Ana Parabucki

Dynamic changes in the expression pattern of ecto‐5′‐nucleotidase in the rat model of cortical stab injury

Traumatic injury induces massive release of ATP in the extracellular space, where it influences numerous aspects of neuronal, astrocytic, and microglial responses to injury by activating P2X and P2Y receptors. The extracellular ATP actions are controlled by the ectonucleotidase enzyme pathway, which hydrolyses ATP to adenosine at all neuronal and nonneuronal cell types. Adenosine activates its P1 receptors, which have important neuroprotective roles. The rate‐limiting enzyme in the ectonucleotidase pathway is ecto‐5′‐nucleotidase (e‐5NT), which catalyzes the final step of dephosphorylation of AMP to adenosine. The aim of the present study was to characterize the expression pattern and cellular distribution of e‐5NT in the perilesioned cortex at 4 hr and 1, 2, 7, and 15 days after unilateral cortical stab injury (CSI). Immunoblot and immunohistochemical studies showed that overall e‐5NT expression was lower 4 hr and 1 day postinjury and then gradually increased above the control levels. Double‐immunofluorescence studies further showed in control tissue the presence of the enzyme in the membranes surrounding neuronal somata and apical dendrites and less frequently in astrocytes. CSI caused a rapid (after 4 hr) and irreversible loss of the enzyme from neurons, accounting for a decrease in the overall enzyme expression. This was accompanied with a gradual increase in e‐5NT‐positive astrocytes, accounting for up‐regulation of the enzyme levels in the injured area. Thus, CSI induced dynamic changes in the expression pattern of e‐5NT that modify the ATP/adenosine ratio and the extent of P1 and P2 receptors activation and, therefore, outcome of the pathological processes after CSI.

Fluctuating vs. Continuous Exposure to H2O2: The Effects on Mitochondrial Membrane Potential, Intracellular Calcium, and NF-κB in Astroglia

The effects of H2O2 are widely studied in cell cultures and other in vitro systems. However, such investigations are performed with the assumption that H2O2 concentration is constant, which may not properly reflect in vivo settings, particularly in redox-turbulent microenvironments such as mitochondria. Here we introduced and tested a novel concept of fluctuating oxidative stress. We treated C6 astroglial cells and primary astrocytes with H2O2, using three regimes of exposure – continuous, as well as fluctuating at low or high rate, and evaluated mitochondrial membrane potential and other parameters of mitochondrial activity – respiration, reducing capacity, and superoxide production, as well as intracellular ATP, intracellular calcium, and NF-κB activation. When compared to continuous exposure, fluctuating H2O2 induced a pronounced hyperpolarization in mitochondria, whereas the activity of electron transport chain appears not to be significantly affected. H2O2 provoked a decrease of ATP level and an increase of intracellular calcium concentration, independently of the regime of treatment. However, fluctuating H2O2 induced a specific pattern of large-amplitude fluctuations of calcium concentration. An impact on NF-κB activation was observed for high rate fluctuations, whereas continuous and low rate fluctuating oxidative stress did not provoke significant effects. Presented results outline the (patho)physiological relevance of redox fluctuations.

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.

Repetitive Hyperbaric Oxygenation Attenuates Reactive Astrogliosis and Suppresses Expression of Inflammatory Mediators in the Rat Model of Brain Injury

The exact mechanisms by which treatment with hyperbaric oxygen (HBOT) exerts its beneficial effects on recovery after brain injury are still unrevealed. Therefore, in this study we investigated the influence of repetitive HBOT on the reactive astrogliosis and expression of mediators of inflammation after cortical stab injury (CSI). CSI was performed on male Wistar rats, divided into control, sham, and lesioned groups with appropriate HBO. The HBOT protocol was as follows: 10 minutes of slow compression, 2.5 atmospheres absolute (ATA) for 60 minutes, and 10 minutes of slow decompression, once a day for 10 consecutive days. Data obtained using real-time polymerase chain reaction, Western blot, and immunohistochemical and immunofluorescence analyses revealed that repetitive HBOT applied after the CSI attenuates reactive astrogliosis and glial scarring, and reduces expression of GFAP (glial fibrillary acidic protein), vimentin, and ICAM-1 (intercellular adhesion molecule-1) both at gene and tissue levels. In addition, HBOT prevents expression of CD40 and its ligand CD40L on microglia, neutrophils, cortical neurons, and reactive astrocytes. Accordingly, repetitive HBOT, by prevention of glial scarring and limiting of expression of inflammatory mediators, supports formation of more permissive environment for repair and regeneration.

Hyperbaric oxygenation alters temporal expression pattern of superoxide dismutase 2 after cortical stab injury in rats.

Aim To evaluate the effect of hyperbaric oxygen therapy (HBOT) on superoxide dismutase 2 (SOD2) expression pattern after the cortical stab injury (CSI). Methods CSI was performed on 88 male Wistar rats, divided into control, sham, lesioned, and HBO groups. HBOT protocol was the following: pressure applied was 2.5 absolute atmospheres, for 60 minutes, once a day for consecutive 3 or 10 days.‎ The pattern of SOD2 expression and cellular localization was analyzed using real-time polymerase chain reaction, Western blot, and double-label fluorescence immunohistochemistry. Neurons undergoing degeneration were visualized with Fluoro-Jade®B. Results CSI induced significant transient increase in SOD2 protein levels at day 3 post injury, which was followed by a reduction toward control levels at post-injury day 10. At the same time points, mRNA levels for SOD2 in the injured cortex were down-regulated. Exposure to HBO for 3 days considerably down-regulated SOD2 protein levels in the injured cortex, while after 10 days of HBOT an up-regulation of SOD2 was observed. HBOT significantly increased mRNA levels for SOD2 at both time points compared to the corresponding L group, but they were still lower than in controls. Double immunofluorescence staining revealed that 3 days after CSI, up-regulation of SOD2 was mostly due to an increased expression in reactive astrocytes surrounding the lesion site. HBOT attenuated SOD2 expression both in neuronal and astroglial cells. Fluoro-Jade®B labeling showed that HBOT significantly decreased the number of degenerating neurons in the injured cortex. Conclusion HBOT alters SOD2 protein and mRNA levels after brain injury in a time-dependent manner.

Ribavirin shows immunomodulatory effects on activated microglia.

Abstract Ribavirin (RBV) is synthetic purine nucleoside analogue, licensed as anti-viral drug that displays immunomodulatory actions on various immune cells. Our previous ex vivo studies have demonstrated immunosuppressive effects of RBV on reactive T-lymphocytes in experimental autoimmune encephalomyelitis. Here, we examined the effects of RBV on inflammatory response of microglia. RBV potency to down-regulate microglia inflammatory response was assessed by measuring microglia cell body size, and the production of nitric oxide (NO) and pro- and anti-inflammatory cytokines. RBV exerted cytotoxic effects on LPS-stimulated microglia, leaving non-stimulated microglia unaffected. The exposure of activated microglia to RBV led to: decrease in the level of NO as a result of decreased cell number, lower average cell surface, the reduction of membrane ruffling, the suppression of interleukin-6 release and promoted interleukin-10 production. On the other hand, RBV promoted LPS-induced interleukin-1 beta release. Our results imply that RBV is a complex immunomodulator showing both anti- and pro-inflammatory effects on activated microglia.

Effect of stab injury in the rat cerebral cortex on temporal pattern of expression of neuronal cytoskeletal proteins: An immunohistochemical study

Compelling evidence now points to the critical role of the cytoskeleton in neurodegeneration. In the present study, using an immunohistochemical approach, we have shown that cortical stab injury (CSI) in adult Wistar rats significantly affects temporal pattern of expression of neurofilament proteins (NFs), a major cytoskeleton components of neurons, and microtubule-associated proteins (MAP2). At 3 days post-injury (dpi) most of the NFs immunoreactivity was found in pyknotic neurons and in fragmentized axonal processes in the perilesioned cortex. These cytoskeletal alterations became more pronounced by 10dpi. At the subcellular level CSI also showed significant impact on NFs and MAP-2 expression. Thus, at 3dpi most of the dendrites disappeared, while large neuronal somata appeared like open circles pointing to membrane disintegration. Conversely, at 10dpi neuronal perikarya and a few new apical dendrites were strongly labeled. Since aberrant NF phosphorylation is a pathological hallmark of many human neurodegenerative disorders, as well as is found after stressor stimuli, the present results shed light into the expression of neurofilaments after the stab brain injury.

Real-Time PCR and Immunocytochemical Study of Chondroitin Sulfate Proteoglycans after Scratch Wounding in Cultured Astrocytes / PCR I IMUNOCITOHEMIJSKA STUDIJA EKSPRESIJE HONDROITIN-SULFATNIH PROTEOGLIKANA NAKON POVREDE ASTROCITA U KULTURI

Summary Background: Various in vivo and in vitro models have been described in order to elucidate the pathobiology underlying the traumatic brain injury (TBI) and test potentially suitable treatments. Since TBI is a complex disease, models differ in regard to the aspect of TBI that is being investigated. One of the used in vitro models is the scratch wound assay, first established as a reproducible, low-cost assay for the analysis of cell migration in vitro. The aim of the present study was to further investigate the relevancy of this model as a counter- part of in vivo TBI models. Methods: We have examined the astrocytic response to a mechanical injury in terms of expression of chondroitin sul- fate proteoglycans (CSPGs) - phosphacan, neurocan and brevican, using real-time PCR and immunocytochemistry. Results: Our results indicate that in vitro scratch wounding alters the expression profile of examined CSPGs. Four hours after the scratch injury of the astrocytic monolayer, real-time PCR analysis revealed upregulation of mRNA levels for phos- phacan (3-fold) and neurocan (2-fold), whereas brevican mRNA was downregulated (2-fold). Immunofluorescent sig- nal for phosphacan and neurocan was more intense in astro- cytes close to the injury site, while brevican was scarcely present in cultured astrocytes. Conclusions: Obtained results indicate that CSPGs are differ- entially expressed by astrocytes after scratch wounding, demonstrating that the scratch wound model might be suit- able for investigation of astrocyte-derived response to injury. Kratak sadržaj Uvod: Brojni in vivo i in vitro modeli opisani su sa ciljem da se rasvetle patobiološki procesi koji su osnova traumatske povrede mozga (TPM) i testiraju potencijalni tretmani. Imajući u vidu da je TPM kompleksno oboljenje, ovi modeli se medusobno razlikuju shodno aspektu TPM koji se ispitu- je. Jedan od in vitro modela je i povreda ćelijskog jednoslo- ja grebanjem (engl. »scratch wound« assay), isprva ustanov- ljen kao ponovljiv, jeftin test za analizu ćelijske migracije in vitro. Cilj ove študije je da se bliže ispita relevantnost ovog modela u odnosu na in vivo modele TPM. Metode: Da bi se istražio odgovor astrocita na mehaničku povredu, pradena je ekspresija odabranih hondroitin-sulfatnih proteoglikana (CSPG) - fosfakana, neurokana i brevikana, koriščenjem PCR u reálnom vremenu i imunocitohemije. Rezultati: Dobijeni rezultati su pokazali da in vitro povreda astrocitnog jednosloja menja profile ekspresije ispitivanih CSPG. Četiri sata nakon povrede, primena PCR u reálnom vremenu analize pokazala je povečanje nivoa iRNKza fos- fakan (trostruko) i neurokan (dvostruko), dok je iRNK za brevikan bila smanjena na polovinu kontrolne vrednosti. Imunofluorescentni signal poreklom od fosfakana i neuro- kana je bio intenzivniji u astrocitima bližim mestu povrede, dok je signal za brevikan bio slab kako u kontrolnoj, tako i u ozledenoj grupi. Zaključak: Dobijeni rezultati pokazuju da povreda izazvana grebanjem različito utiče na ekspresiju ispitivanih CSPG u astrocitima, što ukazuju da ovaj model može biti pogodan za ispitivanje odgovora astrocita na povredu.