Ljubisav Rakic

Behavioral and Adaptive Status in an Experimental Model of Alzheimer's Disease in Rats

Ten days after bilateral electrolytic lesions of nucleus basalis magnocellularis (NBM) we tested behavioral (spontaneous motor activity, acquisition and performance of two-way active avoidance, fear-response in open field test, foot shock induced aggression, depression-response in learned helplessness test) and adaptive status (body temperature at standard, hot and cold environment as well as cold restraint-induced gastric lesions) in adult male Wistar rats. Compared to intact control and sham-operated rats, the bilateral NBM-lesioned rats showed the significant impairment of learning behavior and reduced fear, aggression and depression as well as altered body temperature at standard and stressed conditions. Namely, it was established that body temperature in NBM-lesioned rats was significantly lower at standard laboratory conditions, but in these rats body temperature significantly was raised after exposing to cold and hot environment. On the other hand, spontaneous motor activity and number and length of cold restraint-induced gastric lesions (erosions and petechiae) in NBM-lesioned rats were similarly to those in both controls. It could be concluded that NBM plays a significant role in cognitive, emotional and adaptive processes in the rats.

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.

The combination of sulfinosine and 8-Cl-cAMP induces synergistic cell growth inhibition of the human neuroblastoma cell line in vitro

SummaryTo identify purine analogs that could be effective in treating neuroblastomas, we tested the anticancer properties of sulfinosine, 8-Cl-cAMP and 8-Cl-adenosine in the SK-N-SH cell line. First we examined the effects of these three agents on cell growth inhibition and cell viability by the BrdU and Sulforhodamine B assay. Treatment of SK-N-SH cells with increasing concentrations of these compounds led to a significant inhibition of cell proliferation and decrease of cell viability in a time- and dose-dependant manner at micromolar concentration (<10 μM). Treatment with a combination of sulfinosine and 8-Cl-cAMP resulted in synergistic effects on growth inhibition, cell cycle arrest and induction of apoptosis. Flow-cytometric analysis showed that 8-Cl-cAMP arrested the cells in the G0/G1 phase and sulfinosine blocked cell cycle progression at the G2/M stage, in contrast to the combined effects of both agents that did not arrest growth at any particular phase of the cell cycle. Further analysis of apoptosis induction demonstrated an increase from 17 to 24% of both early and late apoptotic cells and a very low percentage of necrotic cells. These results indicate that apoptosis was the predominant type of cell death after treatment of SK-N-SH cells with both substances, as well as with their combinations.

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.

Purine nucleoside analog--sulfinosine modulates diverse mechanisms of cancer progression in multi-drug resistant cancer cell lines.

Achieving an effective treatment of cancer is difficult, particularly when resistance to conventional chemotherapy is developed. P-glycoprotein (P-gp) activity governs multi-drug resistance (MDR) development in different cancer cell types. Identification of anti-cancer agents with the potential to kill cancer cells and at the same time inhibit MDR is important to intensify the search for novel therapeutic approaches. We examined the effects of sulfinosine (SF), a quite unexplored purine nucleoside analog, in MDR (P-gp over-expressing) non-small cell lung carcinoma (NSCLC) and glioblastoma cell lines (NCI-H460/R and U87-TxR, respectively). SF showed the same efficacy against MDR cancer cell lines and their sensitive counterparts. However, it was non-toxic for normal human keratinocytes (HaCaT). SF induced caspase-dependent apoptotic cell death and autophagy in MDR cancer cells. After SF application, reactive oxygen species (ROS) were generated and glutathione (GSH) concentration was decreased. The expression of key enzyme for GSH synthesis, gamma Glutamyl-cysteine-synthetase (γGCS) was decreased as well as the expression of gst-π mRNA. Consequently, SF significantly decreased the expression of hif-1α, mdr1 and vegf mRNAs even in hypoxic conditions. SF caused the inhibition of P-gp (coded by mdr1) expression and activity. The accumulation of standard chemotherapeutic agent – doxorubicin (DOX) was induced by SF in concentration- and time-dependent manner. The best effect of SF was obtained after 72 h when it attained the effect of known P-gp inhibitors (Dex-verapamil and tariquidar). Accordingly, SF sensitized the resistant cancer cells to DOX in subsequent treatment. Furthermore, SF decreased the experssion of vascular endothelial growth factor (VEGF) on mRNA and protein level and modulated its secretion. In conclusion, the effects on P-gp (implicated in pharmacokinetics and MDR), GSH (implicated in detoxification) and VEGF (implicated in tumor-angiogenesis and progression) qualify SF as multi-potent anti-cancer agent, which use must be considered, in particular for resistant malignancies.

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 Alzheimers 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 Alzheimers disease affect the sodium/potassium pump functionality which might be detected in peripheral blood erythrocyte membranes.

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.

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.

Humoral and cell-mediated immune responses following lesions of the nucleus basalis magnocellularis in the rat

The present study was undertaken to elucidate whether electrolytic lesions of nucleus basalis magnocellularis--NBM (an animal model of Alzheimers disease--AD) may influence humoral and cellular immune responses in adult male Wistar rats. For this purpose intact control (IC), sham-operated (SO) and NBM-lesioned rats were divided into two main groups: (1) rats immunized with sheep red blood cells (SRBC) for plaque-forming cell (PFC) response and anti-SRBC agglutinins, and (2) rats immunized with bovine serum albumin in complete Freunds adjuvant (BSA-CFA) for anti-BSA antibody production, Arthus and delayed hypersensitivity skin reaction to BSA. PFC responses and anti-SRBC agglutinins as well as diameter and expression of edema/induration of Arthus/delayed skin reaction and titer of anti-BSA antibody were significantly lower in NBM lesioned rats (compared to IC and SO). The results showed that in NBM-lesioned rats both the humoral and cellular immune responses were suppressed.

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.