Irena Lavrnja

Up-regulation of ectonucleotidase activity after cortical stab injury in rats

The objective of this study was to examine the changes in the activity and expression of ectonucleotidase enzymes in the model of unilateral cortical stab injury (CSI) in rat. The activities of ecto‐nucleoside triphosphate diphosphohydrolase 1 (NTPDase 1) and ecto 5′‐nucleotidase were assessed by measuring the levels of ATP, ADP and AMP hydrolysis in the crude membrane preparations obtained from injured left cortex, right cortex, left and right caudate nucleus, whole hippocampus and cerebellum. Significant increase in NTPDase and ecto 5′‐nucleotidase activities was observed in the injured cortex following CSI, whereas in other brain areas only an increase in ecto 5′‐nucleotidase activity was seen. Immunohistochemical analysis performed using antibodies specific to NTPDase 1 and ecto 5′‐nucleotidase demonstrated that CSI induced significant changes in enzyme expression around the injury site. Immunoreactivity patterns obtained for NTPDase 1 and ecto 5′‐nucleotidase were compared with those obtained for glial fibrillary acidic protein, as a marker of astrocytes and complement receptor type 3 (OX42), as a marker of microglia. Results suggest that up‐regulation of ectonucleotidase after CSI is catalyzed by cells that activate in response to injury, i.e. cells immunopositive for NTPDase 1 were predominantly microglial cells, whereas cells immunopositive for ecto 5′‐nucleotidase were predominantly astrocytes.

Benfotiamine Attenuates Inflammatory Response in LPS Stimulated BV-2 Microglia

Microglial cells are resident immune cells of the central nervous system (CNS), recognized as key elements in the regulation of neural homeostasis and the response to injury and repair. As excessive activation of microglia may lead to neurodegeneration, therapeutic strategies targeting its inhibition were shown to improve treatment of most neurodegenerative diseases. Benfotiamine is a synthetic vitamin B1 (thiamine) derivate exerting potentially anti-inflammatory effects. Despite the encouraging results regarding benfotiamine potential to alleviate diabetic microangiopathy, neuropathy and other oxidative stress-induced pathological conditions, its activities and cellular mechanisms during microglial activation have yet to be elucidated. In the present study, the anti-inflammatory effects of benfotiamine were investigated in lipopolysaccharide (LPS)-stimulated murine BV-2 microglia. We determined that benfotiamine remodels activated microglia to acquire the shape that is characteristic of non-stimulated BV-2 cells. In addition, benfotiamine significantly decreased production of pro-inflammatory mediators such as inducible form of nitric oxide synthase (iNOS) and NO; cyclooxygenase-2 (COX-2), heat-shock protein 70 (Hsp70), tumor necrosis factor alpha α (TNF-α), interleukin-6 (IL-6), whereas it increased anti-inflammatory interleukin-10 (IL-10) production in LPS stimulated BV-2 microglia. Moreover, benfotiamine suppressed the phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK) and protein kinase B Akt/PKB. Treatment with specific inhibitors revealed that benfotiamine-mediated suppression of NO production was via JNK1/2 and Akt pathway, while the cytokine suppression includes ERK1/2, JNK1/2 and Akt pathways. Finally, the potentially protective effect is mediated by the suppression of translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in the nucleus. Therefore, benfotiamine may have therapeutic potential for neurodegenerative diseases by inhibiting inflammatory mediators and enhancing anti-inflammatory factor production in activated microglia.

Immunohistological Determination of Ecto-nucleoside Triphosphate Diphosphohydrolase1 (NTPDase1) and 5′-nucleotidase in Rat Hippocampus Reveals Overlapping Distribution

Distribution of two enzymes involved in the ectonucleotidase enzyme chain, ecto-nucleoside triphosphate diphosphohydrolase1 (NTPDase1) and ecto-5′-nucleotidase, was assessed by immunohistochemistry in the rat hippocampus. Obtained results have shown co-expression of the enzymes in the hippocampal region, as well as wide and strikingly similar cellular distribution. Both enzymes were expressed at the surface of pyramidal neurons in the CA1 and CA2 sections, while cells in the CA3 section were faintly stained. The granule cell layer of the dentate gyrus was moderately stained for NTPDase1, as well as for ecto-5′-nucleotidase. Glial association for ecto-5′-nucleotidase was also observed, and fiber tracts were intensively stained for both enzymes. This is the first comparative study of NTPDase1 and ecto-5′-nucleotidase distribution in the rat hippocampus. Obtained results suggest that the broad overlapping distribution of these enzymes in neurons and glial cells reflects the functional importance of ectonucleotidase actions in the nervous system.

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.

Application of fractal and grey level co-occurrence matrix analysis in evaluation of brain corpus callosum and cingulum architecture.

This aim of this study was to assess the discriminatory value of fractal and grey level co-occurrence matrix (GLCM) analysis methods in standard microscopy analysis of two histologically similar brain white mass regions that have different nerve fiber orientation. A total of 160 digital micrographs of thionine-stained rat brain white mass were acquired using a Pro-MicroScan DEM-200 instrument. Eighty micrographs from the anterior corpus callosum and eighty from the anterior cingulum areas of the brain were analyzed. The micrographs were evaluated using the National Institutes of Health ImageJ software and its plugins. For each micrograph, seven parameters were calculated: angular second moment, inverse difference moment, GLCM contrast, GLCM correlation, GLCM variance, fractal dimension, and lacunarity. Using the Receiver operating characteristic analysis, the highest discriminatory value was determined for inverse difference moment (IDM) (area under the receiver operating characteristic (ROC) curve equaled 0.925, and for the criterion IDM≤0.610 the sensitivity and specificity were 82.5 and 87.5%, respectively). Most of the other parameters also showed good sensitivity and specificity. The results indicate that GLCM and fractal analysis methods, when applied together in brain histology analysis, are highly capable of discriminating white mass structures that have different axonal orientation.

Discriminatory ability of fractal and grey level co-occurrence matrix methods in structural analysis of hippocampus layers

Fractal and grey level co-occurrence matrix (GLCM) analysis represent two mathematical computer-assisted algorithms that are today thought to be able to accurately detect and quantify changes in tissue architecture during various physiological and pathological processes. However, despite their numerous applications in histology and pathology, their sensitivity, specificity and validity regarding evaluation of brain tissue remain unclear. In this article we present the results indicating that certain parameters of fractal and GLCM analysis have high discriminatory ability in distinguishing two morphologically similar regions of rat hippocampus: stratum lacunosum-moleculare and stratum radiatum. Fractal and GLCM algorithms were performed on a total of 240 thionine-stained hippocampus micrographs of 12 male Wistar albino rats. 120 digital micrographs represented stratum lacunosum-moleculare, and another 120 stratum radiatum. For each image, 7 parameters were calculated: fractal dimension, lacunarity, GLCM angular second moment, GLCM contrast, inverse difference moment, GLCM correlation, and GLCM variance. GLCM variance (VAR) resulted in the largest area under the Receiver operating characteristic (ROC) curve of 0.96, demonstrating an outstanding discriminatory power in analysis of stratum lacunosum-moleculare (average VAR equaled 478.1 ± 179.8) and stratum radiatum (average VAR of 145.9 ± 59.2, p < 0.0001). For the criterion VAR ≤ 227.5, sensitivity and specificity were 90% and 86.7%, respectively. GLCM correlation as a parameter also produced large area under the ROC curve of 0.95. Our results are in accordance with the findings of our previous study regarding brain white mass fractal and textural analysis. GLCM algorithm as an image analysis method has potentially high applicability in structural analysis of brain tissue cytoarcitecture.

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.

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.

Low-Dose Dexamethasone Treatment Promotes the Pro-Survival Signalling Pathway in the Adult Rat Prefrontal Cortex

Synthetic glucocorticoid dexamethasone (DEX), a highly potent anti‐inflammatory and immunosuppressive agent, is widely used in the treatment of brain cancer, as well as for inflammatory and autoimmune diseases. The present study aimed to determine whether low‐dose subchronic DEX treatment (100 μg/kg for eight consecutive days) exerts long‐term effects on apoptosis in the adult rat prefrontal cortex (PFC) by examining the expression of cell death‐promoting molecules [poly(ADP‐ribose) polymerase (PARP), p53, procaspase 3, cleaved caspase 3, Bax] and cell‐survival molecules (AKT, Bcl‐2). The results obtained revealed that body, thymus and adrenal gland weights, as well corticosterone levels, in the serum and PFC were reduced 1 day after the last DEX injection. In the PFC, DEX caused activation of AKT, augmentation of pro‐survival Bcl‐2 protein and an enhanced Bcl‐2/Bax protein ratio, as well Bcl‐2 translocation to the mitochondria. An unaltered profile with respect to the protein expression of apoptotic molecules PARP, procaspase 3 and Bax was detected, whereas p53 protein was decreased. Reverse transcriptase ‐polymerase chain reaction analysis showed a decrease of p53 mRNA levels and no significant difference in Bcl‐2 and Bax mRNA expression in DEX‐treated rats. Finally, a DNA fragmentation assay and Fluoro‐Jade staining demonstrated no considerable changes in apoptosis in the rat PFC. Our findings support the concept that low‐dose DEX creates a hypocorticoid state in the brain and also indicate that subchronic DEX treatment activates the pro‐survival signalling pathway but does not change apoptotic markers in the rat PFC. This mechanism might be relevant for the DEX‐induced apoptosis resistance observed during and after chemotherapy of patients with brain tumours.

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.