Nadežda Nedeljković

Downregulation of Glial Scarring after Brain Injury: The Effect of Purine Nucleoside Analogue Ribavirin

Abstract: 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.

Ontogenetic profile of ecto-5′-nucleotidase in rat brain synaptic plasma membranes

Ecto‐5′‐nucleotidase (CD73; EC 3.1.3.5, e‐5NT) is regarded as the key enzyme in the extracellular formation of adenosine, which acts as a neuromodulator and important trophic and homeostatic factor in the brain. In the present study, we have investigated e‐5NT activity, kinetic properties concerning AMP hydrolysis and the enzyme protein abundance in the purified synaptic plasma membrane (SPM) preparations isolated from whole female rat brain at different ages. We observed pronounced increase in AMP hydrolyzing activity in SPM during maturation, with greatest increment between juvenile (15‐day‐old) and pre‐pubertal (30‐day‐old) rats. Immunodetection of e‐5NT protein in the SPM displayed the reverse pattern of expression, with the maximum relative abundance at juvenile and minimum relative abundance in the adult stage. Negative correlation between the enzyme activity and the enzyme protein abundance in the SPM indicates that e‐5NT has additional roles in the synaptic compartment during postnatal brain development, other than those related to AMP hydrolysis. Determination of kinetic parameters, Km and Vmax, suggested that the increase in the enzyme activity with maturation was entirely due to the increase in the enzyme catalytic efficiency (Vmax/Km). Finally, double immunofluorescence staining against e‐5NT and presynaptic membrane marker syntaxin provided first direct evidence for the existence of this ecto‐enzyme in the presynaptic compartment. The results of the study suggest that e‐5NT may be a part of general scheme of brain development and synapse maturation and provide rationale for the previously reported inconsistencies between enzyme immunohistochemical and biochemical studies concerning localization of e‐5NT in the brain.

Combination of Nucleoside Analogues Tiazofurin and Ribavirin Downregulates Experimental Autoimmune Encephalomyelitis

Abstract: 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 Freunds 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.

Brain Injury Alters Ectonucleotidase Activities and Adenine Nucleotide Levels in Rat Serum / Povreda Mozga Menja Ektonukleotidazne Aktivnosti I Nivo Adeninskih Nukleotida U Serumu Pacova

Summary Background: Cortical stab injury (CSI) induces changes in the activity, expression and cellular distribution of specific ectonucleotidases at the injury site. Also, several experimentally induced neuropathologies are associated with changes in soluble ectonucleotidase activities in the plasma and serum, whilst various insults to the brain alter purine compounds levels in cerebrospinal fluid, but also in serum, indicating that insults to the brain may induce alterations in nucleotides release and rate of their hydrolysis in the vascular system. Since adenine nucleotides and adenosine regulate diverse cellular functions in the vascular system, including vascular tone, platelet aggregation and inflammatory responses of lymphocytes and macrophages, alterations of ectonucleotidase activities in the vascular system may be relevant for the clinical outcome of the primary insult. Methods: We explored ectonucleotidase activities using specific enzyme assays and determined adenine nucleotides concentrations by the UPLC method in the rat serum after cortical stab injury. while phosphodiesterase activity remained unchanged. Also, at 4-h postinjury a marked decrease in ATP concentration and more than 2-fold increase in AMP concentration were recorded. Conclusions: CSI induces rapid up-regulation of nucleotide catabolizing soluble ectonucleotidases in rat serum, which leads to the observed shift in serum nucleotide levels. The results obtained imply that ectonucleotidases and adenine nucleotides participate in the communication between the brain and the vascular system in physiological and pathological conditions and thereby may be involved in the development of various human neuropathologies. Kratak sadržaj Uvod: Ubodna povreda mozga dovodi do promena u aktivnosti, ekspresiji i ćelijskoj distribuciji određenih ektonuk- leotidaza na mestu povrede. Pored toga, neke eksperimen- talno izazvane neuropatologije povezane su sa promenama solubilnih ektonukleotidaznih aktivnosti u plazmi i serumu, dok različite povrede mozga menjaju nivoe purina u cere- brospinalnoj tečnosti, ali i u serumu, ukazujući da povreda mozga može da dovede do promena u oslobađanju nukleo- tida i u stepenu njihove hidrolize u vaskularnom sistemu. Kako adeninski nukleotidi i adenozin regulišu raznovrsne ćelijske funkcije u vaskularnom sistemu, uključujući vasku- larni tonus, agregaciju trombocita i zapaljenski odgovor lim- focita i makrofaga, promene ektonukleotidaznih aktivnosti u vaskularnom sistemu mogu biti značajne za klinički ishod pri- marne povrede. Metode: Primenom specifičnih enzimskih eseja pratili smo ektonukleotidazne aktivnosti, a upotrebom UPLC metode odredili smo koncentracije adeninskih nukleotida u serumu pacova nakon ubodne povrede korteksa. Rezultati: Četiri sata nakon povrede, ATP- i AMP-hidrolizu- juće aktivnosti bile su povišene za približno 60%, odnosno 40%, dok je fosfodiesterazna aktivnost ostala nepromenjena. Pored toga, 4 h nakon povrede zabeleženo je značajno povećanje koncentracije ATP i više nego dvostruko povećanje nivoa АМР Zaključak: Ubodna povreda mozga dovodi do naglog poras- ta hidrolizujućih aktivnosti solubilnih ektonukleotidaza u seru- mu pacova,| što vodi uočenoj promeni nivoa nukleotida. Dobijeni rezultati pokazuju da ektonukleotidaze i adeninski nukleotidi posreduju u komunikaciji između mozga i vasku- larnog sistema, kako u fiziološkim tako i u patološkim uslovi- ma, te stoga mogu biti uključeni u razvoj različitih neuropa- tologija kod čoveka.

Expression of ecto-nucleoside triphosphate diphosphohydrolase3 (NTPDase3) in the female rat brain during postnatal development.

Nucleoside triphosphate diphosphohydrolase3 (NTPDase3) is membrane-bound ecto-enzyme which hydrolyzes extracellular ATP, thus modulating the function of purinergic receptors and the pattern of purinergic signaling. Here we analyzed the developmental expression of NTPDase3 in female hypothalamus, cerebral cortex and hippocampal formation at different postnatal ages (PD7-PD90) by qRT-PCR and immunohistochemistry. In hypothalamus and hippocampus, a similar developmental profile was seen: NTPDase3 gene expression was stable during postnatal development and increased in adults. In the cortex, upregulation of NTPDase3 mRNA expression was seen at PD15 and further increase was evidenced in adults. Immunohistochemical analysis at PD7 revealed faint neuronal NTPDase3 localization in a dorsal hypothalamus. The immunoreactivity (ir) gradually increased in PD15 and PD20, in clusters of cells in the lateral, ventral and dorsomedial hypothalamus. Furthermore, in PD20 animals, NTPDase3-ir was detected on short fibers in the posterior hypothalamic area, while in PD30 the fibers appeared progressively longer and markedly varicose. In adults, the strongest NTPDase3-ir was observed in collections of cells in dorsomedial hypothalamic nucleus, dorsal and lateral hypothalamus and in several thalamic areas, whereas the varicose fibers traversed entire diencephalon, particularly paraventricular thalamic nucleus, ventromedial and dorsomedial hypothalamic nuclei, the arcuate nucleus and the prefornical part of the lateral hypothalamus. The presumably ascending NTPDase3-ir fibers were first observed in PD20; their density and the varicose appearance increased until the adulthood. Prominent enhancement of NTPDase3-ir in the hypothalamus coincides with age when animals acquire diurnal rhythms of sleeping and feeding, supporting the hypothesis that this enzyme may be involved in regulation of homeostatic functions.

Upregulation of Nucleoside Triphosphate Diphosphohydrolase-1 and Ecto-5′-Nucleotidase in Rat Hippocampus after Repeated Low-Dose Dexamethasone Administration

Although dexamethasone (DEX), a synthetic glucocorticoid receptor (GR) analog with profound effects on energy metabolism, immune system, and hypothalamic-pituitary-adrenal axis, is widely used therapeutically, its impact on the brain is poorly understood. The aim of the present study was to explore the effect of repeated low-dose DEX administration on the activity and expression of the ectonucleotidase enzymes which hydrolyze and therefore control extracellular ATP and adenosine concentrations in the synaptic cleft. Ectonucleotidases tested were ectonucleoside triphosphate diphosphohydrolase 1–3 (NTPDase1–3) and ecto-5′-nucleotidase (eN), whereas the effects were evaluated in two brain areas that show different sensitivity to glucocorticoid action, hippocampus, and cerebral cortex. In the hippocampus, but not in cerebral cortex, modest level of neurodegenerative changes as well as increase in ATP, ADP, and AMP hydrolysis and upregulation of NTPDase1 and eN mRNA expression ensued under the influence of DEX. The observed pattern of ectonucleotidase activation, which creates tissue volume with enhanced capacity for adenosine formation, is the hallmark of the response after different insults to the brain.

Spatial Distribution and Expression of Ectonucleotidases in Rat Hippocampus After Removal of Ovaries and Estradiol Replacement

Purinergic signaling is the main synaptic and non-synaptic signaling system in brain. ATP acts as a fast excitatory transmitter, while adenosine sets a global inhibitory tone within hippocampal neuronal networks. ATP and adenosine are interconnected by ectonucleotidase enzymes, which convert ATP to adenosine. Existing data point to the converging roles of ovarian steroids and purinergic signaling in synapse formation and refinement and synapse activity in the hippocampus. Therefore, in the present study, we have used enzyme histochemistry and expression analysis to obtain data on spatial distribution and expression of ecto-enzymes NTPDase1, NTPDase2, and ecto-5′-nucleotidase (eN) after removal of ovaries (OVX) and estradiol replacement (E2) in female rat hippocampus. The results show that target ectonucleotidases are predominantly localized in synapse-rich hippocampal layers. The most represented NTPDase in the hippocampal tissue is NTPDase2, being at the same time the mostly affected ectonucleotidase by OVX and E2. Specifically, OVX decreases the expression of NTPDase2 and eN, whereas E2 restores their expression to control level. Impact of OVX and E2 on ectonucleotidase expression was also examined in purified synaptosome (SYN) and gliosome (GLIO) fractions. Data reveal that SYN expresses NTPDase1 and NTPDase2, both of which are reduced following OVX and restored with E2. GLIO exhibits NTPDase2-mediated ATP hydrolysis, which falls in OVX, and recovers by E2. These changes in the activity occur without parallel changes in NTPDase2-protein abundance. The same holds for eN. The lack of correlation between NTPDase2 and eN activities and their respective protein abundances suggest a non-genomic mode of E2 action, which is studied further in primary astrocyte culture. Since ovarian steroids shape hippocampal synaptic networks and regulate ectonucleotidase activities, it is possible that cognitive deficits seen after ovary removal may arise from the loss of E2 modulatory actions on ectonucleotidase expression in the hippocampus.

Correlation between maternal anxiety, reactivity of fetal cerebral circulation to auditory stimulation, and birth outcome in normotensive and gestational hypertensive women

This study investigated the correlation between maternal anxiety and blood flow changes through the fetal middle cerebral artery (MCA) after defined acoustic stimulation in 43 normotensive (C) and 40 gestational hypertensive (GH) subjects. Neonatal outcomes (gestational age at birth, Apgar score, birth weight) in the C and GH groups were analyzed. State (STAI-S) and trait (STAI-T) anxiety was assessed using Spielbergers questionnaire. The MCA blood flow was assessed once between 28 and 41 weeks of gestation using color Doppler ultrasound before and after application of defined acoustic stimulus. Relative size of the Pulsatility index (Pi) change (RePi) was calculated. The general hypotheses were: (1) women in GH group would have higher anxiety; (2) higher anxiety correlates with higher RePi change and poorer neonatal outcome; (3) fetuses from the GH group would have poorer neonatal outcome. Subjects from the GH group had higher STAI-T and RePi compared to the C group. A positive correlation between RePi and STAI-S, STAI-T, and systolic/diastolic blood pressure was found in both groups. There were more preterm deliveries in the GH group compared to the C group. A significant effect of STAI-T on body weight was observed in the C and GH group. There was a predictive effect of STAI-T and RePi on the C group, and STAI-S, STAI-T, diastolic blood pressure, and RePi on the GH group in terms of neonatal body weight. This study demonstrates an association between antenatal anxiety in GH women and increased fetal cerebral circulation in response to defined auditory stimulation.