Focal Cerebral Ischemia Induces Global Subacute Changes in the Number of Neuroblasts and Neurons and the Angiogenic Factor Density in Mice.

Background and Objectives: Dissecting the complex pathological cascade of an ischemic stroke in preclinical models is highly warranted to understand the course of this disease in humans. Neurogenesis and angiogenesis are integral for post-stroke recovery, yet it is not clear how these processes are altered months after an ischemic stroke. In this study, we investigated the changes that take place subacutely after focal cerebral ischemia in experimental adult male mice. Materials and Methods: Male 12-week-old C57BL/6 mice underwent a 60 min long fMCAo or sham surgery. Two months after the procedure, we examined the immunohistochemistry to assess the changes in neuroblast (DCX) and differentiated neuron (NeuN) numbers, as well as the density of the pro-angiogenic factor VEGF. Results: We found decreased neuroblast numbers in both brain hemispheres of the fMCAo mice: by more than 85% in the dentate gyrus and by more than 70% in the subventricular zone. No neuroblasts were found in the contralateral hemisphere of the fMCAO mice or the sham controls, but a small population was detected in the ipsilateral ischemic core of the fMCAo mice. Intriguingly, the number of differentiated neurons in the ipsilateral ischemic core was lower by 20% compared to the contralateral hemisphere. VEGF expression was diminished in both brain hemispheres of the fMCAo mice. Conclusions: Our current report shows that focal cerebral ischemia induces changes in neuroblast numbers and the pro-angiogenic factor VEGF in both cerebral hemispheres 2 months after an fMCAo in mice. Our data show that focal cerebral ischemia induces a long-term regenerative response in both brain hemispheres.

Intra-Nasally Administered Oligopeptide Lunasin Acts as a Possible Anti-Psychotic Agent in Mice Models.

Background and Objectives: Previously we have shown that synthetic lunasin, a 43 amino acid residue-containing peptide, after its central (intracisternal) administration in mice demonstrated antagonism against dopaminergic drug behavioural effects, indicating a putative antipsychotic/anti-schizophrenic profile of lunasin. The aims of the present studies were: to test whether lunasin would show an influence on the dopaminergic system after intranasal administration, and to examine the effect(s) of lunasin on serotonin and glutamatergic systems, which could play an essential role in antipsychotic action. Materials and Methods: Lunasin was administered intra-nasally at doses 0.1 and 1 nmol/mouse in ICR mice (n = 7-8) and tested in an open field on hyperlocomotion caused by amphetamine; serotonin 5-HT 2A/2C receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)- 2-aminopropane (DOI); and glutamate NMDA receptor antagonist phencyclidine. Following behavioural testing, the contents of neurotransmitters and their metabolites in brain hemispheres (n = 6-8) were assessed by ultra-high-performance liquid chromatography-time of flight mas-spectrometry (UHPLC-TOF-MS) method. Also, lunasin binding to serotonin receptors was assessed. Results: Lunasin intra-nasally fully normalized hyper-locomotion and brain monoamine levels in amphetamine- and DOI-treated mice brains. Phencyclidine behavioural effects were not influenced. In vitro receptor binding data demonstrated a low affinity of lunasin (at µM concentrations) compared with DOI (nM concentrations) for the 5-HT2A and 5-HT2C receptors. Conclusions: These results demonstrated, for the first time, that the intranasal administration of oligopeptide lunasin normalized mice behaviour and brain monoamine levels in experimental psychosis mice models. Its neuro-regulatory effects indicated a usefulness of this peptide molecule for the design of novel psychotropic agents.

Mildronate and its neuroregulatory mechanisms: targeting the mitochondria, neuroinflammation, and protein expression.

This review for the first time summarizes the data obtained in the neuropharmacological studies of mildronate, a drug previously known as a cardioprotective agent. In different animal models of neurotoxicity and neurodegenerative diseases, we demonstrated its neuroprotecting activity. By the use of immunohistochemical methods and Western blot analysis, as well as some selected behavioral tests, the new mechanisms of mildronate have been demonstrated: a regulatory effect on mitochondrial processes and on the expression of nerve cell proteins, which are involved in cell survival, functioning, and inflammation processes. Particular attention is paid to the capability of mildronate to stimulate learning and memory and to the expression of neuronal proteins involved in synaptic plasticity and adult neurogenesis. These properties can be useful in neurological practice to protect and treat neurological disorders, particularly those associated with neurodegeneration and a decline in cognitive functions.

Long-term behavioural alterations in mice following transient cerebral ischemia.

Ischemic stroke is one of the leading causes of disability and mortality worldwide. Acute and chronic post-stroke changes have variable effects on the functional outcomes of the disease. Therefore, it is imperative to identify what daily activities are altered after stroke and to what extent, keeping in mind that ischemic stroke patients often have long-term post-stroke complications. Translational studies in stroke have also been challenging due to inconsistent study design of animal experiments. The objective of this study was to clarify whether and to what extent mouse behaviour was altered during a 6 months period after cerebral stroke. Experimental stroke was induced in mice by intraluminal filament insertion into the middle cerebral artery (fMCAo). Neurological deficits, recovery rate, motor performance, and circadian activity were evaluated following ischemia. We observed severe neurological deficits, motor impairments, and delay in the recovery rate of mice during the first 14 days after fMCAo. Aberrant circadian activity and distorted space map were seen in fMCAo mice starting one month after ischemia, similarly to altered new and familiar cage activity and sucrose preference using the IntelliCage, and was still evident 60- and 180- days following stroke in the voluntary running wheel using the PhenoMaster system. A preference towards ipsilateral side turns was observed in fMCAo mice both acutely and chronically after the stroke induction. Overall, our study shows the importance of determining time-dependent differences in the long-term post-stroke recovery (over 180 days after fMCAo) using multiple behavioural assessments.

Neuroprotective properties of mildronate, a small molecule, in a rat model of Parkinson's disease.

Previously, we have found that mildronate [3-(2,2,2-trimethylhydrazinium) propionate dihydrate], a small molecule with charged nitrogen and oxygen atoms, protects mitochondrial metabolism that is altered by inhibitors of complex I and has neuroprotective effects in an azidothymidine-neurotoxicity mouse model. In the present study, we investigated the effects of mildronate in a rat model of Parkinson's disease (PD) that was generated via a unilateral intrastriatal injection of the neurotoxin 6-hydroxydopamine (6-OHDA). We assessed the expression of cell biomarkers that are involved in signaling cascades and provide neural and glial integration: the neuronal marker TH (tyrosine hydroxylase); ubiquitin (a regulatory peptide involved in the ubiquitin-proteasome degradation system); Notch-3 (a marker of progenitor cells); IBA-1 (a marker of microglial cells); glial fibrillary acidic protein, GFAP (a marker of astrocytes); and inducible nitric oxide synthase, iNOS (a marker of inflammation). The data show that in the 6-OHDA-lesioned striatum, mildronate completely prevented the loss of TH, stimulated Notch-3 expression and decreased the expression of ubiquitin, GFAP and iNOS. These results provide evidence for the ability of mildronate to control the expression of an array of cellular proteins and, thus, impart multi-faceted homeostatic mechanisms in neurons and glial cells in a rat model of PD. We suggest that the use of mildronate provides a protective effect during the early stages of PD that can delay or halt the progression of this neurodegenerative disease.

Gamma1- and gamma2-melanocyte stimulating hormones induce central anxiogenic effects and potentiate ethanol withdrawal responses in the elevated plus-maze test in mice.

Little is known about the endogenous functions of gamma1- and gamma2-melanocyte stimulating hormones (gamma1- and gamma2-MSH). Although gamma-MSHs bind to melanocortin receptor subtypes 3 and 4, we have previously shown that these peptides also influence non-melanocortinergic processes, such as dopaminergic and GABAergic. The aim of this study was to determine the effects of gamma1- and gamma2-MSH (at doses 0.3, 1 and 2 nmol/mouse/5 microl) on the anxiety levels in mice in elevated plus maze. Three experimental paradigms were performed to assess the effects of peptides on: a) ethanol withdrawal; b) acute ethanol-induced anxiolytic action; c) peptides per se. We used ethanol as the model substance, since its action involves either dopaminergic/GABAergic or melanocortinergic processes. gamma-MSHs were administered intracisternally in mice and behavioural responses were assessed in the elevated plus maze test. This study provides the first demonstration of an anxiogenic effect of gamma1- and gamma2-MSH, their synergistic/additive effect on ethanol withdrawal-induced anxiety behaviour, and an antagonism of peptides involved in the anxiolytic action of ethanol. Furthermore, results suggest that gamma-MSHs belong to an anxiogenic peptide family that may play an important role in anxiety disorders as well as in the development of alcohol dependence and/or alcohol withdrawal-induced behaviours.

Intranasal Administration of Extracellular Vesicles Derived from Human Teeth Stem Cells Improves Motor Symptoms and Normalizes Tyrosine Hydroxylase Expression in the Substantia Nigra and Striatum of the 6-Hydroxydopamine-Treated Rats.

Parkinson's disease (PD) is the second most common neurodegenerative disorder affecting millions of people worldwide. At present, there is no effective cure for PD; treatments are symptomatic and do not halt progression of neurodegeneration. Extracellular vesicles (EVs) can cross the blood-brain barrier and represent promising alternative to the classical treatment strategies. In the present study, we examined therapeutic effects of intranasal administration of EVs derived from human exfoliated deciduous teeth stem cells (SHEDs) on unilateral 6-hydroxydopamine (6-OHDA) medial forebrain bundle (MFB) rat model of PD. CatWalk gait tests revealed that EVs effectively suppressed 6-OHDA-induced gait impairments. All tested gait parameters (stand, stride length, step cycle, and duty cycle) were significantly improved in EV-treated animals when compared with 6-OHDA-lesion group rats. Furthermore, EVs slowed down numbers of 6-OHDA-induced contralateral rotations in apomorphine test. Improvements in motor function correlated with normalization of tyrosine hydroxylase expression in the striatum and substantia nigra. In conclusion, we demonstrated, for the first time, the therapeutic efficacy of intranasal administration of EVs derived from SHEDs in a rat model of PD induced by 6-OHDA intra-MFB lesion. Our findings could be potentially exploited for the development of new treatment strategies against PD.

Betulin binds to gamma-aminobutyric acid receptors and exerts anticonvulsant action in mice.

The lupane type pentacyclic triterpenes: lupeol, betulin, and betulinic acid are widely distributed natural compounds. Recently, pharmaceutical compositions from plant extracts (family Marcgraviaceae) containing betulinic acid, have been patented as anxiolytic remedies. To extend our knowledge of the CNS effects of the triterpenes, we suggest here that the chemically related lupeol, betulin and betulinic acid may interact with the brain neurotransmitter gamma-aminobutyric acid (GABA) receptors in vitro and in vivo. Using radioligand receptor-binding assay, we showed that only betulin bound to the GABA(A)-receptor sites in mice brain in vitro and antagonised the GABA(A)-receptor antagonist bicuculline-induced seizures in mice after intracisternal and intraperitoneal administration. Neither betulinic acid nor lupeol bound to GABA(A) receptor nor did they inhibit bicuculline-induced seizures in vivo. These findings demonstrate for the first time the CNS effects of betulin in vivo, and they also show distinct GABA(A)-receptor-related properties of lupane type triterpenes. These findings may open new avenues in understanding the central effects of betulin, and they also indicate possibilities for novel drug design on the basis of betulin structure.

Carnitine congener mildronate protects against stress- and haloperidol-induced impairment in memory and brain protein expression in rats.

The present study investigates the efficacy of mildronate, a carnitine congener, to protect stress and haloperidol-induced impairment of memory in rats and the expression of brain protein biomarkers involved in synaptic plasticity, such as brain-derived neurotrophic factor (BDNF), acetylcholine esterase and glutamate decarboxylase 67 (GAD67). Two amnesia models were used: 2h immobilization stress and 3-week haloperidol treatment. Stress caused memory impairment in the passive avoidance test and induced a significant 2-fold BDNF elevation in hippocampal and striatal tissues that was completely inhibited by mildronate. Mildronate decreased the level of GAD67 (but not acetylcholine esterase) expression by stress. Haloperidol decrease by a third hippocampal BDNF and acetylcholine esterase (but not GAD67) expression, which was normalized by mildronate; it also reversed the haloperidol-induced memory impairment in Barnes test. The results suggest the usefulness of mildronate as protector against neuronal disturbances caused by stress or haloperidol.

Lunasin-induced behavioural effects in mice: focus on the dopaminergic system.

The present study for the first time is devoted to identify central effects of synthetic lunasin, a 43 amino acid peptide. A markedly expressed neuroleptic/cataleptic effect was observed at low (0.1-10 nmol/mouse) centrally administered doses in male C57Bl/6 mice. Lunasin considerably reduced the amphetamine hyperlocomotion but weakly apomorphine climbing behaviour. No influence on ketamine and bicuculline effects was observed. Binding assay studies demonstrated modest affinity of lunasin for the dopamine D1 receptor (Ki=60 ± 15 µM). In a functional assay of cAMP accumulation on live cells lunasin antagonised apomorphine effect on D1 receptor activation (pEC50=6.1 ± 0.3), but had no effect in cells expressing D2 receptors. The obtained data suggest that lunasin's action at least in part is provided via dopaminergic D1 receptor pathways. However, other non-identified mechanisms (probably intracellular) may play an important role in lunasin's central action. Nevertheless further studies of lunasin are promising, particularly taking into account a necessity for novel type of antipsychotic drugs.

Comparative study of taurine and tauropyrone: GABA receptor binding, mitochondrial processes and behaviour.

OBJECTIVES: Taurine, a sulfur-containing amino acid, has high hydrophilicity and is poorly absorbed. Tauropyrone, a taurine-containing 1,4-dihydropyridine derivative, is suggested to have greater activity than taurine owing to improved physicochemical properties that facilitate delivery of the compound to target cells. The aim of this study was to determine whether the 1,4-dihydropyridine moiety in tauropyrone improves the pharmacological efficacy of taurine in vitro and in vivo. METHODS: The effects of taurine and tauropyrone, as well as of the 1,4-dihydropyridine moiety were compared in in-vitro experiments to determine the binding to GABA receptors and influence on mitochondrial processes (isolated rat liver mitochondria), and in in-vivo tests to assess the influence on behavioural effects caused by the GABA-A receptor ligands, bicuculline, diazepam and ethanol. KEY FINDINGS: Unlike taurine, tauropyrone did not display binding activity for the GABA-A receptor, and only taurine (but not tauropyrone) at low doses (0.1, 1.0 and 10 mg/kg) antagonised the bicuculline-induced convulsion effect. Taurine and tauropyrone had no effect on diazepam myorelaxing action, and they both exerted a comparable 'anti-ethanol' effect (shortening of the ethanol-sleeping time). Taurine and tauropyrone did not influence processes of mitochondrial bioenergetics. CONCLUSIONS: The action of tauropyrone at the level of the GABA-A receptor differs qualitatively from that of taurine, probably because of its 1,4-dihydropyridine moiety, which may hinder access to the GABA-A receptor GABA site. Tauropyrone does not show improved pharmacological efficacy in in-vitro and in-vivo studies in comparison with taurine.

Distinct influence of atypical 1,4-dihydropyridine compounds in azidothymidine-induced neuro- and cardiotoxicity in mice ex vivo.

This study demonstrates the effective protection by compounds of atypical 1,4-dihydropyridine (DHP) series cerebrocrast, glutapyrone and tauropyrone against neuro- and cardiotoxicity caused by the model compound azidothymidine, a well-known mitochondria-compromising anti-HIV drug. In previous in vitro experiments, we have demonstrated distinct effects of these DHP compounds to influence mitochondrial functioning. In the present in vivo experiments, DHP compounds were administered intraperitoneally in mice daily for 2 weeks, per se and in combinations with azidothymidine at doses: azidothymidine 50 mg/kg; cerebrocrast 0.1 mg/kg; glutapyrone 1 mg/kg; and tauropyrone 1 mg/kg. At the end of the experiment, mice were killed, heart and brain tissues were removed and examined ex vivo histopathologically and immunohistochemically. NF-kappaBp65 and caspase-3 were used as the markers indicating inflammatory and apoptotic events, respectively. Cerebrocrast (dicyclic structure) was the most potent DHP, which effectively reduced azidothymidine-induced overexpression of NF-kappaBp65 and caspase-3 in mouse myocardium and brain cortex. Glutapyrone per se increased the number of caspase-3-positive cells in the brain, whereas it reduced NF-kappaBp65 and caspase-3 expression in cardiac tissue caused by azidothymidine. Tauropyrone showed dual action: per se it increased caspase-3 in the brain and NF-kappaBp65 expression in the heart, but it considerably reduced these activations in azidothymidine-treated mice. This study provides the first demonstration of a distinct pharmacological action for atypical DHP compounds in cardiac and brain tissues. The dicyclic structure of cerebrocrast is considered beneficial for neuro- and cardioprotection at least in part via mitochondrial targeting and consequent regulation of inflammatory and apoptotic processes.

Potato (Solanum tuberosum) juice exerts an anticonvulsant effect in mice through binding to GABA receptors.

Naturally occurring benzodiazepines have been identified in regular food such as wheat and potato, but there is still no evidence that potato extracts can affect CNS responses in vivo. Here we found that undiluted potato juice and potato juice diluted with saline 1 : 2 administered 10 min intracisternally ( I. C.) and 30 min per os before bicuculline exerted significant anticonvulsant activity in the bicuculline-induced seizure threshold test in mice. In vitro, potato juice from different harvests at dilution series from 10 % to 0.000001 %, diluted 100,000-fold, displaced 50 % of gamma-aminobutyric acid (GABA) receptor ligand [ (3)H]GABA and diluted 40-fold displaced 50 % of [(3)H]flunitrazepam from binding sites in mice forebrain membranes. The low content of diazepam (0.04 +/- 0.01 mg/kg) determined by HPLC and mass spectrometry in the potato extracts could not sustain the anticonvulsant activity of potato juice in vivo; therefore we hypothesized that potato juice might contain GABA (A) receptor GABA-site active compounds. The findings of this study suggest that potato juice as well as potato taken as food may have the capacity of influencing brain GABA-ergic activity.