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.

Plasma oxylipin profiles reflect Parkinson's disease stage.

Derivatives of polyunsaturated fatty acids (PUFAs), also known as oxylipins, are key participants in regulating inflammation. Neuroinflammation is involved in many neurodegenerative diseases, including Parkinson's disease. The development of ultra-high-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) facilitated the study of oxylipins on a system level, i.e., the analysis of oxylipin profiles. We analyzed oxylipin profiles in the blood plasma of 36 healthy volunteers (HC) and 73 patients with Parkinson's disease (PD), divided into early (L\M, 29 patients) or advanced (H, 44 patients) stages based on the Hoehn and Yahr scale. Among the 40 oxylipins detected, we observed a decrease in the concentration of arachidonic acid (AA) and AA derivatives, including anandamide (AEA) and Leukotriene E4 (LTE4), and an increase in the concentration of hydroxyeicosatetraenoic acids 19-HETE and 12-HETE (PD vs HC). Correlation analysis of gender, age of PD onset, and disease stages revealed 20 compounds the concentration of which changed depending on disease stage. Comparison of the acquired oxylipin profiles to openly available PD patient brain transcriptome datasets showed that plasma oxylipins do not appear to directly reflect changes in brain metabolism at different disease stages. However, both the L\M and H stages are characterized by their own oxylipin profiles - in patients with the H stage oxylipin synthesis is increased, while in patients with L\M stages oxylipin synthesis decreases compared to HC. This suggests that different therapeutic approaches may be more effective for patients at early versus late stages of PD.

Impact of manganese accumulation on Na,K-ATPase expression and function in the cerebellum and striatum of C57Bl/6 mice.

Overexposure to Mn causes a neurological disorder-manganism-with motor symptoms that overlap closely with disorders associated with haploinsufficiency in the gene encoding for α3 isoform of Na+,K+-ATPase (NKA). The present study was designed to test the hypothesis that behavioral changes in the mouse model of manganism may be associated with changes in the expression and activity of α3 NKA in the cerebellum (CB) and striatum (STR)-the key brain structures responsible for motor control in adult mice. C57Bl/6 mice were exposed to MnCl2 at 0.5 g/L (in drinking water) for up to eight weeks. After four weeks of Mn consumption, Mn levels were increased in the CB only. Behavioral tests demonstrated decreased performance of Mn-treated mice in the shuttle box test (third through sixth weeks), and the inclined grid walking test (first through sixth weeks), suggesting the development of learning impairment, decreased locomotion, and motor discoordination. The activity of NKA significantly decreased, and the expression of α1-α3 isoforms of NKA increased in the second week in the CB only. Thus, signs of learning and motor disturbances developing in this model of manganism are unlikely to be directly linked to disturbances in the expression or activity of NKA in the CB or STR. Whether these early changes may contribute to the pathogenesis of later behavioral deficits remains to be determined.

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.

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.

Modulation of the Primary Astrocyte-Enriched Cultures' Oxylipin Profiles Reduces Neurotoxicity.

Recently, manipulations with reactive astrocytes have been viewed as a new therapeutic approach that will enable the development of treatments for acute brain injuries and neurodegenerative diseases. Astrocytes can release several substances, which may exert neurotoxic or neuroprotective effects, but the nature of these substances is still largely unknown. In the present work, we tested the hypothesis that these effects may be attributed to oxylipins, which are synthesized from n-3 or n-6 polyunsaturated fatty acids (PUFAs). We used astrocyte-enriched cultures and found that: (1) lipid fractions secreted by lipopolysaccharide (LPS)-stimulated rat primary astrocyte-enriched cultures-possessed neurotoxic activity in rat primary neuronal cultures; (2) both of the tested oxylipin synthesis inhibitors, ML355 and Zileuton, reduce the LPS-stimulated release of interleukin 6 (IL-6) by astrocyte cultures, but only ML355 can change lipid fractions from neurotoxic to non-toxic; and (3) oxylipin profiles, measured by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) from neurotoxic and non-toxic lipid fractions, reveal a group of n-3 docosahexaenoic acid derivatives, hydroxydocosahexaenoic acids (HdoHEs)-4-HdoHE, 8-HdoHE, and 17-HdoHE, which may reflect the neuroprotective features of lipid fractions. Regulating the composition of astrocyte oxylipin profiles may be suggested as an approach for regulation of neurotoxicity in inflammatory processes.

Efficacy and Safety of Divaza for the Correction of Oxidative Disturbances in Patients with Cerebral Atherosclerosis: A Randomized Controlled Trial.

OBJECTIVE: The objective of this study was to determine if Divaza, a drug with nootropic and antioxidant effects, was safe and effective for the correction of oxidative disturbances and to stabilize cognitive impairment in patients with cerebral atherosclerosis. STUDY DESIGN: The study design consisted of a 12-week multicenter, randomized, double-blind, placebo-controlled, prospective trial in parallel groups. SETTING: The setting in which the study was conducted comprised 10 clinical centers across the Russian Federation. INTERVENTIONS: Patients were randomized into 2 groups and instructed to take either 2 tablets of the study drug or a placebo 3 times per day in conjunction with basic therapy. OUTCOMES: The primary outcome was a change in the average endogenous antioxidant potential after the completion of the study. The blood indicators of the oxidative stress (OS) were analyzed at the baseline and then after 12 weeks of therapy using iron-induced chemiluminescence analysis. The Montreal cognitive assessment test was used as a secondary outcome measure to evaluate cognitive impairment at the end of the study. RESULTS: 124 outpatients with a mean age of 60.7 ± 7.6 years were enrolled and randomly assigned to receive Divaza (n = 65) or a placebo (n = 59). An improvement of cognitive function was observed in all patients of the Divaza group at the end of the treatment; this was significantly better than the placebo group (100 [100] vs. 89.5 [89.1]%, respectively, p = 0.0272 [p = 0.0128]). The administration of Divaza restored the activity of the endogenous antioxidant system. The change in the average level of lipoprotein resistance to oxidation after 12 weeks of therapy, compared to the baseline, was significantly higher in the Divaza group (14.8 ± 14.7 [14.8 ± 14.7] seconds latent period vs. 6.4 ± 16.9 [6.9 ± 16.7] seconds in the placebo group (p = 0.007 [p = 0.0107]). CONCLUSIONS: Divaza is a safe and effective therapeutic option for attenuating OS and recovery of cognitive impairment in patients with cerebral atherosclerosis.

Ouabain-Induced Gene Expression Changes in Human iPSC-Derived Neuron Culture Expressing Dopamine and cAMP-Regulated Phosphoprotein 32 and GABA Receptors.

Cardiotonic steroids (CTS) are specific inhibitors and endogenous ligands of a key enzyme in the CNS-the Na+, K+-ATPase, which maintains and creates an ion gradient on the plasma membrane of neurons. CTS cause the activation of various signaling cascades and changes in gene expression in neurons and other cell types. It is known that intracerebroventricular injection of cardiotonic steroid ouabain causes mania-like behavior in rodents, in part due to activation of dopamine-related signaling cascades in the dopamine and cAMP-regulated phosphoprotein 32 (DARPP-32) expressing medium spiny neurons in the striatum. Dopaminergic projections in the striatum innervate these GABAergic medium spiny neurons. The objective of this study was to assess changes in the expression of all genes in human iPSC-derived expressing DARPP-32 and GABA receptors neurons under the influence of ouabain. We noted a large number of statistically significant upregulated and downregulated genes after a 16-h incubation with non-toxic concentration (30 nM) of ouabain. These changes in the transcriptional activity were accomplished with activation of MAP-kinase ERK1/2 and transcriptional factor cAMP response element-binding protein (CREB). Thus, it can be concluded that 30 nM ouabain incubated for 16 h with human iPSC-derived expressing DARPP-32 and GABA receptors neurons activates genes associated with neuronal maturation and synapse formation, by increasing the expression of genes associated with translation, vesicular transport, and increased electron transport chain function. At the same time, the expression of genes associated with proliferation, migration, and early development of neurons decreases. These data indicate that non-toxic concentrations of ouabain may induce neuronal maturation, neurite growth, and increased synaptogenesis in dopamine-receptive GABAergic neurons, suggesting formation of plasticity and the establishment of new neuronal junctions.

Changes in COX histochemistry in the brain of mice and rats exposed to chronic subcutaneous rotenone.

Exposure of experimental animals to the mitochondrial toxin rotenone is considered to be a model of environmental progression of Parkinson's disease (PD). We investigated the differential vulnerability of various brain regions to generalized inhibition of complex I, induced by subcutaneous rotenone injections for the duration of 1, 3 and 7 days in both rats (2 mg/kg dosage) and mice (4 mg/kg dosage). To examine patterns of metabolic activity changes in the brain, histochemical evaluation of cytochrome C oxidase (COX) activity was performed in post mortem brain sections. Animals displayed a similar time course of neuronal loss in substantia nigra pars compacta (SNpc), reaching 44 % in mice and 42 % in rats by the 7th day. The pattern of COX activity changes, however, was different for the two species. In both experiments, metabolic changes were evident not only in the substantia nigra, but also in non-specific structures (cortex and hippocampus). In mice, a decrease in COX activity was shown mostly for the non-specific areas (V1 cortex and ventral hippocampus) after the single exposure to rotenone. Data from the experiment conducted on rats demonstrated both an acute metabolic decrease in mesencephalic structures (SNpc and nucleus ruber) after a single injection of rotenone and secondary changes in cortical structures (S1 cortex and dorsal hippocampus) after chronic 7 day exposure. These changes reflect the general effect of rotenone on neuronal metabolic rate.

An overview of the pathogenic mechanisms involved in severe cases of COVID-19 infection, and the proposal of salicyl-carnosine as a potential drug for its treatment.

Multiple organ failure in COVID-19 patients is a serious problem which can result in a fatal outcome. Damage to organs and tissues, including general lung dysfunction, develops as a consequence of ischemia, which, in turn, is caused by thrombosis in small blood vessels and hypoxia, leading to oxidative stress and inflammation. Currently, research is underway to screen existing drugs for antioxidant, antiplatelet and anti-inflammatory properties. Having studied the available publications concerning the mechanisms of damage to tissues and organs of patients with COVID-19, as well as the available treatment strategies, we propose to investigate salicyl-carnosine as a potential drug for treating COVID-19 patients. In a recent study, we described the drug's synthesis procedure, and showed that salicyl-carnosine possesses antioxidant, anti-inflammatory, and antiplatelet effects. Therefore, it can simultaneously act on the three pathogenetic factors involved in tissue and organ damage in COVID-19. Thus, we propose to consider salicyl-carnosine as a potential drug for the treatment of patients with severe cases of COVID-19 infection.

Oxylipin Profiles in Plasma of Patients with Wilson's Disease.

Wilson's disease (WD) is a rare autosomal recessive metabolic disorder resulting from mutations in the copper-transporting, P-type ATPase gene ATP7B gene, but influences of epigenetics, environment, age, and sex-related factors on the WD phenotype complicate diagnosis and clinical manifestations. Oxylipins, derivatives of omega-3, and omega-6 polyunsaturated fatty acids (PUFAs) are signaling mediators that are deeply involved in innate immunity responses; the regulation of inflammatory responses, including acute and chronic inflammation; and other disturbances related to any system diseases. Therefore, oxylipin profile tests are attractive for the diagnosis of WD. With UPLC-MS/MS lipidomics analysis, we detected 43 oxylipins in the plasma profiles of 39 patients with various clinical manifestations of WD compared with 16 healthy controls (HCs). Analyzing the similarity matrix of oxylipin profiles allowed us to cluster patients into three groups. Analysis of the data by VolcanoPlot and partial least square discriminant analysis (PLS-DA) showed that eight oxylipins and lipids stand for the variance between WD and HCs: eicosapentaenoic acid EPA, oleoylethanolamide OEA, octadecadienoic acids 9-HODE, 9-KODE, 12-hydroxyheptadecatrenoic acid 12-HHT, prostaglandins PGD2, PGE2, and 14,15-dihydroxyeicosatrienoic acids 14,15-DHET. The compounds indicate the involvement of oxidative stress damage, inflammatory processes, and peroxisome proliferator-activated receptor (PPAR) signaling pathways in this disease. The data reveal novel possible therapeutic targets and intervention strategies for treating WD.

A new derivative of acetylsalicylic acid and carnosine: synthesis, physical and chemical properties, biological activity.

PURPOSE: The aim of this study was to create and assess biological activity of a new compound based on carnosine and acetylsalicylic acid (ASA) that will comprise antioxidant effect with antiplatelet activity, while simultaneously preventing side effects on the gastrointestinal tract. METHODS: Salicyl-carnosine (SC) was synthesized by condensation of ASA and carnosine. Antioxidant activity was determined by spectrophotometric and chemiluminescence methods. Antiplatelet activity was carried out by the light transmission-aggregometry method using the inductor ADP. Chronic gastric ulcer in rats was modeled using glacial acetic acid. RESULTS: Using SOD-like activity, iron-induced chemiluminescence, BaSO4-activated respiratory burst, and evaluation of red blood cell structure stabilization during oxidative damage induced by sodium hypochlorite, it was shown that SC possesses antioxidant activity analogous, or better, than that of carnosine. Antiplatelet activity of SC was evaluated in the blood of healthy individuals, and was also shown to be comparable to, or exceeding that of ASA. Also SC demonstrates high resistance to hydrolysis by tissue and serum carnosinases. Most importantly, it was shown that SC has protected the gastric mucosa against the formation of stomach ulcerative lesions and promoted their epithelization, therefore overcoming the undesirable inherent side effects of ASA. CONCLUSIONS: SC preserves pharmacologically significant properties of ASA and carnosine while retaining an anti-ulcer activity and resistance to the carnosinase hydrolysis at the same time. These properties are particularly promising for the potential development of new anti-inflammatory and antithrombotic drugs. Graphical abstract .

A low-cost and customizable alternative for commercial implantable cannula for intracerebral administration in mice.

Stereotaxic intracerebral cannula implantation for neuroactive agent administration is a wide-spread method for chronic experiments requiring bypassing the blood-brain barrier in rodents. However, commercially available cannula are bulky and may interfere with animal movement or lead to their dislodging during grooming. As the number of cannula needed in one experiment, and the accompanying costs can be high, it is in the interest of researchers to produce them on their own. Custom cannula manufacturing also offers the flexibility of different cannula lengths, which is required for agent delivery to various brain structures. In this article we present a protocol for making guide cannula along with the accompanying systems required for injection, which are small, cost-effective, light, easy to make, reusable, and can be made from easily procured materials.

Transcriptomic changes triggered by ouabain in rat cerebellum granule cells: Role of α3- and α1-Na+,K+-ATPase-mediated signaling.

It was shown previously that inhibition of the ubiquitous α1 isoform of Na+,K+-ATPase by ouabain sharply affects gene expression profile via elevation of intracellular [Na+]i/[K+]i ratio. Unlike other cells, neurons are abundant in the α3 isoform of Na+,K+-ATPase, whose affinity in rodents to ouabain is 104-fold higher compared to the α1 isoform. With these sharp differences in mind, we compared transcriptomic changes in rat cerebellum granule cells triggered by inhibition of α1- and α3-Na+,K+-ATPase isoforms. Inhibition of α1- and α3-Na+,K+-ATPase isoforms by 1 mM ouabain resulted in dissipation of transmembrane Na+ and K+ gradients and differential expression of 994 transcripts, whereas selective inhibition of α3-Na+,K+-ATPase isoform by 100 nM ouabain affected expression of 144 transcripts without any impact on the [Na+]i/[K+]i ratio. The list of genes whose expression was affected by 1 mM ouabain by more than 2-fold was abundant in intermediates of intracellular signaling and transcription regulators, including augmented content of Npas4, Fos, Junb, Atf3, and Klf4 mRNAs, whose upregulated expression was demonstrated in neurons subjected to electrical and glutamatergic stimulation. The role [Na+]i/[K+]i-mediated signaling in transcriptomic changes involved in memory formation and storage should be examined further.

Neuroprotective effect of the carnosine - α-lipoic acid nanomicellar complex in a model of early-stage Parkinson's disease.

In a model of early-stage Parkinson's disease induced by a single intranasal administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to Wistar rats, a neuroprotective effect of a new derivative of carnosine and α-lipoic acid (C/LA nanomicellar complex) was demonstrated. Acute intraperitoneal administration of carnosine, α-lipoic acid and C/LA complex following MPTP administration normalized the total antioxidant activity in the brain tissue. Of all the compounds tested only C/LA complex normalized the metabolism of dopamine (DA) and serotonin (5-HT), while its components did not show similar effects when used separately. C/LA complex effectively restored the level of DA metabolites: the level of DOPAC was increased by 24.7 ±â€¯5.6% compared to the animals that had received MPTP only, and the level of HVA was restored to the values observed in the intact animals. Integral metabolic indices of DA (DOPAC/DA and HVA/DA ratios) and 5-HT turnover (5-HIAA/5-HT ratio) in the striatum tended to increase in case of C/LA complex administration.

Ouabain-induced changes in MAP kinase phosphorylation in primary culture of rat cerebellar cells.

Cardiotonic steroid (CTS) ouabain is a well-established inhibitor of Na,K-ATPase capable of inducing signalling processes including changes in the activity of the mitogen activated protein kinases (MAPK) in various cell types. With increasing evidence of endogenous CTS in the blood and cerebrospinal fluid, it is of particular interest to study ouabain-induced signalling in neurons, especially the activation of MAPK, because they are the key kinases activated in response to extracellular signals and regulating cell survival, proliferation and apoptosis. In this study we investigated the effect of ouabain on the level of phosphorylation of three MAPK (ERK1/2, JNK and p38) and on cell survival in the primary culture of rat cerebellar cells. Using Western blotting we described the time course and concentration dependence of phosphorylation for ERK1/2, JNK and p38 in response to ouabain. We discovered that ouabain at a concentration of 1 µM does not cause cell death in cultured neurons while it changes the phosphorylation level of the three MAPK: ERK1/2 is phosphorylated transiently, p38 shows sustained phosphorylation, and JNK is dephosphorylated after a long-term incubation. We showed that ERK1/2 phosphorylation increase does not depend on ouabain-induced calcium increase and p38 activation. Changes in p38 phosphorylation, which is independent from ERK1/2 activation, are calcium dependent. Changes in JNK phosphorylation are calcium dependent and also depend on ERK1/2 and p38 activation. Ten-micromolar ouabain leads to cell death, and we conclude that different effects of 1-µM and 10-µM ouabain depend on different ERK1/2 and p38 phosphorylation profiles. Copyright © 2016 John Wiley & Sons, Ltd.

Oxidative stress induced in rat brain by a combination of 3-nitropropionic acid and global ischemia.

In our investigation, we describe the complex model of brain oxidative stress consisted of combination of experimental brain ischemia and energy metabolism violation induced by irreversible inhibitor of mitochondrial succi-nate dehydrogenase, 3-nitropropionate (3-NPA). 3-NPA causes selective degeneration of striatum neurons, which is extremely sensitive to energy deficit. This complex model allows revealing not only biochemical but also neurological symptoms in experimental animals that permits proper estimation of protective effect of different drugs on animal status. Combination of global ischemia induced by 3-vessel occlusion of major arteries supplys rat brain and subsequent 5-day reperfusion with intraperitoneal injection of 3-nitropropionic acid induces vigorous oxidative stress in brain tissues accompanied by evident neurological symptoms in Wistar rats. Such a combination of damaging factors may be considered as a new complex experimental model of brain oxidative stress permitting the evaluation of neuroprotective effect of potential therapeutic agents. Using this model, protective effect of neuropeptide carnosine was demonstrated which is in agreement with previous data.