Adenosine 5'-triphosphate (ATP) and other nucleotides and nucleosides, such as adenosine, are ver... more Adenosine 5'-triphosphate (ATP) and other nucleotides and nucleosides, such as adenosine, are versatile signaling molecules involved in many physiological processes and pathological conditions in the nervous system, especially those with an inflammatory component. They can be released from nerve cells, glial cells, and vascular cells into the extracellular space where they exert their function via ionotropic (P2X) or metabotropic (P2Y) receptors. Signaling via extracellular nucleotides and adenosine is regulated by cell-surface located enzymes ectonucleotidases that hydrolyze the nucleotide to the respective nucleoside. This review summarizes a histochemical approach for detection of ectonucleotidase activities in the cryo-sections of brain tissue. The enzyme histochemistry (EHC) might be used as suitable replacement for immunohistochemistry, since it gives information about both localization and activity, thus adding a functional component to a classical histological approach. With this technique, it is possible to visualize spatial distribution and cell-specific localization of ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) and ecto-5'-nucleotidase (eN/CD73) activities during brain development, after different hormonal manipulations, during neurodegeneration, etc. EHC is also suitable for investigation of microglial morphology in different (patho)physiological conditions. Furthermore, the review describes how to quantify EHC results.
17β-Estradiol (E2) is a potent steroid hormone of both gonadal and neuronal origin that exerts pr... more 17β-Estradiol (E2) is a potent steroid hormone of both gonadal and neuronal origin that exerts profound effects on neuroplasticity in several brain regions. Dendritic spine and synapse formation and rearrangements are modulated and mediated by estrogens. In this chapter, we highlighted the essential background concerning the effects of E2 on synaptic rearrangements accompanied by synaptic plasticity in E2-sensitive brain regions that mediate learning and memory, i.e., cortex and hippocampus. We also address details of the molecular mechanisms underlying E2 regulation of spine dynamics. The proposed models of action of E2 overlaps with that for well-established synaptic modulators, such as adenosine. Thus, the possible synergistic effects of those two molecules in respect to synaptic rearrangement and plasticity were presented.
Ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2) hydrolyzes extracellular ATP to ADP, ... more Ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2) hydrolyzes extracellular ATP to ADP, which is the ligand for P2Y1,12,13receptors. The present study describes the distribution of NTPDase2 in adult rat brains in physiological conditions, and in hippocampal neurodegeneration induced by trimethyltin (TMT). The study also describes the regulation of NTPDase2 by inflammatory mediators in primary astrocytes and oligodendroglial cell line OLN93. In physiological conditions, NTPDase2 protein was most abundant in the hippocampus, where it was found in fibrous astrocytes and synaptic endings in the synaptic-rich hippocampal layers. In TMT-induced neurodegeneration, NTPDase2-mRNA acutely decreased at 2-dpi and then gradually recovered to the control level at 7-dpi and 21-dpi. As determined by immunohistochemistry and double immunofluorescence, the decrease was most pronounced in the dentate gyrus (DG), where NTPDase2 withdrew from the synaptic boutons in the polymorphic layer of DG,...
Astrocytes are the first responders to noxious stimuli by undergoing cellular and functional tran... more Astrocytes are the first responders to noxious stimuli by undergoing cellular and functional transition referred as reactive gliosis. Every acute or chronic disorder is accompanied by reactive gliosis, which could be categorized as detrimental (A1) of beneficial (A2) for nervous tissue. Another signature of pathological astrocyte activation is disturbed Ca 2+ homeostasis, a common denominator of neurodegenerative diseases. Deregulation of Ca + signaling further contributes to production of pro-inflammatory cytokines and reactive oxygen species. Trimethyltin (TMT) intoxication is a widely used model of hippocampal degeneration, sharing behavioral and molecular hallmarks of Alzheimer’s disease (AD), thus representing a useful model of AD-like pathology. However, the role of astrocyte in the etiopathology of TMT-induced degeneration as well as in AD is not fully understood. In an effort to elucidate the role of astrocytes in such pathological processes, we examined in vitro effects of TMT on primary cortical astrocytes. The application of a range of TMT concentrations (5, 10, 50, and 100 μM) revealed changes in [Ca 2 + ] i in a dose-dependent manner. Specifically, TMT-induced Ca 2 + transients were due to L-type voltage-gated calcium channels (VGCC). Additionally, TMT induced mitochondrial depolarization independent of extracellular Ca 2 + and disturbed antioxidative defense of astrocyte in several time points (4, 6, and 24 h) after 10 μM TMT intoxication, inducing oxidative and nitrosative stress. Chronic exposure (24 h) to 10 μM TMT induced strong upregulation of main pro-inflammatory factors, components of signaling pathways in astrocyte activation, A1 markers, and VGCC. Taken together, our results provide an insight into cellular and molecular events of astrocyte activation in chronic neuroinflammation.
The present study examined the involvement of purinergic signaling components in the rat model of... more The present study examined the involvement of purinergic signaling components in the rat model of hippocampal degeneration induced by trimethyltin (TMT) intoxication (8 mg/kg, single intraperitoneal injection), which results in behavioral and neurological dysfunction similar to neurodegenerative disorders. We investigated spatial and temporal patterns of ecto-nucleoside triphosphate diphosphohydrolase 1 (NTPDase1/CD39) and ecto-5′ nucleotidase (eN/CD73) activity, their cell-specific localization, and analyzed gene expression pattern and/or cellular localization of purinoreceptors and proinflammatory mediators associated with reactive glial cells. Our study demonstrated that all Iba1+ cells at the injured area, irrespective of their morphology, upregulated NTPDase1/CD39, while induction of eN/CD73 has been observed at amoeboid Iba1+ cells localized within the hippocampal neuronal layers with pronounced cell death. Marked induction of P2Y12R, P2Y6R, and P2X4-messenger RNA at the early...
Sustained activation of pro-apoptotic signaling due to a sudden and prolonged disturbance of cere... more Sustained activation of pro-apoptotic signaling due to a sudden and prolonged disturbance of cerebral blood circulation governs the neurodegenerative processes in prefrontal cortex (PFC) of rats whose common carotid arteries are permanently occluded. The adequate neuroprotective therapy should minimize the activation of toxicity pathways and increase the activity of endogenous protective mechanisms. Several neuroprotectants have been proposed, including progesterone (P 4 ). However, the underlying mechanism of its action in PFC following permanent bilateral occlusion of common carotid arteries is not completely investigated. We, thus herein, tested the impact of post-ischemic P 4 treatment (1.7 mg/kg for seven consecutive days) on previously reported aberrant neuronal morphology and amount of DNA fragmentation, as well as the expression of progesterone receptors along with the key elements of Akt/Erk/eNOS signal transduction pathway (Bax, Bcl-2, cytochrome C, caspase 3, PARP, and the level of nitric oxide). The obtained results indicate that potential amelioration of histological changes in PFC might be associated with the absence of activation of Bax/caspase 3 signaling cascade and the decline of DNA fragmentation. The study also provides the evidence that P 4 treatment in repeated regiment of administration might be effective in neuronal protection against ischemic insult due to re-establishment of the compromised action of Akt/Erk/eNOS-mediated signaling pathway and the upregulation of progesterone receptors.
Extracellular purine nucleotides, such as adenosine 5'-triphosphate (ATP), are important modu... more Extracellular purine nucleotides, such as adenosine 5'-triphosphate (ATP), are important modulators of hippocampal function and plasticity. In the extracellular space, ATP is inherently short-lived molecule, which undergoes rapid enzymatic degradation to adenosine by ectonucleotidases. Given that ectonucleotidases have distinct and overlapping distribution in the hippocampus, and as ovarian hormones participate in a formation, maturation, and a refinement of synaptic contacts, both during development and in adulthood, the present chapter summarizes known data about spatial distribution of selected ecto-enzymes and estradiol-induced effects on ectonucleotidases in the rat hippocampus.
Enzyme histochemistry is a valuable histological method which provides a connection between morph... more Enzyme histochemistry is a valuable histological method which provides a connection between morphology, activity, and spatial localization of investigated enzymes. Even though the method relies purely on arbitrary evaluations performed by the human eye, it is still wildly accepted and used in histo(patho)logy. Texture analysis emerged as an excellent tool for image quantification of subtle differences reflected in both spatial discrepancies and gray level values of pixels. The current study of texture analysis utilizes the gray-level co-occurrence matrix as a method for quantification of differences between ecto-5′-nucleotidase activities in healthy hippocampal tissue and tissue with marked neurodegeneration. We used the angular second moment, contrast (CON), correlation, inverse difference moment (INV), and entropy for texture analysis and receiver operating characteristic analysis with immunoblot and qualitative assessment of enzyme histochemistry as a validation. Our results strongly argue that co-occurrence matrix analysis could be used for the determination of fine differences in the enzyme activities with the possibility to ascribe those differences to regions or specific cell types. In addition, it emerged that INV and CON are especially useful parameters for this type of enzyme histochemistry analysis. We concluded that texture analysis is a reliable method for quantification of this descriptive technique, thus removing biases and adding it a quantitative dimension.
Adenosine 5'-triphosphate (ATP) and other nucleotides and nucleosides, such as adenosine, are ver... more Adenosine 5'-triphosphate (ATP) and other nucleotides and nucleosides, such as adenosine, are versatile signaling molecules involved in many physiological processes and pathological conditions in the nervous system, especially those with an inflammatory component. They can be released from nerve cells, glial cells, and vascular cells into the extracellular space where they exert their function via ionotropic (P2X) or metabotropic (P2Y) receptors. Signaling via extracellular nucleotides and adenosine is regulated by cell-surface located enzymes ectonucleotidases that hydrolyze the nucleotide to the respective nucleoside. This review summarizes a histochemical approach for detection of ectonucleotidase activities in the cryo-sections of brain tissue. The enzyme histochemistry (EHC) might be used as suitable replacement for immunohistochemistry, since it gives information about both localization and activity, thus adding a functional component to a classical histological approach. With this technique, it is possible to visualize spatial distribution and cell-specific localization of ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) and ecto-5'-nucleotidase (eN/CD73) activities during brain development, after different hormonal manipulations, during neurodegeneration, etc. EHC is also suitable for investigation of microglial morphology in different (patho)physiological conditions. Furthermore, the review describes how to quantify EHC results.
17β-Estradiol (E2) is a potent steroid hormone of both gonadal and neuronal origin that exerts pr... more 17β-Estradiol (E2) is a potent steroid hormone of both gonadal and neuronal origin that exerts profound effects on neuroplasticity in several brain regions. Dendritic spine and synapse formation and rearrangements are modulated and mediated by estrogens. In this chapter, we highlighted the essential background concerning the effects of E2 on synaptic rearrangements accompanied by synaptic plasticity in E2-sensitive brain regions that mediate learning and memory, i.e., cortex and hippocampus. We also address details of the molecular mechanisms underlying E2 regulation of spine dynamics. The proposed models of action of E2 overlaps with that for well-established synaptic modulators, such as adenosine. Thus, the possible synergistic effects of those two molecules in respect to synaptic rearrangement and plasticity were presented.
Ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2) hydrolyzes extracellular ATP to ADP, ... more Ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2) hydrolyzes extracellular ATP to ADP, which is the ligand for P2Y1,12,13receptors. The present study describes the distribution of NTPDase2 in adult rat brains in physiological conditions, and in hippocampal neurodegeneration induced by trimethyltin (TMT). The study also describes the regulation of NTPDase2 by inflammatory mediators in primary astrocytes and oligodendroglial cell line OLN93. In physiological conditions, NTPDase2 protein was most abundant in the hippocampus, where it was found in fibrous astrocytes and synaptic endings in the synaptic-rich hippocampal layers. In TMT-induced neurodegeneration, NTPDase2-mRNA acutely decreased at 2-dpi and then gradually recovered to the control level at 7-dpi and 21-dpi. As determined by immunohistochemistry and double immunofluorescence, the decrease was most pronounced in the dentate gyrus (DG), where NTPDase2 withdrew from the synaptic boutons in the polymorphic layer of DG,...
Astrocytes are the first responders to noxious stimuli by undergoing cellular and functional tran... more Astrocytes are the first responders to noxious stimuli by undergoing cellular and functional transition referred as reactive gliosis. Every acute or chronic disorder is accompanied by reactive gliosis, which could be categorized as detrimental (A1) of beneficial (A2) for nervous tissue. Another signature of pathological astrocyte activation is disturbed Ca 2+ homeostasis, a common denominator of neurodegenerative diseases. Deregulation of Ca + signaling further contributes to production of pro-inflammatory cytokines and reactive oxygen species. Trimethyltin (TMT) intoxication is a widely used model of hippocampal degeneration, sharing behavioral and molecular hallmarks of Alzheimer’s disease (AD), thus representing a useful model of AD-like pathology. However, the role of astrocyte in the etiopathology of TMT-induced degeneration as well as in AD is not fully understood. In an effort to elucidate the role of astrocytes in such pathological processes, we examined in vitro effects of TMT on primary cortical astrocytes. The application of a range of TMT concentrations (5, 10, 50, and 100 μM) revealed changes in [Ca 2 + ] i in a dose-dependent manner. Specifically, TMT-induced Ca 2 + transients were due to L-type voltage-gated calcium channels (VGCC). Additionally, TMT induced mitochondrial depolarization independent of extracellular Ca 2 + and disturbed antioxidative defense of astrocyte in several time points (4, 6, and 24 h) after 10 μM TMT intoxication, inducing oxidative and nitrosative stress. Chronic exposure (24 h) to 10 μM TMT induced strong upregulation of main pro-inflammatory factors, components of signaling pathways in astrocyte activation, A1 markers, and VGCC. Taken together, our results provide an insight into cellular and molecular events of astrocyte activation in chronic neuroinflammation.
The present study examined the involvement of purinergic signaling components in the rat model of... more The present study examined the involvement of purinergic signaling components in the rat model of hippocampal degeneration induced by trimethyltin (TMT) intoxication (8 mg/kg, single intraperitoneal injection), which results in behavioral and neurological dysfunction similar to neurodegenerative disorders. We investigated spatial and temporal patterns of ecto-nucleoside triphosphate diphosphohydrolase 1 (NTPDase1/CD39) and ecto-5′ nucleotidase (eN/CD73) activity, their cell-specific localization, and analyzed gene expression pattern and/or cellular localization of purinoreceptors and proinflammatory mediators associated with reactive glial cells. Our study demonstrated that all Iba1+ cells at the injured area, irrespective of their morphology, upregulated NTPDase1/CD39, while induction of eN/CD73 has been observed at amoeboid Iba1+ cells localized within the hippocampal neuronal layers with pronounced cell death. Marked induction of P2Y12R, P2Y6R, and P2X4-messenger RNA at the early...
Sustained activation of pro-apoptotic signaling due to a sudden and prolonged disturbance of cere... more Sustained activation of pro-apoptotic signaling due to a sudden and prolonged disturbance of cerebral blood circulation governs the neurodegenerative processes in prefrontal cortex (PFC) of rats whose common carotid arteries are permanently occluded. The adequate neuroprotective therapy should minimize the activation of toxicity pathways and increase the activity of endogenous protective mechanisms. Several neuroprotectants have been proposed, including progesterone (P 4 ). However, the underlying mechanism of its action in PFC following permanent bilateral occlusion of common carotid arteries is not completely investigated. We, thus herein, tested the impact of post-ischemic P 4 treatment (1.7 mg/kg for seven consecutive days) on previously reported aberrant neuronal morphology and amount of DNA fragmentation, as well as the expression of progesterone receptors along with the key elements of Akt/Erk/eNOS signal transduction pathway (Bax, Bcl-2, cytochrome C, caspase 3, PARP, and the level of nitric oxide). The obtained results indicate that potential amelioration of histological changes in PFC might be associated with the absence of activation of Bax/caspase 3 signaling cascade and the decline of DNA fragmentation. The study also provides the evidence that P 4 treatment in repeated regiment of administration might be effective in neuronal protection against ischemic insult due to re-establishment of the compromised action of Akt/Erk/eNOS-mediated signaling pathway and the upregulation of progesterone receptors.
Extracellular purine nucleotides, such as adenosine 5'-triphosphate (ATP), are important modu... more Extracellular purine nucleotides, such as adenosine 5'-triphosphate (ATP), are important modulators of hippocampal function and plasticity. In the extracellular space, ATP is inherently short-lived molecule, which undergoes rapid enzymatic degradation to adenosine by ectonucleotidases. Given that ectonucleotidases have distinct and overlapping distribution in the hippocampus, and as ovarian hormones participate in a formation, maturation, and a refinement of synaptic contacts, both during development and in adulthood, the present chapter summarizes known data about spatial distribution of selected ecto-enzymes and estradiol-induced effects on ectonucleotidases in the rat hippocampus.
Enzyme histochemistry is a valuable histological method which provides a connection between morph... more Enzyme histochemistry is a valuable histological method which provides a connection between morphology, activity, and spatial localization of investigated enzymes. Even though the method relies purely on arbitrary evaluations performed by the human eye, it is still wildly accepted and used in histo(patho)logy. Texture analysis emerged as an excellent tool for image quantification of subtle differences reflected in both spatial discrepancies and gray level values of pixels. The current study of texture analysis utilizes the gray-level co-occurrence matrix as a method for quantification of differences between ecto-5′-nucleotidase activities in healthy hippocampal tissue and tissue with marked neurodegeneration. We used the angular second moment, contrast (CON), correlation, inverse difference moment (INV), and entropy for texture analysis and receiver operating characteristic analysis with immunoblot and qualitative assessment of enzyme histochemistry as a validation. Our results strongly argue that co-occurrence matrix analysis could be used for the determination of fine differences in the enzyme activities with the possibility to ascribe those differences to regions or specific cell types. In addition, it emerged that INV and CON are especially useful parameters for this type of enzyme histochemistry analysis. We concluded that texture analysis is a reliable method for quantification of this descriptive technique, thus removing biases and adding it a quantitative dimension.
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