in brain frontal lobe tissue homogenates, as compared to intact rats. Thus, in the used model of rotenone-induced parkinsonism, the dynamics of neuropathology development are described and the premotor stage of the disease is highlighted, which allows future using of this model in developing new approaches for treatment of parkinsonism at an early stage.

Parkinson's Disease: Available Clinical and Promising Omics Tests for Diagnostics, Disease Risk Assessment, and Pharmacotherapy Personalization.

Parkinson's disease is the second most frequent neurodegenerative disease, representing a significant medical and socio-economic problem. Modern medicine still has no answer to the question of why Parkinson's disease develops and whether it is possible to develop an effective system of prevention. Therefore, active work is currently underway to find ways to assess the risks of the disease, as well as a means to extend the life of patients and improve its quality. Modern studies aim to create a method of assessing the risk of occurrence of Parkinson's disease (PD), to search for the specific ways of correction of biochemical disorders occurring in the prodromal stage of Parkinson's disease, and to personalize approaches to antiparkinsonian pharmacotherapy. In this review, we summarized all available clinically approved tests and techniques for PD diagnostics. Then, we reviewed major improvements and recent advancements in genomics, transcriptomics, and proteomics studies and application of metabolomics in PD research, and discussed the major metabolomics findings for diagnostics and therapy of the disease.

Intracerebroventricular injection of ouabain causes mania-like behavior in mice through D2 receptor activation.

Intracerebroventricular (ICV) administration of ouabain, an inhibitor of the Na, K-ATPase, is an approach used to study the physiological functions of the Na, K-ATPase and cardiotonic steroids in the central nervous system, known to cause mania-like hyperactivity in rats. We describe a mouse model of ouabain-induced mania-like behavior. ICV administration of 0.5 µl of 50 µM (25 pmol, 14.6 ng) ouabain into each lateral brain ventricle results in increased locomotor activity, stereotypical behavior, and decreased anxiety level an hour at minimum. Fast-scan cyclic voltammetry showed that administration of 50 µM ouabain causes a drastic drop in dopamine uptake rate, confirmed by elevated concentrations of dopamine metabolites detected in the striatum 1 h after administration. Ouabain administration also caused activation of Akt, deactivation of GSK3ß and activation of ERK1/2 in the striatum of ouabain-treated mice. All of the abovementioned effects are attenuated by haloperidol (70 µg/kg intraperitoneally). Observed effects were not associated with neurotoxicity, since no dystrophic neuron changes in brain structures were demonstrated by histological analysis. This newly developed mouse model of ouabain-induced mania-like behavior could provide a perspective tool for studying the interactions between the Na,K-ATPase and the dopaminergic system.