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1. Astrocytes of the eye and optic nerve: heterogeneous populations with unique functions mediate axonal resilience and vulnerability to glaucoma

Author

Paul F. Cullen and Daniel Sun

Subjects
optic nerve head astrocytes, glaucoma, reactive astrocytes, neurodegeneration, astrocyte heterogeneity, astrocyte metabolism, Medicine
Abstract

The role of glia, particularly astrocytes, in mediating the central nervous system’s response to injury and neurodegenerative disease is an increasingly well studied topic. These cells perform myriad support functions under physiological conditions but undergo behavioral changes – collectively referred to as ‘reactivity’ – in response to the disruption of neuronal homeostasis from insults, including glaucoma. However, much remains unknown about how reactivity alters disease progression – both beneficially and detrimentally – and whether these changes can be therapeutically modulated to improve outcomes. Historically, the heterogeneity of astrocyte behavior has been insufficiently addressed under both physiological and pathological conditions, resulting in a fragmented and often contradictory understanding of their contributions to health and disease. Thanks to increased focus in recent years, we now know this heterogeneity encompasses both intrinsic variation in physiological function and insult-specific changes that vary between pathologies. Although previous studies demonstrate astrocytic alterations in glaucoma, both in human disease and animal models, generally these findings do not conclusively link astrocytes to causative roles in neuroprotection or degeneration, rather than a subsequent response. Efforts to bolster our understanding by drawing on knowledge of brain astrocytes has been constrained by the primacy in the literature of findings from peri-synaptic ‘gray matter’ astrocytes, whereas much early degeneration in glaucoma occurs in axonal regions populated by fibrous ‘white matter’ astrocytes. However, by focusing on findings from astrocytes of the anterior visual pathway – those of the retina, unmyelinated optic nerve head, and myelinated optic nerve regions – we aim to highlight aspects of their behavior that may contribute to axonal vulnerability and glaucoma progression, including roles in mitochondrial turnover and energy provisioning. Furthermore, we posit that astrocytes of the retina, optic nerve head and myelinated optic nerve, although sharing developmental origins and linked by a network of gap junctions, may be best understood as distinct populations residing in markedly different niches with accompanying functional specializations. A closer investigation of their behavioral repertoires may elucidate not only their role in glaucoma, but also mechanisms to induce protective behaviors that can impede the progressive axonal damage and retinal ganglion cell death that drive vision loss in this devastating condition.

Published
2023
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2. Lysyl oxidase like-1 deficiency in optic nerve head astrocytes elicits reactive astrocytosis and alters functional effects of astrocyte derived exosomes.

Author

Hariani, Harsh N., Ghosh, Anita K., Rosen, Sasha M., Tso, Huen-Yee, Kessinger, Cassidy, Zhang, Chongyu, Jones, W. Keith, Sappington, Rebecca M., Mitchell, Claire H., Stubbs, Evan B., Rao, Vidhya R., and Kaja, Simon

Subjects
*LYSYL oxidase, *OPTIC nerve, *GLIOSIS, *EXOSOMES, *ASTROCYTES
Abstract

Glaucoma is a multifactorial progressive ocular pathology that manifests clinically with damage to the optic nerve (ON) and the retina, ultimately leading to blindness. The optic nerve head (ONH) shows the earliest signs of glaucoma pathology, and therefore, is an attractive target for drug discovery. The goal of this study was to elucidate the effects of reactive astrocytosis on the elastin metabolism pathway in primary rat optic nerve head astrocytes (ONHA), the primary glial cell type in the unmyelinated ONH. Following exposure to static equibiaxial mechanical strain, we observed prototypic molecular and biochemical signatures of reactive astrocytosis that were associated with a decrease in lysyl oxidase like 1 (Loxl1) expression and a concomitant decrease in elastin (Eln) gene expression. We subsequently investigated the role of Loxl1 in reactive astrocytosis by generating primary rat ONHA cultures with ∼50% decreased Loxl1 expression. Our results suggest that reduced Loxl1 expression is sufficient to elicit molecular signatures of elastinopathy in ONHA. Astrocyte derived exosomes (ADE) significantly increased the length of primary neurites of primary neurons in vitro. In contrast, ADE from Loxl1-deficient ONHA were deficient of trophic effects on neurite outgrowth in vitro , positing that Loxl1 dysfunction and the ensuing impaired elastin synthesis during reactive astrocytosis in the ONH may contribute to impaired neuron-glia signaling in glaucoma. Our data support a role of dysregulated Loxl1 function in eliciting reactive astrocytosis in glaucoma subtypes associated with increased IOP, even in the absence of genetic polymorphisms in LOXL1 typically associated with exfoliation glaucoma. This suggests the need for a paradigm shift toward considering lysyl oxidase activity and elastin metabolism and signaling as contributors to an altered secretome of the ONH that may lead to the progression of glaucomatous changes. Future research is needed to investigate cargo of exosomes in the context of reactive astrocytosis and identify the pathways leading to the observed transcriptome changes during reactive astrocytosis. • Reactive astrocytosis reduces expression of lysyl oxidase like 1 (Loxl1) in ONHA. • Loxl1 knock-down is sufficient to induce reactive astrocytosis in ONHA. • ONHA-derived exosomes are readily taken up by ONHA and cultured neurons. • ONHA-derived exosomes increase neurite outgrowth of cultured primary neurons. • Exosomes from Loxl1-deficient ONHA have diminished trophic effects on neurons. [ABSTRACT FROM AUTHOR]

Published
2024
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3. Inhibition of oxidative stress by coenzyme Q10 increases mitochondrial mass and improves bioenergetic function in optic nerve head astrocytes.

Author

Noh, YH, Kim, K-Y, Shim, MS, Choi, S-H, Choi, S, Ellisman, MH, Weinreb, RN, Perkins, GA, and Ju, W-K

Subjects
Astrocytes, Optic Disk, Cells, Cultured, Mitochondria, Animals, Rats, Rats, Sprague-Dawley, Ubiquinone, Multiprotein Complexes, Mitochondrial Proteins, Transcription Factors, Energy Metabolism, Oxidative Phosphorylation, Oxidative Stress, Image Processing, Computer-Assisted, Female, Mitochondrial Turnover, oxidative stress, optic nerve head astrocytes, mitochondrial dysfunction, coenzyme Q10, glaucoma, OXPHOS complex, Cells, Cultured, Sprague-Dawley, Image Processing, Computer-Assisted, Neurosciences, Neurodegenerative, Eye Disease and Disorders of Vision, 5.1 Pharmaceuticals, Generic Health Relevance, Neurological, Oncology and Carcinogenesis, Biochemistry and Cell Biology
Abstract

Oxidative stress contributes to dysfunction of glial cells in the optic nerve head (ONH). However, the biological basis of the precise functional role of mitochondria in this dysfunction is not fully understood. Coenzyme Q10 (CoQ10), an essential cofactor of the electron transport chain and a potent antioxidant, acts by scavenging reactive oxygen species (ROS) for protecting neuronal cells against oxidative stress in many neurodegenerative diseases. Here, we tested whether hydrogen peroxide (100 μM H2O2)-induced oxidative stress alters the mitochondrial network, oxidative phosphorylation (OXPHOS) complex (Cx) expression and bioenergetics, as well as whether CoQ10 can ameliorate oxidative stress-mediated alterations in mitochondria of the ONH astrocytes in vitro. Oxidative stress triggered the activation of ONH astrocytes and the upregulation of superoxide dismutase 2 (SOD2) and heme oxygenase-1 (HO-1) protein expression in the ONH astrocytes. In contrast, CoQ10 not only prevented activation of ONH astrocytes but also significantly decreased SOD2 and HO-1 protein expression in the ONH astrocytes against oxidative stress. Further, CoQ10 prevented a significant loss of mitochondrial mass by increasing mitochondrial number and volume density and by preserving mitochondrial cristae structure, as well as promoted mitofilin and peroxisome-proliferator-activated receptor-γ coactivator-1 protein expression in the ONH astrocyte, suggesting an induction of mitochondrial biogenesis. Finally, oxidative stress triggered the upregulation of OXPHOS Cx protein expression, as well as reduction of cellular adeonsine triphosphate (ATP) production and increase of ROS generation in the ONH astocytes. However, CoQ10 preserved OXPHOS protein expression and cellular ATP production, as well as decreased ROS generation in the ONH astrocytes. On the basis of these observations, we suggest that oxidative stress-mediated mitochondrial dysfunction or alteration may be an important pathophysiological mechanism in the dysfunction of ONH astrocytes. CoQ10 may provide new therapeutic potentials and strategies for protecting ONH astrocytes against oxidative stress-mediated mitochondrial dysfunction or alteration in glaucoma and other optic neuropathies.

Published
2013

4. Optimization of Cell Viability Assays for Screening Glioprotective Compounds in Primary Rat Optic Nerve Head Astrocytes

Author

Yuliya A. Naumchuk, Vitaliy B. Maksymenko, and Simon Kaja

Subjects
Glaucoma, Optic nerve head astrocytes, Oxidative stress, Reactive oxygen species, Viability assays, Chemical technology, TP1-1185, Science
Abstract

Background. Optic nerve head astrocytes (ONHAs) are the major glia cell type in the non-myelinated optic nerve head where they contribute to extracellular matrix synthesis. Pathological changes in glaucoma include reactive astrocytosis, a process characterized by altered astrocyte gene and protein expression and extracellular matrix remodeling. ONHAs are highly sensitive to mechanical and oxidative stress resulting in the initiation of axon damage early during pathogenesis. Furthermore, ONHAs are crucial for the maintenance of retinal ganglion cell physiology and function. Therefore, glioprotective strategies with the goal to preserve and/or restore the structural and functional viability of ONHA to slow glaucoma and related pathologies are of high clinical relevance. Objective. The aim of the paper is the development of standardized methods for the systematic advancement of glioprotective strategies using plate reader-based assays determining cellular viability, plate-reader based proliferation and the intracellular redox state. Methods. In our work we used primary culture of OHNAs as a model system. The oxidative stress was induced by tBHP. Reactive oxygen species (ROS) were measured by the DCDFA assay. For cell viability tests we used the optimized lactate dehydrogenase (LDH) release assay, as well as the MTT and Calcein-AM uptake assays. Results. The half-maximal effect (EC50) of tBHP on OHNAs ROS levels in the DCFDA assay was 192.1 ± 15.7 μM. The measurement of cellular viability of OHNAs after tBHP-induced oxidative stress showed EC50 = 156.9 ± 3.8 µM in the Calcein-AM uptake assay. In the MTT assay, the EC50 for tBHP was 138.1 ± 1.4 µM, and shifted to 192.7 ± 2.8 µM with 100 μM Trolox pre-treatment of OHNA. In the LDH release assay, the EC50 for tBHP was 146.9 ± 4.9 µM and 246.3 ± 7.3 µM for the control and Trolox conditions respectively. Conclusions. Our results provide feasibility data for the plate-reader based screening for novel glioprotectants using primary ONHA culture.

Published
2017
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5. Differential Activation of Glioprotective Intracellular Signaling Pathways in Primary Optic Nerve Head Astrocytes after Treatment with Different Classes of Antioxidants

Author

Anita K. Ghosh, Vidhya R. Rao, Victoria J. Wisniewski, Alexandra D. Zigrossi, Jamie Floss, Peter Koulen, Evan B Stubbs, and Simon Kaja

Subjects
glioprotection, xanthohumol, resveratrol, manganese porphyrin, optic nerve head astrocytes, phase II antioxidant enzymes, Therapeutics. Pharmacology, RM1-950
Abstract

Optic nerve head astrocytes are the specialized glia cells that provide structural and trophic support to the optic nerve head. In response to cellular injury, optic nerve head astrocytes undergo reactive astrocytosis, the process of cellular activation associated with cytoskeletal remodeling, increases in the rate of proliferation and motility, and the generation of Reactive Oxygen Species. Antioxidant intervention has previously been proposed as a therapeutic approach for glaucomatous optic neuropathy, however, little is known regarding the response of optic nerve head astrocytes to antioxidants under physiological versus pathological conditions. The goal of this study was to determine the effects of three different antioxidants, manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin (Mn-TM-2-PyP), resveratrol and xanthohumol in primary optic nerve head astrocytes. Effects on the expression of the master regulator nuclear factor erythroid 2-related factor 2 (Nrf2), the antioxidant enzyme, manganese-dependent superoxide dismutase 2 (SOD2), and the pro-oxidant enzyme, nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4), were determined by quantitative immunoblotting. Furthermore, efficacy in preventing chemically and reactive astrocytosis-induced increases in cellular oxidative stress was quantified using cell viability assays. The results were compared to the effects of the prototypic antioxidant, Trolox. Antioxidants elicited highly differential changes in the expression levels of Nrf2, SOD2, and NOX4. Notably, Mn-TM-2-PyP increased SOD2 expression eight-fold, while resveratrol increased Nrf2 expression three-fold. In contrast, xanthohumol exerted no statistically significant changes in expression levels. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) uptake and lactate dehydrogenase (LDH) release assays were performed to assess cell viability after chemically and reactive astrocytosis-induced oxidative stress. Mn-TM-2-PyP exerted the most potent glioprotection by fully preventing the loss of cell viability, whereas resveratrol and xanthohumol partially restored cell viability. Our data provide the first evidence for a well-developed antioxidant defense system in optic nerve head astrocytes, which can be pharmacologically targeted by different classes of antioxidants.

Published
2020
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6. Relationship between Sigma-1 receptor and BDNF in the visual system.

Author

Mysona, Barbara A., Zhao, Jing, Smith, Sylvia, and Bollinger, Kathryn E.

Subjects
*GLAUCOMA, *BRAIN-derived neurotrophic factor, *NEUROPATHY, *RETINAL ganglion cells, *PROTEIN expression
Abstract

Glaucoma is an incurable optic neuropathy characterized by dysfunction and death of retinal ganglion cells (RGCs). Brain derived neurotrophic factor (BDNF) is an essential neurotrophin that supports RGC function and survival. Despite BDNF's importance, our knowledge of molecular mechanisms that modulate BDNF processing and secretion is incomplete. Sigma-1 receptor (S1R) is associated with increased BDNF in hippocampus and with BDNF secretion by brain-derived astrocytes and neuronal cell lines. Much less is known about the relationship between S1R and BDNF in the visual system. Here, we examine how S1R activation and deletion alter expression of mature BDNF (mBDNF) and proBDNF in retina and cultured optic nerve head (ONH) astrocytes. For S1R activation, the S1R agonist (+)-pentazocine (PTZ, 0.5 mg/kg) was administered by intraperitoneal injection to C57BL/6J mice, 3 times per week, for 5 weeks. Expression of proBDNF and mBDNF was also examined in S1R knockout and age-matched C57BL/6J mice. In vitro , cultured ONH astrocytes were treated with 3 μM PTZ for 24 h followed by collection of media and ONH astrocyte lysates. Results showed that treatment with (+)-PTZ increased mBDNF protein in both retina and hippocampus. In contrast, S1R deletion was associated with retinal mBDNF deficits. In ONH astrocytes S1R agonist (+)-PTZ significantly increased levels of secreted BDNF and proBDNF in cell lysates. These findings support a role for S1R in the modulation of BDNF levels within the retina and optic nerve head. Treatment with S1R agonists might provide benefit in diseases such as glaucoma by increasing BDNF levels from endogenous sources. [ABSTRACT FROM AUTHOR]

Published
2018
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7. ОПТИМІЗАЦІЯ ТЕСТІВ ЖИТТЄЗДАТНОСТІ КЛІ&#1058...

Author

Наумчук, Ю. А., Максименко, В. Б., and Кая, С.

Abstract

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Published
2017
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8. Identification and localization of lamina cribrosa cells in the human optic nerve head.

Author

Tovar-Vidales, Tara, Wordinger, Robert J., and Clark, Abbot F.

Subjects
*GLAUCOMA diagnosis, *OPTIC disc abnormalities, *RETINAL ganglion cells, *LAMINA epithelialis, *GLIAL fibrillary acidic protein, *EXTRACELLULAR matrix
Abstract

One of the central features of glaucoma is progressive cupping and excavation of the optic nerve head (ONH). Unmyelinated retinal ganglion cell (RGC) axons exit the eye through the ONH, which is supported by the lamina cribrosa (LC) consisting of plates of connective tissue with channels for bundles of RGC axons. The LC progressively remodels during glaucoma, but the cellular and molecular mechanisms responsible for this remodeling are poorly understood. Two major cell types have been isolated and cultured from the human ONH, which differ in their characteristics. Glial fibrillary acidic protein (GFAP) positive ONH astrocytes are the major cell type and are reactive in glaucoma. GFAP negative LC cells are the second major cell type isolated from the human ONH, and in contrast to ONH astrocytes, are α-smooth muscle actin (α-SMA) positive. Although a number of in vitro studies have been conducted with LC cells, to date there has been no direct evidence for these cells in situ in the human ONH. We used GFAP and α-SMA immunofluorescent staining of human eyes to clearly demonstrate the presence of not only ONH astrocytes within the human ONH, but also LC cells within the cribriform (e.g. laminar) plates/beams of the LC region. Both of these cell types likely play important roles in the homeostatic maintenance of the ONH and pathogenic changes that occur in primary open angle glaucoma (POAG). [ABSTRACT FROM AUTHOR]

Published
2016
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9. Altered expression of 3α-hydroxysteroid dehydrogenases in human glaucomatous optic nerve head astrocytes

Author

O.A Agapova, P Yang, W-H Wang, D.A Lane, A.F Clark, B.I Weinstein, and M.R Hernandez

Subjects
Optic nerve head astrocytes, Glaucoma, Optic nerve transection, 3α-HSD, Neurosteroid, AKR1C1, Neurosciences. Biological psychiatry. Neuropsychiatry, RC321-571
Abstract

3α-Hydroxysteroid dehydrogenase (3α-HSD) isoforms (AKR1C1–AKR1C4) are aldo-keto reductases that metabolize steroids and other substances in many tissues including the CNS. Here we demonstrated that in glaucomatous human optic nerve heads, increased expression of 3α-HSD was localized to reactive astrocytes in the lamina cribrosa. Similar, optic nerve head astrocytes exhibited increased expression of 3α-HSD in response to elevated intraocular pressure in a monkey model of experimental glaucoma, but not in monkeys with unilateral optic nerve transection. In vitro, glaucomatous optic nerve head astrocytes expressed higher levels of AKR1C1, AKR1C2, and AKR1C3 mRNA, than normal astrocytes, with significant differential increase of AKR1C2 expression, and exhibited higher enzymatic activity forming 3α-androstanediol a well-recognized neurosteroid. Normal astrocytes exposed to elevated hydrostatic pressure selectively increased AKR1C2 expression. Our findings of increased expression of 3α-HSDs in glaucomatous optic nerve head astrocytes offer new insights into possible roles for neurosteroids in the pathophysiology of glaucoma.

Published
2003
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10. Astrocytes of the eye and optic nerve: heterogeneous populations with unique functions mediate axonal resilience and vulnerability to glaucoma.

Author

Cullen PF and Sun D

Abstract

The role of glia, particularly astrocytes, in mediating the central nervous system's response to injury and neurodegenerative disease is an increasingly well studied topic. These cells perform myriad support functions under physiological conditions but undergo behavioral changes - collectively referred to as 'reactivity' - in response to the disruption of neuronal homeostasis from insults, including glaucoma. However, much remains unknown about how reactivity alters disease progression - both beneficially and detrimentally - and whether these changes can be therapeutically modulated to improve outcomes. Historically, the heterogeneity of astrocyte behavior has been insufficiently addressed under both physiological and pathological conditions, resulting in a fragmented and often contradictory understanding of their contributions to health and disease. Thanks to increased focus in recent years, we now know this heterogeneity encompasses both intrinsic variation in physiological function and insult-specific changes that vary between pathologies. Although previous studies demonstrate astrocytic alterations in glaucoma, both in human disease and animal models, generally these findings do not conclusively link astrocytes to causative roles in neuroprotection or degeneration, rather than a subsequent response. Efforts to bolster our understanding by drawing on knowledge of brain astrocytes has been constrained by the primacy in the literature of findings from peri-synaptic 'gray matter' astrocytes, whereas much early degeneration in glaucoma occurs in axonal regions populated by fibrous 'white matter' astrocytes. However, by focusing on findings from astrocytes of the anterior visual pathway - those of the retina, unmyelinated optic nerve head, and myelinated optic nerve regions - we aim to highlight aspects of their behavior that may contribute to axonal vulnerability and glaucoma progression, including roles in mitochondrial turnover and energy provisioning. Furthermore, we posit that astrocytes of the retina, optic nerve head and myelinated optic nerve, although sharing developmental origins and linked by a network of gap junctions, may be best understood as distinct populations residing in markedly different niches with accompanying functional specializations. A closer investigation of their behavioral repertoires may elucidate not only their role in glaucoma, but also mechanisms to induce protective behaviors that can impede the progressive axonal damage and retinal ganglion cell death that drive vision loss in this devastating condition., Competing Interests: Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Published
2023
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11. Plate reader-based cell viability assays for glioprotection using primary rat optic nerve head astrocytes.

Author

Kaja, Simon, Payne, Andrew J., Yuliya Naumchuk, Levy, Deborah, Zaidi, Danish H., Altman, Alexa M., Nawazish, Saba, Ghuman, Jasleen K., Gerdes, Bryan C., Moore, Mark A., and Koulen, Peter

Subjects
*CELL survival, *OPTIC nerve, *ASTROCYTES, *EXTRACELLULAR matrix, *PROTEIN expression, *LABORATORY rats
Abstract

Optic nerve head astrocytes (ONHAs) are the major glia cell type in the non-myelinated optic nerve head where they contribute critically to extracellular matrix synthesis during development and throughout life. In glaucoma, and in related disorders affecting the optic nerve and the optic nerve head, pathological changes include altered astrocyte gene and protein expression resulting in their activation and extracellular matrix remodeling. ONHAs are highly sensitive to mechanical and oxidative stress resulting in the initiation of axon damage early during pathogenesis. Furthermore, ONHAs are crucial for the maintenance of retinal ganglion cell physiology and function. Therefore, glioprotective strategies with the goal to preserve and/or restore the structural and functional viability of ONHA in order to slow glaucoma and related pathologies are of high clinical relevance. Herein, we describe the development of standardized methods that will allow for the systematic advancement of such glioprotective strategies. These include isolation, purification and culture of primary adult rat ONHAs, optimized immunocytochemical protocols for cell type validation, as well as plate reader-based assays determining cellular viability, proliferation and the intracellular redox state. We validated and standardized our protocols by performing a glioprotection study using primary ONHAs. Specifically, we measured protection against exogenously-applied oxidative stress using tert-butylhydroperoxide (tBHP) as a model of disease-mediated oxidative stress in the retina and optic nerve head by the prototypic antioxidant, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox). Levels of oxidative stress were increased in the response to exogenously applied tBHP and were assessed by 6-carboxy-2', 7' dichlorodihydrofluorescein diacetate (DCFDA) fluorescence. Normalized DCFDA fluorescence showed a maximal 5.1-fold increase; the half-maximal effect (EC50) for tBHP was 212 ± 25 µM. This was paralleled very effectively in the assays measuring cell death and cell viability with half-maximal effects of 241 ± 20 µM and 194 ± 5 µM for tBHP in the lactate dehydrogenase (LDH) release and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) conversion assays, respectively. Pre-treatment with 100 µM Trolox decreased the sensitivity of ONHAs to tBHP. Half-maximal effects increased to 396 ± 12 µM tBHP in the LDH release assay and to 383 ± 3 µM tBHP in the MTT assay. Vehicle treatment (0.1% v/v ethanol) did not significantly affect cellular responses to tBHP. Antioxidant treatment increases ONHA viability and reduces the deleterious effects of oxidative stress. Our experiments provide important feasibility data for utilizing primary rat ONHAs in plate reader-based assays assessing novel therapeutics for glioprotection of the optic nerve and the optic nerve head in glaucoma and related disorders. Furthermore, our novel, standardized protocols have the potential to be readily adapted to high-throughput and high-content testing strategies. [ABSTRACT FROM AUTHOR]

Published
2015
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12. Histone Deacetylases Regulation by δ-Opioids in Human Optic Nerve Head Astrocytes

Author

Syed A H, Zaidi, Nakul, Thakore, Sudha, Singh, Wendy, Guzman, Shikhar, Mehrotra, Vamsi, Gangaraju, and Shahid, Husain

Subjects
Aged, 80 and over, Male, Retinal Ganglion Cells, Physiology and Pharmacology, Blotting, Western, Optic Disk, protein acetylation, Acetylation, optic nerve head astrocytes, Middle Aged, Piperazines, Rats, glaucoma, Astrocytes, Rats, Inbred BN, Receptors, Opioid, delta, Benzamides, Cadaver, Electroretinography, Animals, Humans, Female, Cells, Cultured, histone deacetylases, Aged
Abstract

Purpose We determined whether δ-opioid receptor agonist (SNC-121) regulates acetylation homeostasis via controlling histone deacetylases (HDACs) activity and expression in optic nerve head (ONH) astrocytes. Methods ONH astrocytes were treated with SNC-121 (1 µM) for 24 hours. The HDAC activity was measured using HDAC-specific fluorophore-conjugated synthetic substrates, Boc-Lys(Ac)-AMC and (Boc-Lys(Tfa)-AMC). Protein and mRNA expression of each HDAC was determined by Western blotting and quantitative real-time PCR. IOP in rats was elevated by injecting 2.0 M hypertonic saline into the limbal veins. Results Delta opioid receptor agonist, SNC-121 (1 µM), treatment increased acetylation of histone H3, H2B, and H4 by 128 ± 3%, 45 ± 1%, and 68 ± 2%, respectively. The addition of Garcinol, a histone-acetyltransferase inhibitor, fully blocked SNC-121–induced histone H3 acetylation. SNC-121 reduced the activities of class I and IIb HDACs activities significantly (17 ± 3%) and this decrease in HDACs activities was fully blocked by a selective δ-opioid receptors antagonist, naltrindole. SNC-121 also decrease the mRNA expression of HDAC-3 and HDAC-6 by 19% and 18%, respectively. Furthermore, protein expression of HDAC 1, 2, 3, and 6 was significantly (P < 0.05) decreased by SNC-121 treatment. SNC-121 treatment also reduced lipopolysaccharide-induced TNF-α production from ONH astrocytes and glial fibrillary acidic protein immunostaining in the optic nerve of ocular hypertensive animals. Conclusions We provided evidence that δ-opioid receptor agonist activation increased histone acetylation, decrease HDACs class I and class IIb activities, mRNA, and protein expression, lipopolysaccharide-induced TNF-α production in ONH astrocytes. Our data also demonstrate that SNC-121 treatment decrease glial fibrillary acidic protein immunostaining in the optic nerves of animals with ocular hypertension.

Published
2020

13. Optimization of Cell Viability Assays for Screening Glioprotective Compounds in Primary Rat Optic Nerve Head Astrocytes

Author

Vitaliy Maksymenko, Yuliya Naumchuk, and Simon Kaja

Subjects
chemistry.chemical_classification, Reactive oxygen species, Glaucoma, General Medicine, Optic nerve head astrocytes, Viability assays, lcsh:Chemical technology, medicine.disease_cause, Cell biology, chemistry.chemical_compound, medicine.anatomical_structure, chemistry, Oxidative stress, Lactate dehydrogenase, medicine, lcsh:TP1-1185, lcsh:Q, MTT assay, Viability assay, Trolox, lcsh:Science, Intracellular, Astrocyte
Abstract

Background. Optic nerve head astrocytes (ONHAs) are the major glia cell type in the non-myelinated optic nerve head where they contribute to extracellular matrix synthesis. Pathological changes in glaucoma include reactive astrocytosis, a process characterized by altered astrocyte gene and protein expression and extracellular matrix remodeling. ONHAs are highly sensitive to mechanical and oxidative stress resulting in the initiation of axon damage early during pathogenesis. Furthermore, ONHAs are crucial for the maintenance of retinal ganglion cell physiology and function. Therefore, glioprotective strategies with the goal to preserve and/or restore the structural and functional viability of ONHA to slow glaucoma and related pathologies are of high clinical relevance. Objective. The aim of the paper is the development of standardized methods for the systematic advancement of glioprotective strategies using plate reader-based assays determining cellular viability, plate-reader based proliferation and the intracellular redox state. Methods. In our work we used primary culture of OHNAs as a model system. The oxidative stress was induced by tBHP. Reactive oxygen species (ROS) were measured by the DCDFA assay. For cell viability tests we used the optimized lactate dehydrogenase (LDH) release assay, as well as the MTT and Calcein-AM uptake assays. Results. The half-maximal effect (EC50) of tBHP on OHNAs ROS levels in the DCFDA assay was 192.1 ± 15.7 μM. The measurement of cellular viability of OHNAs after tBHP-induced oxidative stress showed EC50 = 156.9 ± 3.8 µM in the Calcein-AM uptake assay. In the MTT assay, the EC50 for tBHP was 138.1 ± 1.4 µM, and shifted to 192.7 ± 2.8 µM with 100 μM Trolox pre-treatment of OHNA. In the LDH release assay, the EC50 for tBHP was 146.9 ± 4.9 µM and 246.3 ± 7.3 µM for the control and Trolox conditions respectively. Conclusions. Our results provide feasibility data for the plate-reader based screening for novel glioprotectants using primary ONHA culture.

Published
2017

14. Myelination transition zone astrocytes: a novel cell type in the optic nerve with a putative role in glaucoma.

Author

Prasanna, Ganesh and Iok-Hou Pang

Subjects
GLAUCOMA, EYE diseases, ASTROCYTES, PHAGOCYTOSIS, ALTERNATIVE medicine
Abstract

Glaucomatous optic neuropathy is characterized by retinal ganglion cell death and optic nerve axon loss. Optic nerve head astrocytes are involved in regulating physiologic and pathophysiologic effects on the unmyelinated axons of the optic nerve. Presently we evaluate an article that has identified a novel subpopulation of optic nerve head astrocytes termed myelination transition zone (MTZ) astrocytes. Based on the specific expression of phagocytic marker for myelin (Mac-2), MTZ astrocytes appear to possess a constitutive role in clearing axonal debris via phagocytosis, which is proposed to protect the nerve from further damage. Mac-2 expression is elevated in glaucomatous mice and regulated by γ-synuclein, a chaperone protein and marker for retinal ganglion cells. Additional characterization of phagocytosis via direct interactions between γ-synuclein/Mac-2 in MTZ astrocytes could be useful while confirming the presence of MTZ astrocytes in human optic nerve tissues. Characterization of the role of MTZ astrocytes in regulating mitochondrial transport/turnover and release on neurite inhibitory factors in the optic nerve in glaucoma may be highly relevant. [ABSTRACT FROM AUTHOR]

Published
2011
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15. A neuroactive steroid 5α-androstane-3α,17β-diol regulates androgen receptor level in astrocytes.

Author

Agapova, Olga A., Malone, Paula E., and Hernandez, M. Rosario

Subjects
*ANDROSTANE, *ANDROGENS, *STEROIDS, *ASTROCYTES, *GLAUCOMA
Abstract

Optic nerve head (ONH) astrocytes from patients with glaucomatous optic neuropathy exhibit increased production of 5α-androstane-3α,17β-diol (3α-diol), a neuroactive metabolite of 5α-dihydrotestosterone (5α-DHT). To determine whether ONH astrocytes are androgen target cells, and whether 3α-diol is capable of regulating astrocyte functions, we studied the response of human ONH astrocytes to 3α-diol compared with 17β-hydroxy-17α-methyl-estra-4,9,11-trien-3-one (R1881), a synthetic 5α-DHT agonist. In ONH astrocytes, both 3α-diol and R1881 increased protein levels of androgen receptor (AR) and glial fibrillary acidic protein (GFAP), however, only R1881 also increased the AR mRNA level and astrocyte proliferation. Both R1881 and 3α-diol rapidly activate the mitogen-activated protein kinase (MAPK) signaling pathway in ONH astrocytes, as confirmed by phosphorylation of extracellular signal-regulated kinase (ERK). 3α-diol also activates the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway. 3α-diol regulates the increase of AR protein level and the phosphorylation through the PI3K/Akt pathway, whereas R1881 regulates them through the MAPK/ERK pathway. Our findings demonstrate that human ONH astrocytes are androgen target cells and respond to androgens by the rapid activation of cell signaling. The activation of the PI3K/Akt pathway by 3α-diol may regulate various properties of astrocytes, including cell motility and survival, and may play a role in the formation and maintenance of the reactive phenotype of ONH astrocytes in glaucoma. [ABSTRACT FROM AUTHOR]

Published
2006
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16. Effect of elevated intraocular pressure on endothelin-1 in a rat model of glaucoma

Author

Prasanna, Ganesh, Hulet, Christina, Desai, Devashish, Krishnamoorthy, Raghu R., Narayan, Santosh, Brun, Anne-Marie, Suburo, Angela M., and Yorio, Thomas

Subjects
*ENDOTHELINS, *PEPTIDES, *ASTROCYTES, *NERVES
Abstract

The role of endothelin-1 (ET-1) a potent vasoactive peptide, in glaucoma pathogenesis is receiving increasing attention, particularly in astroglial activation in optic nerve damage. Our laboratory has also shown that ET-1 treatment causes proliferation of cultured human optic nerve head astrocytes to possibly initiate astrogliosis. ET-1 is distributed in retina, optic nerve, and ciliary epithelium, however the effects of elevated intraocular pressure (IOP) (as seen in glaucoma) on ET-1 and ETB receptors are not clearly understood. In the present study, the levels of immunoreactive ET-1 (ir-ET-1) in aqueous humour (AH) and optic nerve head (ONH) were determined in the Morrison elevated IOP model of glaucoma. Additionally in the ONH of these rats, immunohistochemical analyses of ETB receptors and glial fibrillary acidic protein (GFAP; a marker for astroglial cells and for astrogliosis) were performed. There was 2- to 2.5-fold increase in AH ir-ET-1 levels for rats subjected to elevated IOP, compared to their respective controls. In the Morrison rat model of glaucoma, elevated IOP increased optic nerve ir-ET-1 with concomitant increases in ir-ETB and ir-GFAP labelling (possibly indicative of astrogliosis and hypertrophy). As seen in brain astrocytes subjected to neurotrauma, the present findings are suggestive of ET-1’s role in astroglial activation, particularly in response to elevated IOP in glaucoma. [Copyright &y& Elsevier]

Published
2005
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17. Altered expression of 3α-hydroxysteroid dehydrogenases in human glaucomatous optic nerve head astrocytes

Author

Agapova, O.A., Yang, P., Wang, W-H., Lane, D.A., Clark, A.F., Weinstein, B.I., and Hernandez, M.R.

Subjects
*STEROIDS, *DEHYDROGENASES, *CENTRAL nervous system, *OPTIC nerve
Abstract

3α-Hydroxysteroid dehydrogenase (3α-HSD) isoforms (AKR1C1–AKR1C4) are aldo-keto reductases that metabolize steroids and other substances in many tissues including the CNS. Here we demonstrated that in glaucomatous human optic nerve heads, increased expression of 3α-HSD was localized to reactive astrocytes in the lamina cribrosa. Similar, optic nerve head astrocytes exhibited increased expression of 3α-HSD in response to elevated intraocular pressure in a monkey model of experimental glaucoma, but not in monkeys with unilateral optic nerve transection. In vitro, glaucomatous optic nerve head astrocytes expressed higher levels of AKR1C1, AKR1C2, and AKR1C3 mRNA, than normal astrocytes, with significant differential increase of AKR1C2 expression, and exhibited higher enzymatic activity forming 3α-androstanediol a well-recognized neurosteroid. Normal astrocytes exposed to elevated hydrostatic pressure selectively increased AKR1C2 expression. Our findings of increased expression of 3α-HSDs in glaucomatous optic nerve head astrocytes offer new insights into possible roles for neurosteroids in the pathophysiology of glaucoma. [Copyright &y& Elsevier]

Published
2003
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18. Біотехнологічні основи лактатдегідрогеназної системи для тестування гліопротекторів астроцитів зорового нерву

Author

Наумчук, Юлія Анатоліївна and Наумчук, Юлія Анатоліївна

Abstract

У дисертації представлені результати розробки та оптимізації платформи скринінгу гліопротекторних засобів для лікування глаукоми. Для створення модельної системи тестування гліопротекції був розроблений протокол отримання і культивування первинних астроцитів голівки зорового нерву (АГЗН) з підтвердженими маркерами астроцитів. Для отриманої культури АГЗН були оптимізовані умови культивування та покриття 21поверхні. Була проведена модифікація ЛДГ тесту та його адаптація для отриманої культури АГЗН, в рамках якої були оптимізовані періоди інкубації з індуктором оксидативного стресу tBHP і тест-реагентами. Ефективність tBHP як індуктора оксидативного стресу була підтверджена в тестах оцінки рівня реактивних форм кисню (РФК) і виживання клітин. Також було показано, що гіпербарично тиск не впливає на виживаність АГЗН, але при цьому істотно підвищує їх чутливість до оксидативного стресу. Це уможливило подальше використання оксидативного стресу (tBHP) як патогенного фактору впливу при тестуванні засобів гліопротекціі. Нарешті, була проведена порівняльна оцінка розробленої ЛДГ системи з комерційним тестом виживання клітин МТТ з використанням прототипного антиоксиданта Тролокса та нутрціветика Ресвератролу. Значення EC50 (tBHP), отримані в ЛДГ і МТТ тестах, підтвердили точність розробленої системи., This thesis presents the results of the development and optimization of a standardized plate reader-based screening platform for glioprotective compounds in optic nerve head astrocytes. Optic nerve head astrocytes (ONHAs) are the major glia cell type in the nonmyelinated optic nerve head where they contribute to extracellular matrix synthesis. Pathological changes in glaucoma include reactive astrocytosis, a process characterized by altered astrocyte gene and protein expression and extracellular matrix remodeling. ONHAs are highly sensitive to mechanical and oxidative stresses and crucial for the maintenance of retinal ganglion cell physiology and function. Therefore, glioprotective strategies with the goal to preserve and/or restore the structural and functional viability of ONHAs to slow progression of glaucoma are of high clinical relevance. Available methods for the evaluation of drug candidates with glioprotective potential in ONHAs are poorly developed, laborious and costly, thus causing a challenge for the development of the novel glioprotective strategies. Therefore, the objective of this work was to establish the scientific premise for glioprotection in primary ONHA culture and to develop a biotechnological method for the screening of glioprotective compounds based on their efficacy in reducing or preventing the deleterious effects of oxidative stress in ONHAs. We formulated the following five specific aims to reach our objective: 1) optimization of the isolation and culturing techniques for primary rat ONHAs; 2) development and validation of a lactate dehydrogenase (LDH) release assay for ONHAs; 3) optimization of chemically-induced oxidative stress to use as an insult in primary rat ONHAs; 4) evaluation of the effects of oxidative stress and hyperbaric pressure on ONHAs; 5) validation of the glioprotective screening platform using antioxidants in primary rat ONHA culture. We devised a novel protocol for the primary culture of ONHAs derived from adult rats, В диссертации представлены результаты разработки и оптимизации платформы скрининга глиопротекторных средств для лечения глаукомы. Для создания модельной системы тестирования глиопротекции был разработан протокол получения и культивирования первичных астроцитов головки зрительного нерва (АГЗН) с подтвержденными маркерами астроцитов. Для полученной культуры АГЗН были оптимизированы условия культивирования и покрытия поверхности. Была проведена модификация ЛДГ теста и его адаптация для полученной культуры АГЗН, в рамках которой были оптимизированы периоды инкубации с индуктором оксидативного стресса tBHP и тест-реагентами. Эффективность tBHP в качестве индуктора оксидативного стресса была подтверждена в тестах оценки уровня реактивных форм кислорода (РФК) и выживаемости клеток. Также было показано, что гипербарическое давление не влияет на выживаемость АГЗН, но при этом существенно повышает их чувствительность к оксидативному стрессу. Это позволило дальнейшее использования оксидативного стресса (tBHP) в качестве фактора влияния при тестировании средств глиопротекции. Наконец, была проведена сравнительная оценка разработанной ЛДГ системы с коммерческим тестом выживаемости клеток МТТ с использованием прототипного антиоксиданта Тролокса и нутрицевтика Ресвератрола. Значения EC50 (tBHP), полученные в ЛДГ и МТТ тестах, подтвердили точность разработанной системы.

Published
2018

19. Differential Activation of Glioprotective Intracellular Signaling Pathways in Primary Optic Nerve Head Astrocytes after Treatment with Different Classes of Antioxidants

Author

Vidhya R. Rao, Alexandra D. Zigrossi, Jamie C. Floss, Simon Kaja, Peter Koulen, Evan B. Stubbs, Victoria J. Wisniewski, and Anita K. Ghosh

Subjects
0301 basic medicine, glioprotection, Physiology, Clinical Biochemistry, SOD2, optic nerve head astrocytes, resveratrol, Resveratrol, medicine.disease_cause, Biochemistry, Article, Superoxide dismutase, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, medicine, Viability assay, manganese porphyrin, Molecular Biology, cell viability, chemistry.chemical_classification, Reactive oxygen species, biology, phase II antioxidant enzymes, lcsh:RM1-950, Cell Biology, xanthohumol, Cell biology, lcsh:Therapeutics. Pharmacology, 030104 developmental biology, chemistry, 030221 ophthalmology & optometry, biology.protein, Optic nerve, Nicotinamide adenine dinucleotide phosphate, Oxidative stress
Abstract

Optic nerve head astrocytes are the specialized glia cells that provide structural and trophic support to the optic nerve head. In response to cellular injury, optic nerve head astrocytes undergo reactive astrocytosis, the process of cellular activation associated with cytoskeletal remodeling, increases in the rate of proliferation and motility, and the generation of Reactive Oxygen Species. Antioxidant intervention has previously been proposed as a therapeutic approach for glaucomatous optic neuropathy, however, little is known regarding the response of optic nerve head astrocytes to antioxidants under physiological versus pathological conditions. The goal of this study was to determine the effects of three different antioxidants, manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin (Mn-TM-2-PyP), resveratrol and xanthohumol in primary optic nerve head astrocytes. Effects on the expression of the master regulator nuclear factor erythroid 2-related factor 2 (Nrf2), the antioxidant enzyme, manganese-dependent superoxide dismutase 2 (SOD2), and the pro-oxidant enzyme, nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4), were determined by quantitative immunoblotting. Furthermore, efficacy in preventing chemically and reactive astrocytosis-induced increases in cellular oxidative stress was quantified using cell viability assays. The results were compared to the effects of the prototypic antioxidant, Trolox. Antioxidants elicited highly differential changes in the expression levels of Nrf2, SOD2, and NOX4. Notably, Mn-TM-2-PyP increased SOD2 expression eight-fold, while resveratrol increased Nrf2 expression three-fold. In contrast, xanthohumol exerted no statistically significant changes in expression levels. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) uptake and lactate dehydrogenase (LDH) release assays were performed to assess cell viability after chemically and reactive astrocytosis-induced oxidative stress. Mn-TM-2-PyP exerted the most potent glioprotection by fully preventing the loss of cell viability, whereas resveratrol and xanthohumol partially restored cell viability. Our data provide the first evidence for a well-developed antioxidant defense system in optic nerve head astrocytes, which can be pharmacologically targeted by different classes of antioxidants.

Published
2020

20. Біотехнологічні основи лактатдегідрогеназної системи для тестування гліопротекторів астроцитів зорового нерву

Subjects
616.08](043.3) 616.7-007.681:616.08](043.3) [617.731+612.843]], glioprotection, оксидативний стрес, астроцити голівки зорового нерву, optic nerve head astrocytes, астроциты головки зрительного нерва, глаукома, lactatdehydrogenase assay, лактатдегідрогеназний тест, glaucoma, гліопротекція, лактатдегидрогеназный тест, oxidative stress, оксидативный стресс, глиопротекция
Abstract

У дисертації представлені результати розробки та оптимізації платформи скринінгу гліопротекторних засобів для лікування глаукоми. Для створення модельної системи тестування гліопротекції був розроблений протокол отримання і культивування первинних астроцитів голівки зорового нерву (АГЗН) з підтвердженими маркерами астроцитів. Для отриманої культури АГЗН були оптимізовані умови культивування та покриття 21поверхні. Була проведена модифікація ЛДГ тесту та його адаптація для отриманої культури АГЗН, в рамках якої були оптимізовані періоди інкубації з індуктором оксидативного стресу tBHP і тест-реагентами. Ефективність tBHP як індуктора оксидативного стресу була підтверджена в тестах оцінки рівня реактивних форм кисню (РФК) і виживання клітин. Також було показано, що гіпербарично тиск не впливає на виживаність АГЗН, але при цьому істотно підвищує їх чутливість до оксидативного стресу. Це уможливило подальше використання оксидативного стресу (tBHP) як патогенного фактору впливу при тестуванні засобів гліопротекціі. Нарешті, була проведена порівняльна оцінка розробленої ЛДГ системи з комерційним тестом виживання клітин МТТ з використанням прототипного антиоксиданта Тролокса та нутрціветика Ресвератролу. Значення EC50 (tBHP), отримані в ЛДГ і МТТ тестах, підтвердили точність розробленої системи. This thesis presents the results of the development and optimization of a standardized plate reader-based screening platform for glioprotective compounds in optic nerve head astrocytes. Optic nerve head astrocytes (ONHAs) are the major glia cell type in the nonmyelinated optic nerve head where they contribute to extracellular matrix synthesis. Pathological changes in glaucoma include reactive astrocytosis, a process characterized by altered astrocyte gene and protein expression and extracellular matrix remodeling. ONHAs are highly sensitive to mechanical and oxidative stresses and crucial for the maintenance of retinal ganglion cell physiology and function. Therefore, glioprotective strategies with the goal to preserve and/or restore the structural and functional viability of ONHAs to slow progression of glaucoma are of high clinical relevance. Available methods for the evaluation of drug candidates with glioprotective potential in ONHAs are poorly developed, laborious and costly, thus causing a challenge for the development of the novel glioprotective strategies. Therefore, the objective of this work was to establish the scientific premise for glioprotection in primary ONHA culture and to develop a biotechnological method for the screening of glioprotective compounds based on their efficacy in reducing or preventing the deleterious effects of oxidative stress in ONHAs. We formulated the following five specific aims to reach our objective: 1) optimization of the isolation and culturing techniques for primary rat ONHAs; 2) development and validation of a lactate dehydrogenase (LDH) release assay for ONHAs; 3) optimization of chemically-induced oxidative stress to use as an insult in primary rat ONHAs; 4) evaluation of the effects of oxidative stress and hyperbaric pressure on ONHAs; 5) validation of the glioprotective screening platform using antioxidants in primary rat ONHA culture. We devised a novel protocol for the primary culture of ONHAs derived from adult rats and identified the optimal culture and seeding conditions for the experiments conducted in this thesis. In order to validate the astrocytic identity of the obtained primary cultures, expression of astrocyte specific markers (excitatory amino acid transporter 1 (EAAT1), glial fibrillary acidic protein (GFAP), and the glial calcium binding protein S100β) was confirmed by immunocytochemistry. Minimal to no contamination by other cell types was found. We next developed a protocol for a custom LDH assay as biotechnological platform for the screening of glioprotective compounds using chemically-induced oxidative stress, tert-butylhydroperoxide (tBHP), as an inducer of reactive oxygen species (ROS). Our comparison of the newly developed LDH release assay with a commercially available test kit revealed no statistically significant differences in sensitivity while being far less expensive when compared to the commercial assay. Another important advantage of our custom protocol is the ability to refine the protocol for specific compounds given the known formulation of all reagents. Next, we optimized the conditions for the use of tBHP as an inducer of oxidative stress in primary rat ONHA culture. The generation of ROS was confirmed using a fluorescent ROS sensor, 2',7'–dichlorofluorescein diacetate (DCFDA). tBHP-induced oxidative stress resulted in a dose-dependent loss of cell viability that was assessed using three different assays, each quantifying a different physiological mechanism that can serve as correlate of cell viability: calcein-AM uptake test, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) assay and LDH assay. Data from calcein-AM uptake test and MTT assay were compared to the results of the newly developed LDH assay. All three assays yielded similar EC50/IC50 values for tBHP. Therefore, screening of glioprotective compounds using the developed LDH assay is predicted to provide an accurate reflection of the effects of compounds on the physiological function and survival of ONHAs. Lastly, we validated our biotechnological LDH assay platform for the screening of glioprotective compounds using the prototypic antioxidant Trolox and the nutraceutical Resveratrol. Both compounds exerted potent glioprotective effects with no statistically significant difference between EC50/IC50 values for tBHP obtained from LDH and MTT assays. In conclusion, we developed and validated a standardized biotechnological screening platform for the quantitative assessment of glioprotective properties in primary adult rat ONHA culture. We recommend the use of our standardized screening platform for advancing the development of novel glioprotective therapeutic strategies for the management of glaucoma. В диссертации представлены результаты разработки и оптимизации платформы скрининга глиопротекторных средств для лечения глаукомы. Для создания модельной системы тестирования глиопротекции был разработан протокол получения и культивирования первичных астроцитов головки зрительного нерва (АГЗН) с подтвержденными маркерами астроцитов. Для полученной культуры АГЗН были оптимизированы условия культивирования и покрытия поверхности. Была проведена модификация ЛДГ теста и его адаптация для полученной культуры АГЗН, в рамках которой были оптимизированы периоды инкубации с индуктором оксидативного стресса tBHP и тест-реагентами. Эффективность tBHP в качестве индуктора оксидативного стресса была подтверждена в тестах оценки уровня реактивных форм кислорода (РФК) и выживаемости клеток. Также было показано, что гипербарическое давление не влияет на выживаемость АГЗН, но при этом существенно повышает их чувствительность к оксидативному стрессу. Это позволило дальнейшее использования оксидативного стресса (tBHP) в качестве фактора влияния при тестировании средств глиопротекции. Наконец, была проведена сравнительная оценка разработанной ЛДГ системы с коммерческим тестом выживаемости клеток МТТ с использованием прототипного антиоксиданта Тролокса и нутрицевтика Ресвератрола. Значения EC50 (tBHP), полученные в ЛДГ и МТТ тестах, подтвердили точность разработанной системы.

Published
2018

21. Оптимізація тестів життєздатності клітин для скринінгу гліопротекторних сполук у культурі первинних астроцитів головки зорового нерву щура

Author

Naumchuk, Yuliya A., Maksymenko, Vitaliy B., and Kaja, Simon

Subjects
Глаукома, Астроцити головки зорового нерву, Оксидативний стрес, Реактивні форми кисню, Тести на визначення життєздатності клітин, Биотехнологии, Астроциты головки зрительного нерва, Оксидативный стресс, Реактивные формы кислорода, Тесты на определение жизнеспособности клеток, Glaucoma, Optic nerve head astrocytes, Oxidative stress, Reactive oxygen species, Viability assays, Біотехнології, Biotechnology
Abstract

Background. Optic nerve head astrocytes (ONHAs) are the major glia cell type in the non-myelinated optic nerve head where they contribute to extracellular matrix synthesis. Pathological changes in glaucoma include reactive astrocytosis, a process characterized by altered astrocyte gene and protein expression and extracellular matrix remodeling. ONHAs are highly sensitive to mechanical and oxidative stress resulting in the initiation of axon damage early during pathogenesis. Furthermore, ONHAs are crucial for the maintenance of retinal ganglion cell physiology and function. Therefore, glioprotective strategies with the goal to preserve and/or restore the structural and functional viability of ONHA to slow glaucoma and related pathologies are of high clinical relevance.Objective. The aim of the paper is the development of standardized methods for the systematic advancement of glioprotective strategies using plate reader-based assays determining cellular viability, plate-reader based proliferation and the intracellular redox state.Methods. In our work we used primary culture of OHNAs as a model system. The oxidative stress was induced by tBHP. Reactive oxygen species (ROS) were measured by the DCDFA assay. For cell viability tests we used the optimized lactate dehydrogenase (LDH) release assay, as well as the MTT and Calcein-AM uptake assays.Results. The half-maximal effect (EC50) of tBHP on OHNAs ROS levels in the DCFDA assay was 192.1 ± 15.7 μM. The measurement of cellular viability of OHNAs after tBHP-induced oxidative stress showed EC50 = 156.9 ± 3.8 µM in the Calcein-AM uptake assay. In the MTT assay, the EC50 for tBHP was 138.1 ± 1.4 µM, and shifted to 192.7 ± 2.8 µM with 100 μM Trolox pre-treatment of OHNA. In the LDH release assay, the EC50 for tBHP was 146.9 ± 4.9 µM and 246.3 ± 7.3 µM for the control and Trolox conditions respectively.Conclusions. Our results provide feasibility data for the plate-reader based screening for novel glioprotectants using primary ONHA culture., Проблематика. Астроциты головки зрительного нерва (АГЗН) являются основным типом глиальных клеток в немиелинированной головке зрительного нерва, где они участвуют в синтезе внешнеклеточного матрикса. Патологические изменения при глаукоме включают реактивный астроцитоз – процесс, который характеризуется изменениями в экспрессии астроцитарных генов и белков, а также ремоделингом внешнеклеточного матрикса. АГЗН являются чрезвычайно чувствительными к механическому и оксидативному стрессу, что приводит к инициации повреждения аксонов при раннем патогенезе. Кроме того, АГЗН играют ключевую роль в поддержке физиологии и функций ганглионарных клеток сетчатки. Именно поэтому глиопротекторные стратегии с целью сохранения и/или возобновления структурной и функциональной жизнеспособности АГЗН для замедления глаукомы и сродных патологий являются чрезвычайно важными для клинической практики.Цель исследования. Разработка стандартизированных методов для систематического внедрения глиопротекторных стратегий с использованием тестов определения жизнеспособности, пролиферации и внутриклеточного редокс-состояния клеток с помощью планшетного ридера.Методика реализации. В нашей работе мы использовали первичную культуру АГЗН как модельную систему. Оксидативный стресс был индуцирован с помощью tBHP. Реактивные формы кислорода (РФК) были измерены с помощью теста DCFDA. Для тестов жизнеспособности клеток мы использовали оптимизированный нами тест высвобождения лактатдегидрогеназы (ЛДГ), а также тест МТТ и тест поглощения Кальцеина-АМ.Результаты исследования. Полумаксимальный эффект (EC50) tBHP для уровней РФК в АГЗН в тесте DCFDA составил 192,1 ± 15,7 мкМ. Измерение клеточной жизнеспособности АГЗН после оксидативного стресса, индуцированного tBHP, показало EC50 = 156,9 ± 3,8 мкМ в тесте поглощения Кальцеина-АМ. В тесте МТТ EC50 для tBHP составило 138,1 ± 1,4 мкМ и смести­лось до 192,7 ± 2,8 мкМ после предварительной обработки АГЗН 100 мкМ Тролоксом. В тесте высвобождения ЛДГ EC50 для tBHP составило 146,9 ± 4,9 и 246,3 ± 7,3 мкМ для контрольного и обработанного Тролоксом образцов соответственно.Выводы. Наши результаты предоставляют данные о возможности проводить скрининг новейших глиопротекторов с помощью планшетного ридера, используя первичную культуру АГЗН., Проблематика. Астроцити головки зорового нерву (АГЗН) є основним типом гліальних клітин у немієлінованій головці зорового нерву, де вони беруть участь у синтезі зовнішньоклітинного матриксу. Патологічні зміни при глаукомі включають реактивний астроцитоз – процес, що характеризується змінами в експресії астроцитарних генів і білків, а також ремоделінгом зов­нішньоклітинного матриксу. АГЗН є надзвичайно чутливими до механічного та оксидативного стресу, що призводить до ініціації ушкодження аксонів при ранньому патогенезі. Крім того, АГЗН відіграють ключову роль у підтримці фізіології та функцій гангліо­нарних клітин сітківки. Саме тому гліопротекторні стратегії з метою збереження і/або відновлення структурної та функціональної життєздатності АГЗН для сповільнення глаукоми та споріднених патологій є надзвичайно важливими для клінічної практики.Мета дослідження. Розробка стандартизованих методів для систематичного впровадження гліопротекторних стратегій із використанням тестів з визначення життєздатності, проліферації та внутрішньоклітинного редокс-стану клітин за допомогою план­шет-рідера.Методика реалізації. У нашій роботі ми використовували первинну культуру АГЗН як модельну систему. Оксидативний стрес було індуковано за допомогою tBHP. Реактивні форми кисню (РФК) було виміряно за допомогою тесту DCFDA. Для тестів життєздатності клітин ми використовували оптимізований нами тест вивільнення лактатдегідрогенази (ЛДГ), а також тест МТТ і тест поглинання Кальцеїну-АМ.Результати дослідження. Напівмаксимальний ефект (EC50) tBHP для рівнів РФК у АГЗН у тесті DCFDA становив 192,1 ± 15,7 мкМ. Вимірювання клітинної життєздатності АГЗН після оксидативного стресу, індукованого tBHP, показало EC50 = 156,9 ± 3,8 мкМ у тесті поглинання Кальцеїну-АМ. У тесті МТТ EC50 для tBHP становило 138,1 ± 1,4 мкМ і змістилося до 192,7 ± 2,8 мкМ після попередньої обробки АГЗН 100 мкМ Тролоксом. У тесті вивільнення ЛДГ EC50 для tBHP становило 146,9 ± 4,9 і 246,3 ± 7,3 мкМ для контрольного та обробленого Тролоксом зразків відповідно.Висновки. Наші результати надають данні про можливість проводити скринінг новітніх гліопротекторів з допомогою план­шет-рідера, використовуючи первинну культуру АГЗН.

Published
2017

22. Оптимізація тестів життєздатності клітин для скринінгу гліопротекторних сполук у культурі первинних астроцитів головки зорового нерву щура

Author

Naumchuk, Yuliya A.; Igor Sikorsky Kyiv Polytechnic Institute, Loyola University Chicago, Maksymenko, Vitaliy B.; Igor Sikorsky Kyiv Polytechnic Institute, Kaja, Simon; Loyola University Chicago, Naumchuk, Yuliya A.; Igor Sikorsky Kyiv Polytechnic Institute, Loyola University Chicago, Maksymenko, Vitaliy B.; Igor Sikorsky Kyiv Polytechnic Institute, and Kaja, Simon; Loyola University Chicago

Abstract

Проблематика. Астроцити головки зорового нерву (АГЗН) є основним типом гліальних клітин у немієлінованій головці зорового нерву, де вони беруть участь у синтезі зовнішньоклітинного матриксу. Патологічні зміни при глаукомі включають реактивний астроцитоз – процес, що характеризується змінами в експресії астроцитарних генів і білків, а також ремоделінгом зов­нішньоклітинного матриксу. АГЗН є надзвичайно чутливими до механічного та оксидативного стресу, що призводить до ініціації ушкодження аксонів при ранньому патогенезі. Крім того, АГЗН відіграють ключову роль у підтримці фізіології та функцій гангліо­нарних клітин сітківки. Саме тому гліопротекторні стратегії з метою збереження і/або відновлення структурної та функціональної життєздатності АГЗН для сповільнення глаукоми та споріднених патологій є надзвичайно важливими для клінічної практики.Мета дослідження. Розробка стандартизованих методів для систематичного впровадження гліопротекторних стратегій із використанням тестів з визначення життєздатності, проліферації та внутрішньоклітинного редокс-стану клітин за допомогою план­шет-рідера.Методика реалізації. У нашій роботі ми використовували первинну культуру АГЗН як модельну систему. Оксидативний стрес було індуковано за допомогою tBHP. Реактивні форми кисню (РФК) було виміряно за допомогою тесту DCFDA. Для тестів життєздатності клітин ми використовували оптимізований нами тест вивільнення лактатдегідрогенази (ЛДГ), а також тест МТТ і тест поглинання Кальцеїну-АМ.Результати дослідження. Напівмаксимальний ефект (EC50) tBHP для рівнів РФК у АГЗН у тесті DCFDA становив 192,1 ± 15,7 мкМ. Вимірювання клітинної життєздатності АГЗН після оксидативного стресу, індукованого tBHP, показало EC50 = 156,9 ± 3,8 мкМ у тесті поглинання Кальцеїну-АМ. У тесті МТТ EC50 для tBHP становило 138,1 ± 1,4 мкМ і змістилося до 192,7 ± 2,8 мкМ після попередньої обробки АГЗН 100 мкМ Тролоксом. У тесті вивільнення ЛДГ EC50 для tBHP становило 146,9 ± 4,9 і 246,3 ± 7,3 мкМ для контрольного та обро, Проблематика. Астроциты головки зрительного нерва (АГЗН) являются основным типом глиальных клеток в немиелинированной головке зрительного нерва, где они участвуют в синтезе внешнеклеточного матрикса. Патологические изменения при глаукоме включают реактивный астроцитоз – процесс, который характеризуется изменениями в экспрессии астроцитарных генов и белков, а также ремоделингом внешнеклеточного матрикса. АГЗН являются чрезвычайно чувствительными к механическому и оксидативному стрессу, что приводит к инициации повреждения аксонов при раннем патогенезе. Кроме того, АГЗН играют ключевую роль в поддержке физиологии и функций ганглионарных клеток сетчатки. Именно поэтому глиопротекторные стратегии с целью сохранения и/или возобновления структурной и функциональной жизнеспособности АГЗН для замедления глаукомы и сродных патологий являются чрезвычайно важными для клинической практики.Цель исследования. Разработка стандартизированных методов для систематического внедрения глиопротекторных стратегий с использованием тестов определения жизнеспособности, пролиферации и внутриклеточного редокс-состояния клеток с помощью планшетного ридера.Методика реализации. В нашей работе мы использовали первичную культуру АГЗН как модельную систему. Оксидативный стресс был индуцирован с помощью tBHP. Реактивные формы кислорода (РФК) были измерены с помощью теста DCFDA. Для тестов жизнеспособности клеток мы использовали оптимизированный нами тест высвобождения лактатдегидрогеназы (ЛДГ), а также тест МТТ и тест поглощения Кальцеина-АМ.Результаты исследования. Полумаксимальный эффект (EC50) tBHP для уровней РФК в АГЗН в тесте DCFDA составил 192,1 ± 15,7 мкМ. Измерение клеточной жизнеспособности АГЗН после оксидативного стресса, индуцированного tBHP, показало EC50 = 156,9 ± 3,8 мкМ в тесте поглощения Кальцеина-АМ. В тесте МТТ EC50 для tBHP составило 138,1 ± 1,4 мкМ и смести­лось до 192,7 ± 2,8 мкМ после предварительной обработки АГЗН 100 мкМ Тролоксом. В тесте высвобождения ЛДГ EC50 для tBHP сос, Background. Optic nerve head astrocytes (ONHAs) are the major glia cell type in the non-myelinated optic nerve head where they contribute to extracellular matrix synthesis. Pathological changes in glaucoma include reactive astrocytosis, a process characterized by altered astrocyte gene and protein expression and extracellular matrix remodeling. ONHAs are highly sensitive to mechanical and oxidative stress resulting in the initiation of axon damage early during pathogenesis. Furthermore, ONHAs are crucial for the maintenance of retinal ganglion cell physiology and function. Therefore, glioprotective strategies with the goal to preserve and/or restore the structural and functional viability of ONHA to slow glaucoma and related pathologies are of high clinical relevance.Objective. The aim of the paper is the development of standardized methods for the systematic advancement of glioprotective strategies using plate reader-based assays determining cellular viability, plate-reader based proliferation and the intracellular redox state.Methods. In our work we used primary culture of OHNAs as a model system. The oxidative stress was induced by tBHP. Reactive oxygen species (ROS) were measured by the DCDFA assay. For cell viability tests we used the optimized lactate dehydrogenase (LDH) release assay, as well as the MTT and Calcein-AM uptake assays.Results. The half-maximal effect (EC50) of tBHP on OHNAs ROS levels in the DCFDA assay was 192.1 ± 15.7 μM. The measurement of cellular viability of OHNAs after tBHP-induced oxidative stress showed EC50 = 156.9 ± 3.8 µM in the Calcein-AM uptake assay. In the MTT assay, the EC50 for tBHP was 138.1 ± 1.4 µM, and shifted to 192.7 ± 2.8 µM with 100 μM Trolox pre-treatment of OHNA. In the LDH release assay, the EC50 for tBHP was 146.9 ± 4.9 µM and 246.3 ± 7.3 µM for the control and Trolox conditions respectively.Conclusions. Our results provide feasibility data for the plate-reader based screening for novel glioprotectants using primary

Published
2017

23. Optimization of Cell Viability Assays for Screening Glioprotective Compounds in Primary Rat Optic Nerve Head Astrocytes

Author

Naumchuk, Yuliya A., Maksymenko, Vitaliy B., and Kaja, Simon

Subjects
тести на визначення життєздатності клітин, астроцити головки зорового нерву, оксидативний стрес, Glaucoma, Optic nerve head astrocytes, Viability assays, 54 [616-018], глаукома, фстроциты головки зрительного нерва, Oxidative stress, тесты на определение жизнеспособности клеток, реактивні форми кисню, реактивные формы кислорода, Reactive oxygen species, оксидативный стресс
Abstract

Проблематика. Астроцити головки зорового нерву (АГЗН) є основним типом гліальних клітин у немієлінованій головці зорового нерву, де вони беруть участь у синтезі зовнішньоклітинного матриксу. Патологічні зміни при глаукомі включають реактивний астроцитоз – процес, що характеризується змінами в експресії астроцитарних генів і білків, а також ремоделінгом зов­нішньоклітинного матриксу. АГЗН є надзвичайно чутливими до механічного та оксидативного стресу, що призводить до ініціації ушкодження аксонів при ранньому патогенезі. Крім того, АГЗН відіграють ключову роль у підтримці фізіології та функцій гангліо­нарних клітин сітківки. Саме тому гліопротекторні стратегії з метою збереження і/або відновлення структурної та функціональної життєздатності АГЗН для сповільнення глаукоми та споріднених патологій є надзвичайно важливими для клінічної практики. Мета дослідження. Розробка стандартизованих методів для систематичного впровадження гліопротекторних стратегій із використанням тестів з визначення життєздатності, проліферації та внутрішньоклітинного редокс-стану клітин за допомогою план­шет-рідера. Методика реалізації. У нашій роботі ми використовували первинну культуру АГЗН як модельну систему. Оксидативний стрес було індуковано за допомогою tBHP. Реактивні форми кисню (РФК) було виміряно за допомогою тесту DCFDA. Для тестів життєздатності клітин ми використовували оптимізований нами тест вивільнення лактатдегідрогенази (ЛДГ), а також тест МТТ і тест поглинання Кальцеїну-АМ. Результати дослідження. Напівмаксимальний ефект (EC50) tBHP для рівнів РФК у АГЗН у тесті DCFDA становив 192,1 ± 15,7 мкМ. Вимірювання клітинної життєздатності АГЗН після оксидативного стресу, індукованого tBHP, показало EC50 = 156,9 ± 3,8 мкМ у тесті поглинання Кальцеїну-АМ. У тесті МТТ EC50 для tBHP становило 138,1 ± 1,4 мкМ і змістилося до 192,7 ± 2,8 мкМ після попередньої обробки АГЗН 100 мкМ Тролоксом. У тесті вивільнення ЛДГ EC50 для tBHP становило 146,9 ± 4,9 і 246,3 ± 7,3 мкМ для контрольного та обробленого Тролоксом зразків відповідно. Висновки. Наші результати надають данні про можливість проводити скринінг новітніх гліопротекторів з допомогою план­шет-рідера, використовуючи первинну культуру АГЗН. Background. Optic nerve head astrocytes (ONHAs) are the major glia cell type in the non-myelinated optic nerve head where they contribute to extracellular matrix synthesis. Pathological changes in glaucoma include reactive astrocytosis, a process characterized by altered astrocyte gene and protein expression and extracellular matrix remodeling. ONHAs are highly sensitive to mechanical and oxidative stress resulting in the initiation of axon damage early during pathogenesis. Furthermore, ONHAs are crucial for the maintenance of retinal ganglion cell physiology and function. Therefore, glioprotective strategies with the goal to preserve and/or restore the structural and functional viability of ONHA to slow glaucoma and related pathologies are of high clinical relevance. Objective. The aim of the paper is the development of standardized methods for the systematic advancement of glioprotective strategies using plate reader-based assays determining cellular viability, plate-reader based proliferation and the intracellular redox state. Methods. In our work we used primary culture of OHNAs as a model system. The oxidative stress was induced by tBHP. Reactive oxygen species (ROS) were measured by the DCDFA assay. For cell viability tests we used the optimized lactate dehydrogenase (LDH) release assay, as well as the MTT and Calcein-AM uptake assays. Results. The half-maximal effect (EC50) of tBHP on OHNAs ROS levels in the DCFDA assay was 192.1 ± 15.7 μM. The measurement of cellular viability of OHNAs after tBHP-induced oxidative stress showed EC50 = 156.9 ± 3.8 µM in the Calcein-AM uptake assay. In the MTT assay, the EC50 for tBHP was 138.1 ± 1.4 µM, and shifted to 192.7 ± 2.8 µM with 100 μM Trolox pre-treatment of OHNA. In the LDH release assay, the EC50 for tBHP was 146.9 ± 4.9 µM and 246.3 ± 7.3 µM for the control and Trolox conditions respectively. Conclusions. Our results provide feasibility data for the plate-reader based screening for novel glioprotectants using primary ONHA culture. Проблематика. Астроциты головки зрительного нерва (АГЗН) являются основным типом глиальных клеток в немиелинированной головке зрительного нерва, где они участвуют в синтезе внешнеклеточного матрикса. Патологические изменения при глаукоме включают реактивный астроцитоз – процесс, который характеризуется изменениями в экспрессии астроцитарных генов и белков, а также ремоделингом внешнеклеточного матрикса. АГЗН являются чрезвычайно чувствительными к механическому и оксидативному стрессу, что приводит к инициации повреждения аксонов при раннем патогенезе. Кроме того, АГЗН играют ключевую роль в поддержке физиологии и функций ганглионарных клеток сетчатки. Именно поэтому глиопротекторные стратегии с целью сохранения и/или возобновления структурной и функциональной жизнеспособности АГЗН для замедления глаукомы и сродных патологий являются чрезвычайно важными для клинической практики. Цель исследования. Разработка стандартизированных методов для систематического внедрения глиопротекторных стратегий с использованием тестов определения жизнеспособности, пролиферации и внутриклеточного редокс-состояния клеток с помощью планшетного ридера. Методика реализации. В нашей работе мы использовали первичную культуру АГЗН как модельную систему. Оксидативный стресс был индуцирован с помощью tBHP. Реактивные формы кислорода (РФК) были измерены с помощью теста DCFDA. Для тестов жизнеспособности клеток мы использовали оптимизированный нами тест высвобождения лактатдегидрогеназы (ЛДГ), а также тест МТТ и тест поглощения Кальцеина-АМ. Результаты исследования. Полумаксимальный эффект (EC50) tBHP для уровней РФК в АГЗН в тесте DCFDA составил 192,1 ± 15,7 мкМ. Измерение клеточной жизнеспособности АГЗН после оксидативного стресса, индуцированного tBHP, показало EC50 = 156,9 ± 3,8 мкМ в тесте поглощения Кальцеина-АМ. В тесте МТТ EC50 для tBHP составило 138,1 ± 1,4 мкМ и смести­лось до 192,7 ± 2,8 мкМ после предварительной обработки АГЗН 100 мкМ Тролоксом. В тесте высвобождения ЛДГ EC50 для tBHP составило 146,9 ± 4,9 и 246,3 ± 7,3 мкМ для контрольного и обработанного Тролоксом образцов соответственно. Выводы. Наши результаты предоставляют данные о возможности проводить скрининг новейших глиопротекторов с помощью планшетного ридера, используя первичную культуру АГЗН.

Published
2017

24. Differential Activation of Glioprotective Intracellular Signaling Pathways in Primary Optic Nerve Head Astrocytes after Treatment with Different Classes of Antioxidants.

Author

Ghosh, Anita K., Rao, Vidhya R., Wisniewski, Victoria J., Zigrossi, Alexandra D., Floss, Jamie, Koulen, Peter, Stubbs, Evan B, and Kaja, Simon

Subjects
OPTIC nerve, NICOTINAMIDE adenine dinucleotide phosphate, LACTATE dehydrogenase, RESVERATROL, MANGANESE porphyrins
Abstract

Optic nerve head astrocytes are the specialized glia cells that provide structural and trophic support to the optic nerve head. In response to cellular injury, optic nerve head astrocytes undergo reactive astrocytosis, the process of cellular activation associated with cytoskeletal remodeling, increases in the rate of proliferation and motility, and the generation of Reactive Oxygen Species. Antioxidant intervention has previously been proposed as a therapeutic approach for glaucomatous optic neuropathy, however, little is known regarding the response of optic nerve head astrocytes to antioxidants under physiological versus pathological conditions. The goal of this study was to determine the effects of three different antioxidants, manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin (Mn-TM-2-PyP), resveratrol and xanthohumol in primary optic nerve head astrocytes. Effects on the expression of the master regulator nuclear factor erythroid 2-related factor 2 (Nrf2), the antioxidant enzyme, manganese-dependent superoxide dismutase 2 (SOD2), and the pro-oxidant enzyme, nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4), were determined by quantitative immunoblotting. Furthermore, efficacy in preventing chemically and reactive astrocytosis-induced increases in cellular oxidative stress was quantified using cell viability assays. The results were compared to the effects of the prototypic antioxidant, Trolox. Antioxidants elicited highly differential changes in the expression levels of Nrf2, SOD2, and NOX4. Notably, Mn-TM-2-PyP increased SOD2 expression eight-fold, while resveratrol increased Nrf2 expression three-fold. In contrast, xanthohumol exerted no statistically significant changes in expression levels. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) uptake and lactate dehydrogenase (LDH) release assays were performed to assess cell viability after chemically and reactive astrocytosis-induced oxidative stress. Mn-TM-2-PyP exerted the most potent glioprotection by fully preventing the loss of cell viability, whereas resveratrol and xanthohumol partially restored cell viability. Our data provide the first evidence for a well-developed antioxidant defense system in optic nerve head astrocytes, which can be pharmacologically targeted by different classes of antioxidants. [ABSTRACT FROM AUTHOR]

Published
2020
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25. Altered expression of 3α-hydroxysteroid dehydrogenases in human glaucomatous optic nerve head astrocytes

Author

Bernard I. Weinstein, M R Hernandez, W.-H. Wang, Olga A. Agapova, Ping Yang, AF Clark, and Lane Da

Subjects
Adult, Male, medicine.medical_specialty, endocrine system, Neuroactive steroid, 3-Hydroxysteroid Dehydrogenases, genetic structures, Hydrostatic pressure, Optic Disk, Glaucoma, In situ hybridization, Biology, Optic nerve head astrocytes, 3α-HSD, Gene Expression Regulation, Enzymologic, lcsh:RC321-571, Internal medicine, medicine, Neurosteroid, Animals, Humans, RNA, Messenger, lcsh:Neurosciences. Biological psychiatry. Neuropsychiatry, Cells, Cultured, Aged, Regulation of gene expression, Aged, 80 and over, AKR1C1, Middle Aged, medicine.disease, 3-alpha-Hydroxysteroid Dehydrogenase (B-Specific), Macaca mulatta, Pathophysiology, eye diseases, Isoenzymes, Endocrinology, Optic nerve transection, Neurology, Astrocytes, Optic nerve, Immunohistochemistry, Female, sense organs, Neuroscience
Abstract

3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) isoforms (AKR1C1-AKR1C4) are aldo-keto reductases that metabolize steroids and other substances in many tissues including the CNS. Here we demonstrated that in glaucomatous human optic nerve heads, increased expression of 3alpha-HSD was localized to reactive astrocytes in the lamina cribrosa. Similar, optic nerve head astrocytes exhibited increased expression of 3alpha-HSD in response to elevated intraocular pressure in a monkey model of experimental glaucoma, but not in monkeys with unilateral optic nerve transection. In vitro, glaucomatous optic nerve head astrocytes expressed higher levels of AKR1C1, AKR1C2, and AKR1C3 mRNA, than normal astrocytes, with significant differential increase of AKR1C2 expression, and exhibited higher enzymatic activity forming 3alpha-androstanediol a well-recognized neurosteroid. Normal astrocytes exposed to elevated hydrostatic pressure selectively increased AKR1C2 expression. Our findings of increased expression of 3alpha-HSDs in glaucomatous optic nerve head astrocytes offer new insights into possible roles for neurosteroids in the pathophysiology of glaucoma.

Published
2003

26. Minocycline is cytoprotective in human trabecular meshwork cells and optic nerve head astrocytes by increasing expression of XIAP, survivin, and Bcl-2

Author

Cristoph Hirneiss, Michael W. Ulbig, Marcus Kernt, Aljoscha S. Neubauer, Armin Wolf, Kirsten H. Eibl, and Anselm Kampik

Subjects
Pathology, medicine.medical_specialty, genetic structures, Glaucoma, optic nerve head astrocytes, medicine.disease_cause, minocycline, Ophthalmology, Survivin, medicine, Original Research, biology, business.industry, trabecular meshwork, apoptosis, Clinical Ophthalmology, Minocycline, Transforming growth factor beta, medicine.disease, eye diseases, XIAP, medicine.anatomical_structure, glaucoma, Optic nerve, Cancer research, biology.protein, sense organs, Trabecular meshwork, business, Oxidative stress, medicine.drug
Abstract

Marcus Kernt, Aljoscha S Neubauer, Kirsten H Eibl, Armin Wolf, Michael W Ulbig, Anselm Kampik, Cristoph HirneissDepartment of Ophthalmology, Ludwig-Maximilians-University, Munich, GermanyIntroduction: Primary open-angle glaucoma (POAG) is one of the leading causes of blindness. Activation of optic nerve head astrocytes (ONHA) and loss of trabecular meshwork cells (TMC) are pathognomonic for this neurodegenerative disease. Oxidative stress and elevated levels of transforming growth factor beta (TGFβ) play an important role in the pathogenesis of POAG. This study investigates the possible antiapoptotic and cytoprotective effects of minocycline on TMC and ONHA under oxidative stress and increased TGFβ levels.Methods: TMC and ONHA were treated with minocycline 1–150 μM. Possible toxic effects and IC50 were evaluated after 48 hours. Cell proliferation and viability were examined in order to assess the protective effects of minocycline on TMC and ONHA. Expression of Bcl-2, XIAP, and survivin, as well as their mRNA expression, were assessed by real time polymerase chain reaction (RT-PCR) and Western Blot analysis 48 hours after treatment with minocycline alone and additional incubation with TGFβ-2 or oxidative stress.Results: Minocycline 1–75 μM showed no toxic effects on TMC and ONHA. Under conditions of oxidative stress, both TMC and ONHA showed an increase in viability and an ability to proliferate when treated with minocycline 20–40 μM. RT-PCR and Western blotting yielded an overexpression of Bcl-2, XIAP, and survivin when TMC or ONHA were treated with minocycline 20–40 μM under conditions of oxidative stress and when additionally incubated with TGFβ-2.Conclusion: Minocycline up to 75 μM does not have toxic effects on TMC and ONHA. Treatment with minocycline 20–40 μM led to increased viability and proliferation under oxidative stress and TGFβ-2, as well as overexpression of Bcl-2, XIAP, and survivin. This protective pathway may help to prevent apoptotic cell death of TMC and ONHA and therefore be a promising approach to avoidance of progression of glaucomatous degeneration.Keywords: glaucoma, apoptosis, minocycline, trabecular meshwork, optic nerve head astrocytes

Published
2010

27. Inhibition of oxidative stress by coenzyme Q10 increases mitochondrial mass and improves bioenergetic function in optic nerve head astrocytes

Author

Robert N. Weinreb, Guy Perkins, Soo-Ho Choi, Choi S, Mark H. Ellisman, Myoung Sup Shim, You Hyun Noh, Won-Kyu Ju, and Keunyoung Kim

Subjects
Cancer Research, genetic structures, Mitochondrial Turnover, Ubiquinone, Image Processing, Optic disk, Mitochondrion, Neurodegenerative, medicine.disease_cause, Oxidative Phosphorylation, Rats, Sprague-Dawley, 0302 clinical medicine, Computer-Assisted, OXPHOS complex, Image Processing, Computer-Assisted, oxidative stress, Cells, Cultured, 0303 health sciences, Cultured, biology, Cell biology, Mitochondria, 5.1 Pharmaceuticals, Generic Health Relevance, Neurological, Original Article, Female, Development of treatments and therapeutic interventions, Cells, Immunology, Oncology and Carcinogenesis, Optic Disk, SOD2, optic nerve head astrocytes, Oxidative phosphorylation, Superoxide dismutase, Mitochondrial Proteins, 03 medical and health sciences, Cellular and Molecular Neuroscience, coenzyme Q10, mitochondrial dysfunction, medicine, Animals, Eye Disease and Disorders of Vision, 030304 developmental biology, Neurosciences, Cell Biology, eye diseases, Rats, Oxidative Stress, glaucoma, Mitochondrial biogenesis, Astrocytes, Multiprotein Complexes, biology.protein, Sprague-Dawley, Biochemistry and Cell Biology, sense organs, Energy Metabolism, 030217 neurology & neurosurgery, Oxidative stress, Transcription Factors
Abstract

Oxidative stress contributes to dysfunction of glial cells in the optic nerve head (ONH). However, the biological basis of the precise functional role of mitochondria in this dysfunction is not fully understood. Coenzyme Q10 (CoQ10), an essential cofactor of the electron transport chain and a potent antioxidant, acts by scavenging reactive oxygen species (ROS) for protecting neuronal cells against oxidative stress in many neurodegenerative diseases. Here, we tested whether hydrogen peroxide (100 μM H2O2)-induced oxidative stress alters the mitochondrial network, oxidative phosphorylation (OXPHOS) complex (Cx) expression and bioenergetics, as well as whether CoQ10 can ameliorate oxidative stress-mediated alterations in mitochondria of the ONH astrocytes in vitro. Oxidative stress triggered the activation of ONH astrocytes and the upregulation of superoxide dismutase 2 (SOD2) and heme oxygenase-1 (HO-1) protein expression in the ONH astrocytes. In contrast, CoQ10 not only prevented activation of ONH astrocytes but also significantly decreased SOD2 and HO-1 protein expression in the ONH astrocytes against oxidative stress. Further, CoQ10 prevented a significant loss of mitochondrial mass by increasing mitochondrial number and volume density and by preserving mitochondrial cristae structure, as well as promoted mitofilin and peroxisome-proliferator-activated receptor-c coactivator-1 protein expression in the ONH astrocyte, suggesting an induction of mitochondrial biogenesis. Finally, oxidative stress triggered the upregulation of OXPHOS Cx protein expression, as well as reduction of cellular adeonsine triphosphate (ATP) production and increase of ROS generation in the ONH astocytes. However, CoQ10 preserved OXPHOS protein expression and cellular ATP production, as well as decreased ROS generation in the ONH astrocytes. On the basis of these observations, we suggest that oxidative stress-mediated mitochondrial dysfunction or alteration may be an important pathophysiological mechanism in the dysfunction of ONH astrocytes. CoQ10 may provide new therapeutic potentials and strategies for protecting ONH astrocytes against oxidative stress-mediated mitochondrial dysfunction or alteration in glaucoma and other optic neuropathies. © 2013 Macmillan Publishers Limited. All rights reserved.

Published
2013

28. Minocycline is cytoprotective in human trabecular meshwork cells and optic nerve head astrocytes by increasing expression of XIAP, survivin, and Bcl-2.

Author

Kernt M, Neubauer AS, Eibl KH, Wolf A, Ulbig MW, Kampik A, and Hirneiss C

Abstract

Introduction: Primary open-angle glaucoma (POAG) is one of the leading causes of blindness. Activation of optic nerve head astrocytes (ONHA) and loss of trabecular meshwork cells (TMC) are pathognomonic for this neurodegenerative disease. Oxidative stress and elevated levels of transforming growth factor beta (TGFbeta) play an important role in the pathogenesis of POAG. This study investigates the possible antiapoptotic and cytoprotective effects of minocycline on TMC and ONHA under oxidative stress and increased TGFbeta levels., Methods: TMC and ONHA were treated with minocycline 1-150 muM. Possible toxic effects and IC(50) were evaluated after 48 hours. Cell proliferation and viability were examined in order to assess the protective effects of minocycline on TMC and ONHA. Expression of Bcl-2, XIAP, and survivin, as well as their mRNA expression, were assessed by real time polymerase chain reaction (RT-PCR) and Western Blot analysis 48 hours after treatment with minocycline alone and additional incubation with TGFbeta-2 or oxidative stress., Results: Minocycline 1-75 muM showed no toxic effects on TMC and ONHA. Under conditions of oxidative stress, both TMC and ONHA showed an increase in viability and an ability to proliferate when treated with minocycline 20-40 muM. RT-PCR and Western blotting yielded an overexpression of Bcl-2, XIAP, and survivin when TMC or ONHA were treated with minocycline 20-40 muM under conditions of oxidative stress and when additionally incubated with TGFbeta-2., Conclusion: Minocycline up to 75 muM does not have toxic effects on TMC and ONHA. Treatment with minocycline 20-40 muM led to increased viability and proliferation under oxidative stress and TGFbeta-2, as well as overexpression of Bcl-2, XIAP, and survivin. This protective pathway may help to prevent apoptotic cell death of TMC and ONHA and therefore be a promising approach to avoidance of progression of glaucomatous degeneration.

Published
2010
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