Expression and functional activity of neurotransmitter system components in sea urchins' early development.

Reverse-transcription polymerase chain reaction (RT-PCR) investigation of the expression of the components supposedly taking part in serotonin regulation of the early development of Paracentrotus lividus has shown the presence of transcripts of five receptors, one of which has conservative amino acid residues characteristic of monoaminergic receptors. At the early stages of embryogenesis the expressions of serotonin transporter (SERT) and noradrenaline transporter (NET) were also recognized. The activities of the enzymes of serotonin synthesis and serotonin transporter were shown using immunohistochemistry and incubation with para-chlorophenylalanine (PСРА) and 5-hydroxytryptophan (HTP). Pharmacological experiments have shown a preferential cytostatic activity of ligands characterized as mammalian 5-hydroxytryptamine (5-HT)1-antagonists. On the basis of the sum of the data from molecular biology and embryo physiological experiments, it is suggested that metabotropic serotonin receptors and membrane transporters take part in the regulatory processes of early sea urchin embryogenesis.

The effect of ribavirin on reactive astrogliosis in experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is an animal model of CNS inflammatory and demyelinating disease multiple sclerosis. Microglia and astrocytes represent two related cell types involved in the brain pathology in EAE. Accumulations of hypertrophic reactive astrocytes, intensely stained with glial fibrillary acidic protein (GFAP), which also expressed vimentin, are prominent features of EAE lesions. Recent studies from our laboratory reported that ribavirin attenuated the disease process in EAE by reducing clinical and histological manifestations. EAE was induced in genetically susceptible Dark Agouti rats with syngeneic spinal cord homogenate in complete Freund's adjuvant. Real time PCR and immunohistochemistry were used for determination of GFAP and vimentin gene and tissue expression. We have observed the increased gene and tissue expression of GFAP and vimentin in EAE rats. Ribavirin treatment significantly decreased the number of reactive astrocytes at the peak of disease. At the end of the disease, we have observed reactive GFAP(+) and vimentin(+) astrocytes in both immunized and ribavirin-treated groups, accompanied by increased level of GFAP mRNA. The present study indicates that ribavirin may have the ability to attenuate astrocyte proliferation and glial scaring at the peak of the disease and modulate the astroglial response to EAE during the time-course of the disease.

Hyperbaric oxygenation improves locomotor ability by enhancing neuroplastic responses after cortical ablation in rats.

OBJECTIVE: To investigate whether hyperbaric oxygenation (HBO) can improve the recovery of motor functions in rats after suction ablation of the right sensorimotor cortex. METHODS: The experimental paradigm implies the following groups: Control animals (C), Control + HBO (CHBO), Sham controls (S), Sham control + HBO (SHBO), Lesion group (L), right sensorimotor cortex was removed by suction, Lesion + HBO (LHBO). Hyperbaric protocol: pressure applied 2.5 atmospheres absolute, for 60 minutes, once a day for 10 days. A beam walking test and grip strength meter were used to evaluate the recovery of motor functions. Expression profiles of growth-associated protein 43 (GAP43) and synaptophysin (SYP) were detected using immunohistochemistry. RESULTS: The LHBO group achieved statistically superior scores in the beam walking test compared to the L group. Additionally, the recovery of muscle strength of the affected hindpaw was significantly enhanced after HBO treatment. Hyperbaric oxygenation induced over-expression of GAP43 and SYP in the neurons surrounding the lesion site. CONCLUSIONS: Data presented suggest that hyperbaric oxygen therapy can intensify neuroplastic responses by promoting axonal sprouting and synapse remodelling, which contributes to the recovery of locomotor performances in rats. This provides the perspective for implementation of HBO in clinical strategies for treating traumatic brain injuries.

Biochemical characterization of soluble nucleotide pyrophosphatase/phosphodiesterase activity in rat serum.

Biochemical properties of nucleotide pyrophosphatase/phosphodiesterase (NPP) in rat serum have been described by assessing its nucleotide phosphodiesterase activity, using p-nitrophenyl-5'-thymidine monophosphate (p-Nph-5'-TMP) as a substrate. It was demonstrated that NPP activity shares some typical characteristics described for other soluble NPP, such as divalent cation dependence, strong alkaline pH optimum (pH 10.5), inhibition by glycosaminoglycans, and K (m) for p-Nph-5'-TMP hydrolysis of 61.8 +/- 5.2 microM. In order to characterize the relation between phosphodiesterase and pyrophosphatase activities of NPP, we have analyzed the effects of different natural nucleotides and nucleotide analogs. ATP, ADP, and AMP competitively inhibited p-Nph-5'-TMP hydrolysis with K (i) values ranging 13-43 microM. Nucleotide analogs, alpha,beta-metATP, BzATP, 2-MeSATP, and dialATP behaved as competitive inhibitors, whereas alpha,beta-metADP induced mixed inhibition, with K (i) ranging from 2 to 20 microM. Chromatographic analysis revealed that alpha,beta-metATP, BzATP, and 2-MeSATP were catalytically degraded in the serum, whereas dialATP and alpha,beta-metADP resisted hydrolysis, implying that the former act as substrates and the latter as true competitive inhibitors of serum NPP activity. Since NPP activity is involved in generation, breakdown, and recycling of extracellular adenine nucleotides in the vascular compartment, the results suggest that both hydrolyzable and non-hydrolyzable nucleotide analogs could alter the amplitude and direction of ATP actions and could have potential therapeutic application.

Expression of cholesterol homeostasis genes in the brain of the male rat is affected by age and dietary restriction.

Expression profiles of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), apolipoprotein E (ApoE) and cholesterol 24S-hydroxylase (CYP46), proteins involved in cholesterol biosynthesis, transport and excretion from the CNS, were analyzed in the rat cortex, hippocampus and cerebellum as a function of aging (6­24 months) and in response to long-term dietary restriction (DR). Age-related increases for all three mRNAs were observed, with the highest induction found for Cyp46 in the cortex and hippocampus of 24-month-old animals. DR maintained stable levels of Cyp46, HMGR, and ApoE mRNAs during aging, exhibiting an attenuating effect on age-related changes through specific temporal and regional pattern. Neither age nor DR had any prominent effects at the protein level, except for Cyp46 and ApoE protein levels in the hippocampus and cerebellum, respectively. Overall, the changes in the cerebellum were different from those in the cortex and hippocampus. Our results demonstrated a modulatory effect of DR on agerelated changes of CYP46, HMGR, and ApoE and suggest that the anti-aging effect of DR is in part mediated though transcriptional modulation of cholesterol metabolism genes in the rat brain.

Regional changes in ectonucleotidase activity after cortical stab injury in rat.

During a variety of insults to the brain adenine nucleotides are released in large quantities from damaged cells, triggering local cellular and biochemical responses to injury. Different models of brain injury reveal that the local increase in adenine nucleotides levels is followed by a compensatory up-regulation of ectonucleotidase enzymes that catalyze sequential hydrolysis of ATP to ADP, AMP and adenosine. However, recent studies imply that changes in adenine nucleotides release may also occur in the areas distant from the site of direct damage. Therefore, in the present study we have used the model of cortical stab injury to analyze extracellular ATP, ADP and AMP hydrolysis in the membrane preparations obtained from the brain regions that were not subjected to direct tissue damage. The brain regions analyzed were contralateral cortex, hippocampus, caudate nucleus, thalamus and hypothalamus. It was evidenced that cortical stab injury induced early widespread decrease in AMP hydrolysis in all brain areas tested, except in the hypothalamus, without changes in ATP hydrolysis. These findings imply that brain injury affects global extracellular adenine nucleotide and nucleoside levels, consequently affecting neuronal function in the regions distant to the primary damage.

Ribavirin administration alters ectonucleotidase activities in experimental autoimmune encephalomyelitis.

The role of extracellular purines and purinoreceptors in the pathophysiology of different neurological disorders is the focus of rapidly expanding area of research. Ectonucleotidases are the enzymes with multiple roles in extracellular nucleotides metabolism and regulation of nucleotidebased intercellular signaling. The aim of present study was to investigate the changes in the ATP, ADP and AMP hydrolyzing activities after ribavirin treatment in spinal cord during experimental autoimmune encephalomyelitis (EAE). Our results demonstrate that ribavirin itself had no significant effect on ectoenzyme activities, when tested in vitro and in vivo on spinal cord crude membrane preparation of intact animals. We observed significant increase in ATP, ADP and AMP hydrolyzing activity in the spinal cord crude membrane preparation in EAE animals at 15 days post immunization compared to control animals. The increase was registered at 28 days post immunization, as well. At same time points, ribavirin treatment decreased ATP, ADP and AMP hydrolyzing activity compared to EAE animals. In addition, no significant changes 8 days post immunization was observed between EAE-induced and ribavirin- treated EAE animals and these levels were similar to control level. Thus, we suppose that ribavirin-induced alteration in ectonucleotidase activities is rather due to its suppression of inflammation, than to its direct action on ATP, ADP and AMP hydrolysis.

Aging, aluminium and basal forebrain lesions modify substrate kinetics of erythrocyte membrane Na,K-ATPase in the rat.

Several studies suggested that the activity of erythrocyte Na,K-ATPase declines with aging. Here, it is postulated that alterations in the substrate kinetics of the erythrocyte membrane Na,K-ATPase could be more aggravated in conditions of brain cholinergic dysfunction seen in Alzheimer's disease than in normal aging. To test this hypothesis, we compared the Na,K-ATPase activity (Vmax/Km parameters) in aged rats with those in young rats with brain cholinergic dysfunction induced by electrolytic-, kainic acid-lesioned nucleus basalis magnocellularis (NBM) or by intracerebroventricular AlCl_{3} administration. In the above mentioned groups, Vmax values were significantly lower in comparison to the control animals. Furthermore, Km values were significantly higher in animals with electrolytic-induced NBM lesions, AlCl_{3} treated rats and aged animals. However, Km was significantly lower in kainic acid-induced NBM lesions compared to the control group. The Na,K-ATPase catalytic efficiency, estimated by the ratio Vm/Km, decreased as followed: young animals > aged animals > kainic acid lesion > electrolityc lesion > AlCl_{3}. Our data suggest that neurodegenerative processes similar to those seen in Alzheimer's disease affect the sodium/potassium pump functionality which might be detected in peripheral blood erythrocyte membranes.

Therapeutic effects of combined treatment with ribavirin and tiazofurin on experimental autoimmune encephalomyelitis development: clinical and histopathological evaluation.

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS) and the helpful tool in preclinical testing of various substances considered for treatment of this human CNS disease. Ribavirin (R) and tiazofurin (T) are purine nucleoside analogues, with the broad spectrum of anti-viral, anti-tumoral and anti-inflammatory properties. We proposed that combined treatment with RT, administrated during the effector phase of EAE, would attenuate disease severity, both clinically and pathologically. Ribavirin was given daily at a dosage of 30 mg/kg and tiazofurin was given at a dosage of 10 mg/kg every other day for 15 days. We detected amelioration of clinical signs and faster recovery in the RT group compared to the control group. Immunohistochemical analyses revealed that RT treatment decrease the number of T cells, macrophages and microglia. In the controls, we detected reactive type of microglia, while in the RT group we noticed ramified/resting form. Demyelination areas and axonal damage were not recorded in the RT group, in contrast to the control group where multiple areas of demyelination zones and axonal loss were found. RT combination treatment suppresses ongoing EAE, prevents demyelination and axonal loss, and therefore may well be the potential therapy for the treatment of MS.

Ribavirin ameliorates experimental autoimmune encephalomyelitis in rats and modulates cytokine production.

To determine the mechanism underlying ribavirin induced amelioration of experimental autoimmune encephalomyelitis (EAE), cytokine profiles were evaluated in draining lymph node (DLN) cell culture supernatants and spinal cord obtained from EAE and/or ribavirin-treated EAE Dark Agouti rats. Administration of ribavirin to EAE rats markedly affected the production of pro-inflammatory cytokines IFN-gamma, IL-1beta and TNF-alpha in DLN and spinal cord, thus shifting the balance towards the anti-inflammatory cytokines IL-10 and TGF-beta. These findings suggest that ribavirin attenuates EAE by limiting cytokine-mediated immunoinflammatory events leading to CNS destruction. The conducted experiments provide rationale for ribavirin to be considered as a candidate drug in the development of new therapeutic strategies for the treatment of autoimmune diseases in humans, such as multiple sclerosis.

Enhancement of AP-1 DNA-binding activity during amphetamine- and phencyclidine-mediated behaviour in rats.

Amphetamine (AMPH) and phencyclidine (PCP) induce a variety of behavioural and synaptic changes in the brain, many of which are believed to involve the regulation of gene expression. In this study, we examined the effects of AMPH (5mg/kg), PCP (5mg/kg) and their combination (5mg/kg each) on rat motor activity as well as on the activation of the AP-1 transcription factor in rat brains. AMPH administration, followed by PCP, led to a statistically significant elevation of locomotor activity. It was found that the behavioural response of rats was more pronounced when the two drugs were administered together. The electrophoretic mobility shift assay (EMSA) revealed a significant increase in AP-1-binding activity after treatments with AMPH, PCP or their combination. Super shift/shift inhibition analysis demonstrated the presence of c-Fos and c-Jun protein families in the transcriptional complex bound to AP-1 sequences. Further, our results suggest that the enhanced behavioural changes after AMPH and PCP administration were associated with increased expression of AP-1 proteins (Fos and Jun) in the cortex, striatum and hippocampus and that their binding to AP-1 sites on the DNA contributes to long-term changes in rat brain.

Immunolocalization of ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) in the rat forebrain.

Immunohistochemical study was performed to determine distribution of ecto-nucleotide pyrophosphatase/phosphodiesterase1 (NPP1) in adult rat forebrain. The study revealed widespread distribution of NPP1 in rat forebrain, yet with regional differences in the expression pattern and abundance. Strong NPP1 immunoreaction was detected in pyramidal cell layer of cerebral cortex and hippocampus, and in the midline regions of hypothalamus and thalamus. In many immunopositive forebrain areas, NPP1 was mainly localized at neuronal cell bodies. However, prominent immunoreaction was also detected at ependymal cells, tanycytes, endothelial cells of the capillaries and cells of the choroid plexus, suggesting that NPP1 could be involved in some highly specialized transport process.

Different effects of adenosine A1 agonist ribavirin on amphetamine-induced total locomotor and stereotypic activities in rats.

Pretreatment with a single dose of ribavirin (10, 20, and 30 mg/kg b.w., i.p.) decreased amphetamine (1.5 mg/kg b.w., i.p.)-induced total locomotor activity (distance traveled) compared to amphetamine alone, but the most significant effect was observed with 30 mg/kg dose. In contrast, total stereotypic activities (such as sniffing, self-grooming, licking, and head weaving) of these animals were unchanged. These findings demonstrate the ability of ribavirin to modulate in a different way central regulation of locomotor and stereotypic activities that in the future could have a practical application in target therapy of some movement disorders.

Downregulation of glial scarring after brain injury: the effect of purine nucleoside analogue ribavirin.

The weak regenerative capacity of self-repair after injury to the adult brain is caused by the formation of glial scar due to reactive astrogliosis. In the present study the beginning of reactive astrogliosis in the adult, as shown immunocytochemically by upregulation of glial fibrillary acidic protein (GFAP) and vimentin, was seen two days after the left sensorimotor cortex lesion, being maximal during the first two weeks and declining by 30 days after the lesion. This was accompanied by intensive glial scarring. Conversely, after the neonatal lesion a lack of gliotic scar was seen until 30 days postsurgery, although the pattern of GFAP and vimentin expression during recovery period was the same. The aim of the study was to define an appropriate therapeutic intervention that could modulate astrocyte proliferation and diminish glial scar formation after adult brain lesion. For this purpose the effects of an antiproliferative agent, the purine nucleoside analogue ribavirin was examined. It was shown that daily injection of ribavirin for 5 and 10 days considerably decreased the number of reactive astrocytes, while slight GFAP labeling was restricted to the lesion site. Obtained results show that ribavirin treatment downregulates the process of reactive astrogliosis after adult brain injury, and thus may be a useful approach for improving neurological recovery from brain damage.

Combination of nucleoside analogues tiazofurin and ribavirin downregulates experimental autoimmune encephalomyelitis.

The effect of combined treatment with ribavirin and tiazofurin on the development of experimental autoimmune encephalomyelitis, the best characterized animal model for human autoimmune disease multiple sclerosis, was investigated. The disease was induced in highly susceptible Dark Agouti rats with spinal cord homogenate in complete Freund's adjuvant. Although ribavirin or tiazofurin alone reduced the clinical and histopathological signs of experimental autoimmune encephalomyelitis, the combination of drugs achieved the same effect with significantly lower doses.

Long-term intermittent feeding restores impaired GR signaling in the hippocampus of aged rat.

Diminished glucocorticoid signaling is associated with an age-related decline in hippocampal functioning. In this study we demonstrate the effect of intermittent, every other day (EOD) feeding on the glucocorticoid hormone/glucocorticoid receptor (GR) system in the hippocampus of middle-aged (18-month-old) and aged (24-month-old) Wistar rats. In aged ad libitum-fed rats, a decrease in the level of total GR and GR phosphorylated at Ser(232) (pGR) was detected. Conversely, aged rats subjected to EOD feeding, starting from 6 months of age, showed an increase in GR and pGR levels and a higher content of hippocampal corticosterone. Furthermore, prominent nuclear staining of pGR was observed in CA1 pyramidal and DG granule neurons of aged EOD-fed rats. These changes were accompanied by increased Sgk-1 and decreased GFAP transcription, pointing to upregulated transcriptional activity of GR. EOD feeding also induced an increase in the expression of the mineralocorticoid receptor. Our results reveal that intermittent feeding restores impaired GR signaling in the hippocampus of aged animals by inducing rather than by stabilizing GR signaling during aging.

Alpha-synuclein is expressed in different tissues during human fetal development.

Alpha-synuclein is a small presynaptic protein associated with both normal synaptic plasticity and neurodegenerative processes. Its normal cellular function, however, remains unknown. Even though it is highly enriched in the brain, its presence was reported in other human adult tissues. In the present study, we examined tissue expression of alpha-synuclein in human and rat prenatal development. Using Western blot analysis, various peripheral tissues from 15 to 23 gestational weeks, human and E19 rat fetuses, along with human and rat adult tissues, were assayed. alpha-Synuclein expression was observed in all fetal human organs examined. In adult human tissues the high expression of alpha-synuclein was maintained in the brain, whereas in other organs the expression was greatly reduced. In contrast, both in fetal and adult rat tissues, alpha-synuclein was only detected in the brain. In addition to a 19-kDa alpha-synuclein band, 36- and 52-kDa immunoreactive bands were observed in all fetal and adult human organs, with the exception of the brain, but their identity remains to be determined. These findings suggest that apart from its function in development of the nervous system, alpha-synuclein has an important function in peripheral tissues as well during normal human prenatal development.

Effect of amphetamine and phencyclidine on DNA-binding activities of serum response and dyad symmetry elements.

Acute administration of D-amphetamine sulphate (AMPH) and (1-[1-phenylcyclohexyl]piperidine hydrochloride) (phencyclidine; PCP) produces a characteristic spatio-temporal distribution of c-Fos protein in the brain. As transcriptional mechanisms underlying the induction of c-fos gene expression may be regulated in a stimulus-specific manner, we have analyzed the binding activities of serum response element (SRE), dyad symmetry element (DSE) and calcium response element (CRE), the major regulatory sites of the c-fos promoter. Electrophoretic mobility shift showed that SRE binding activity was increased for 50-60%, 2-6h after AMPH, while treatment with PCP resulted in light decrease of SRE binding activity throughout the same time period. Co-administration of AMPH and PCP induced gradual increase of SRE binding activity, reaching maximum (86%) at 6h. Binding of nuclear proteins to DSE sequence was increased 1-2h after administration of AMPH (72-87%) and remained elevated till the end of the time window observed. PCP and AMPH/PCP caused different temporal profile of DSE binding with peak (40-54%) 4-6h after administration. In contrast, DNA-binding activity of the CRE sequences remained unchanged throughout the time period of 6h under all conditions. Finally, supershift analysis clearly demonstrated presence of SRF and c-Fos protein in the transcriptional complexes bound to SRE and DSE sequences irrespective to AMPH, PCP or combined treatment. These findings also showed that the presence of c-Fos protein in SRE and DSE nucleocomplex support the hypothesis concerning autoregulation of c-fos gene expression during psychostimulant action in vivo.

Motor effects of amphetamine in rats pretreated with either dizocilpine or phencyclidine.

The aim of the present study was to examine motor effects of amphetamine (AMPH) in rats pretreated with either dizocilpine (MK-801) or phencyclidine (PCP), and to estimate possible differences in these effects. Our results showed that AMPH increases the duration of motor effects of PCP, while it does not change motor effects of MK-801. These findings may reflect different mechanisms of action of MK-801 and PCP, as well as selective influence of AMPH on metabolism of these drugs.

6-Hydroxydopamine increases the level of TNFalpha and bax mRNA in the striatum and induces apoptosis of dopaminergic neurons in hemiparkinsonian rats.

This study was focused on the apoptosis (programmed cell death) induction involved in the loss of dopaminergic (DA-ergic) neurons in 6-hydroxydopamine (6-OHDA) hemiparkinsonian rats. The apoptosis in the striatum and substantia nigra pars compacta (SNpc), was examined 6, 24 h and 7 days after the 6-OHDA lesions employing the TUNEL method. The changes in mRNA levels of pro-apoptotic protein tumor necrosis factor alpha (TNFalpha) and its "death receptor" TNFalphaRI and then bax mRNA, as an important regulator of apoptotic neurodegeneration were followed by RT-PCR procedure. In situ analysis revealed an increased number of TUNEL-positive neurons in 6-OHDA-treated animals in all examined time points. The highest number of apoptotic neurons was detected 24 h after the lesion, both in the ipsilateral striatum (3.41+/-0.18) and SNpc (5.8+/-0.79). A significant increase in the level of TNFalpha mRNA was observed in 6-OHDA-lesioned striatum, with maximal value after 24 h (46%) comparing to the control. In contrast, 6-OHDA did not significantly change the level of TNFalphaRI mRNA in any time point. Six and 24 h post-operatively, we observed a significant increase of bax mRNA expression (40% and 45%, respectively) in the ipsilateral striatum of treated animals in comparison with the right striatum of the controls. However, the highest level of the bax mRNA expression was reached 7 days after the surgery (94%) in the ipsilateral striatum of 6-OHDA-treated animals. These results suggest that striatal injection of 6-OHDA can induce early changes that would be an important regulator of apoptotic neurodegeneration of dopamine-producing neurons, during the first post-operative week.