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• W2054724063 abstract "Stressed cells release ATP, which participates in neurodegenerative processes through the specific ligation of P2RX7 purinergic receptors. Here, we demonstrate that extracellular ATP and the more specific P2RX7 agonist, 2′- and 3′-O-(4-benzoylbenzoyl)-ATP, both induce photoreceptor cell death when added to primary retinal cell cultures or when injected into the eyes from wild-type mice, but not into the eyes from P2RX7−/− mice. Photoreceptor cell death was accompanied by the activation of caspase-8 and -9, translocation of apoptosis-inducing factor from mitochondria to nuclei, and TUNEL-detectable chromatin fragmentation. All hallmarks of photoreceptor apoptosis were prevented by premedication or co-application of Brilliant Blue G, a selective P2RX7 antagonist that is already approved for the staining of internal limiting membranes during ocular surgery. ATP release is up-regulated by nutrient starvation in primary retinal cell cultures and seems to be an initializing event that triggers primary and/or secondary cell death via the positive feedback loop on P2RX7. Our results encourage the potential application of Brilliant Blue G as a novel neuroprotective agent in retinal diseases or similar neurodegenerative pathologies linked to excessive extracellular ATP. Stressed cells release ATP, which participates in neurodegenerative processes through the specific ligation of P2RX7 purinergic receptors. Here, we demonstrate that extracellular ATP and the more specific P2RX7 agonist, 2′- and 3′-O-(4-benzoylbenzoyl)-ATP, both induce photoreceptor cell death when added to primary retinal cell cultures or when injected into the eyes from wild-type mice, but not into the eyes from P2RX7−/− mice. Photoreceptor cell death was accompanied by the activation of caspase-8 and -9, translocation of apoptosis-inducing factor from mitochondria to nuclei, and TUNEL-detectable chromatin fragmentation. All hallmarks of photoreceptor apoptosis were prevented by premedication or co-application of Brilliant Blue G, a selective P2RX7 antagonist that is already approved for the staining of internal limiting membranes during ocular surgery. ATP release is up-regulated by nutrient starvation in primary retinal cell cultures and seems to be an initializing event that triggers primary and/or secondary cell death via the positive feedback loop on P2RX7. Our results encourage the potential application of Brilliant Blue G as a novel neuroprotective agent in retinal diseases or similar neurodegenerative pathologies linked to excessive extracellular ATP. Photoreceptor degeneration involves the activation of several pathways of regulated cell death that may constitute potential therapeutic targets. Accordingly, attempts have been undertaken to inhibit caspases, which play a central role in the acquisition of the apoptotic structure,1Ellis H.M. Horvitz H.R. Genetic control of programmed cell death in the nematode C. elegans.Cell. 1986; 44: 817-829Abstract Full Text PDF PubMed Scopus (1356) Google Scholar, 2Yuan J. Shaham S. Ledoux S. Ellis H.M. Horvitz H.R. The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1 beta-converting enzyme.Cell. 1993; 75: 641-652Abstract Full Text PDF PubMed Scopus (2248) Google Scholar although pharmacologic pan-caspase inhibitors have largely failed to preserve the structure and function of photoreceptors.3Green D.R. Kroemer G. Pharmacological manipulation of cell death: clinical applications in sight?.J Clin Invest. 2005; 115: 2610-2617Crossref PubMed Scopus (218) Google Scholar, 4Hisatomi T. Sakamoto T. Murata T. Yamanaka I. Oshima Y. Hata Y. Ishibashi T. Inomata H. Susin S.A. Kroemer G. Relocalization of apoptosis-inducing factor in photoreceptor apoptosis induced by retinal detachment in vivo.Am J Pathol. 2001; 158: 1271-1278Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar Caspases can be activated as the result of mitochondrial outer membrane permeabilization (MOMP) in thus far that the mitochondrial release of cytochrome c results in the Apaf-1 apoptosome-dependent activation of caspase-9. MOMP also results in the mitochondrial release of apoptosis-inducing factor (AIF), which then translocates to the nucleus and participates in caspase-independent peripheral chromatin condensation and large-scale DNA fragmentation,5Susin S.A. Lorenzo H.K. Zamzami N. Marzo I. Snow B.E. Brothers G.M. Mangion J. Jacotot E. Costantini P. Loeffler M. Larochette N. Goodlett D.R. Aebersold R. Siderovski D.P. Penninger J.M. Kroemer G. Molecular characterization of mitochondrial apoptosis-inducing factor.Nature. 1999; 397: 441-446Crossref PubMed Scopus (3450) Google Scholar suggesting the existence of redundant cell death mechanisms downstream of MOMP.4Hisatomi T. Sakamoto T. Murata T. Yamanaka I. Oshima Y. Hata Y. Ishibashi T. Inomata H. Susin S.A. Kroemer G. Relocalization of apoptosis-inducing factor in photoreceptor apoptosis induced by retinal detachment in vivo.Am J Pathol. 2001; 158: 1271-1278Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 6Hisatomi T. Nakazawa T. Noda K. Almulki L. Miyahara S. Nakao S. Ito Y. She H. Kohno R. Michaud N. Ishibashi T. Hafezi-Moghadam A. Badley A.D. Kroemer G. Miller J.W. HIV protease inhibitors provide neuroprotection through inhibition of mitochondrial apoptosis in mice.J Clin Invest. 2008; 118: 2025-2038PubMed Google Scholar, 7Murakami Y. Ikeda Y. Yonemitsu Y. Onimaru M. Nakagawa K. Kohno R. Miyazaki M. Hisatomi T. Nakamura M. Yabe T. Hasegawa M. Ishibashi T. Sueishi K. Inhibition of nuclear translocation of apoptosis-inducing factor is an essential mechanism of the neuroprotective activity of pigment epithelium-derived factor in a rat model of retinal degeneration.Am J Pathol. 2008; 173: 1326-1338Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar Pharmacologic inhibition of MOMP has indeed been shown to confer some degree of neuroprotection in a model of photoreceptor degeneration induced by retinal detachment.4Hisatomi T. Sakamoto T. Murata T. Yamanaka I. Oshima Y. Hata Y. Ishibashi T. Inomata H. Susin S.A. Kroemer G. Relocalization of apoptosis-inducing factor in photoreceptor apoptosis induced by retinal detachment in vivo.Am J Pathol. 2001; 158: 1271-1278Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar Upstream of or independently from MOMP, death receptors from the tumor necrosis factor (TNF) receptor family proteins, including Fas receptor (Apo-1/CD95) or death receptor 4, can transmit apoptotic and/or necrotic signals initiated by specific “death ligands.”8Tartaglia L.A. Ayres T.M. Wong G.H. Goeddel D.V. A novel domain within the 55 kd TNF receptor signals cell death.Cell. 1993; 74: 845-853Abstract Full Text PDF PubMed Scopus (1168) Google Scholar Increased TNF receptor or CD95 signaling can participate in photoreceptor death induced by retinal detachment,9Nakazawa T. Matsubara A. Noda K. Hisatomi T. She H. Skondra D. Miyahara S. Sobrin L. Thomas K.L. Chen D.F. Grosskreutz C.L. Hafezi-Moghadam A. Miller J.W. Characterization of cytokine responses to retinal detachment in rats.Mol Vis. 2006; 12: 867-878PubMed Google Scholar, 10Zacks D.N. Zheng Q.D. Han Y. Bakhru R. Miller J.W. FAS-mediated apoptosis and its relation to intrinsic pathway activation in an experimental model of retinal detachment.Invest Ophthalmol Vis Sci. 2004; 45: 4563-4569Crossref PubMed Scopus (88) Google Scholar in which case these receptors may either stimulate the initiation of apoptosis (via the activation of caspase-8) or programed necrosis (via the activation of RIP1 kinase). Indeed, simultaneous inhibition of caspases (with Z-VAD-fmk) and RIP1 kinase (with necrostatin 1) has a more pronounced neuroprotective effect on photoreceptors in retinal detachment than either treatment alone.11Trichonas G. Murakami Y. Thanos A. Morizane Y. Kayama M. Debouck C.M. Hisatomi T. Miller J.W. Vavvas D.G. Receptor interacting protein kinases mediate retinal detachment-induced photoreceptor necrosis and compensate for inhibition of apoptosis.Proc Natl Acad Sci U S A. 2010; 107: 21695-21700Crossref PubMed Scopus (251) Google Scholar Taken together, these results underscore the existence of multiple cell death mechanisms that have to be inhibited simultaneously to confer optimal neuroprotection. An alternative to the inhibition of executioner pathways consists in intercepting the initiating events that account for upstream damage signals. Recently, ATP has been discovered as a main extracellular messenger that can contribute to lethal signaling.12Dubyak G.R. el-Moatassim C. Signal transduction via P2-purinergic receptors for extracellular ATP and other nucleotides.Am J Physiol. 1993; 265: C577-C606PubMed Google Scholar ATP, which can be released via exocytosis, anion channels, or transporters, can act in autocrine and paracrine signaling pathways,13Sabirov R.Z. Okada Y. ATP release via anion channels.Purinergic Signal. 2005; 1: 311-328Crossref PubMed Scopus (134) Google Scholar for instance by evoking action potentials in brain slices, isolated nerves, and glial cells,14Burnstock G. Purinergic receptors in the nervous system.Curr Top Membr. 2003; 54: 307-368Crossref Scopus (43) Google Scholar indicating that ATP acts as a physiological mediator of neurotransmission and neuron-glia communication.15Abbracchio M.P. Burnstock G. Verkhratsky A. Zimmermann H. Purinergic signalling in the nervous system: an overview.Trends Neurosci. 2009; 32: 19-29Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar Moreover, extracellular ATP concentrations increase in spinal cord injury,16Wang X. Arcuino G. Takano T. Lin J. Peng W.G. Wan P. Li P. Xu Q. Liu Q.S. Goldman S.A. Nedergaard M. P2X7 receptor inhibition improves recovery after spinal cord injury.Nat Med. 2004; 10: 821-827Crossref PubMed Scopus (429) Google Scholar chronic neuropathic pain,17Khakh B.S. North R.A. P2X receptors as cell-surface ATP sensors in health and disease.Nature. 2006; 442: 527-532Crossref PubMed Scopus (707) Google Scholar and brain ischemia,18Melani A. Turchi D. Vannucchi M.G. Cipriani S. Gianfriddo M. Pedata F. ATP extracellular concentrations are increased in the rat striatum during in vivo ischemia.Neurochem Int. 2005; 47: 442-448Crossref PubMed Scopus (202) Google Scholar suggesting a pathologic role for extracellular ATP as well. Extracellular ATP can act on purinergic receptors, which are classified into two classes, the ionotropic, ligand-gated P2X receptors and the metabotropic, G protein–coupled P2Y receptors.19Abbracchio M.P. Burnstock G. Purinoceptors: are there families of P2X and P2Y purinoceptors?.Pharmacol Ther. 1994; 64: 445-475Crossref PubMed Scopus (988) Google Scholar Among the seven mammalian P2X receptors,20Ralevic V. Burnstock G. Receptors for purines and pyrimidines.Pharmacol Rev. 1998; 50: 413-492PubMed Google Scholar the P2X7 receptor (P2RX7) has the highest affinity for ATP. P2RX7 differs from other P2X receptor subtypes by its long cytoplasmic, carboxy-terminal tail (240 amino acids). Brief application of agonists renders P2RX7 permeable to small cations (K+, Na+, Ca2+) similar to other P2X receptors,12Dubyak G.R. el-Moatassim C. Signal transduction via P2-purinergic receptors for extracellular ATP and other nucleotides.Am J Physiol. 1993; 265: C577-C606PubMed Google Scholar whereas repeated or prolonged exposure to agonists can lead to the formation of P2RX7-dependent pores that become permeable to solutes ≤900 Da, hence triggering cell death.21Surprenant A. Rassendren F. Kawashima E. North R.A. Buell G. The cytolytic P2Z receptor for extracellular ATP identified as a P2X receptor (P2X7).Science. 1996; 272: 735-738Crossref PubMed Scopus (1505) Google Scholar Thus, extracellular ATP can induce apoptotic and/or necrotic cell death by acting on P2RX7.22Ferrari D. Los M. Bauer M.K. Vandenabeele P. Wesselborg S. Schulze-Osthoff K. P2Z purinoreceptor ligation induces activation of caspases with distinct roles in apoptotic and necrotic alterations of cell death.FEBS Lett. 1999; 447: 71-75Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar Despite the great interest in the carboxyl-terminal region of P2RX7, the mechanism by which P2RX7 mediates apoptotic signaling is largely unknown. Of note, the residues 436 to 531 of P2RX7 are similar to a region of TNF receptor 1 that overlaps its death domain,23Denlinger L.C. Fisette P.L. Sommer J.A. Watters J.J. Prabhu U. Dubyak G.R. Proctor R.A. Bertics P.J. Cutting edge: the nucleotide receptor P2X7 contains multiple protein- and lipid-interaction motifs including a potential binding site for bacterial lipopolysaccharide.J Immunol. 2001; 167: 1871-1876PubMed Google Scholar which has led to the proposal of a potential mechanism for P2RX7-induced caspase-8 activity and apoptosis.24Kong Q. Wang M. Liao Z. Camden J.M. Yu S. Simonyi A. Sun G.Y. Gonzalez F.A. Erb L. Seye C.I. Weisman G.A. P2X(7) nucleotide receptors mediate caspase-8/9/3-dependent apoptosis in rat primary cortical neurons.Purinergic Signal. 2005; 1: 337-347Crossref PubMed Scopus (54) Google Scholar Thus, the plasma membrane receptor, P2RX7, may cleave and activate caspase-8 during extrinsic apoptotic pathway. Importantly, P2RX7 is widely expressed in various organs, including the immune system (thymus or spleen)21Surprenant A. Rassendren F. Kawashima E. North R.A. Buell G. The cytolytic P2Z receptor for extracellular ATP identified as a P2X receptor (P2X7).Science. 1996; 272: 735-738Crossref PubMed Scopus (1505) Google Scholar and the central nervous system (cortex, hippocampus,25Sperlagh B. Szabo G. Erdelyi F. Baranyi M. Vizi E.S. Homo- and heteroexchange of adenine nucleotides and nucleosides in rat hippocampal slices by the nucleoside transport system.Br J Pharmacol. 2003; 139: 623-633Crossref PubMed Scopus (29) Google Scholar and spinal cord26Deuchars S.A. Atkinson L. Brooke R.E. Musa H. Milligan C.J. Batten T.F. Buckley N.J. Parson S.H. Deuchars J. Neuronal P2X7 receptors are targeted to presynaptic terminals in the central and peripheral nervous systems.J Neurosci. 2001; 21: 7143-7152Crossref PubMed Google Scholar). P2RX7 is expressed on astrocytes, microglial cells, and neurons.14Burnstock G. Purinergic receptors in the nervous system.Curr Top Membr. 2003; 54: 307-368Crossref Scopus (43) Google Scholar In the retina, P2RX7 is expressed on Müller glia,27Pannicke T. Fischer W. Biedermann B. Schadlich H. Grosche J. Faude F. Wiedemann P. Allgaier C. Illes P. Burnstock G. Reichenbach A. P2X7 receptors in Muller glial cells from the human retina.J Neurosci. 2000; 20: 5965-5972Crossref PubMed Google Scholar and in both inner and outer retinal neurons, including retinal ganglion cells28Brandle U. Kohler K. Wheeler-Schilling T.H. Expression of the P2X7-receptor subunit in neurons of the rat retina.Brain Res Mol Brain Res. 1998; 62: 106-109Crossref PubMed Scopus (99) Google Scholar, 29Zhang X. Zhang M. Laties A.M. Mitchell C.H. Stimulation of P2X7 receptors elevates Ca2+ and kills retinal ganglion cells.Invest Ophthalmol Vis Sci. 2005; 46: 2183-2191Crossref PubMed Scopus (117) Google Scholar and photoreceptors.30Puthussery T. Fletcher E.L. Synaptic localization of P2X7 receptors in the rat retina.J Comp Neurol. 2004; 472: 13-23Crossref PubMed Scopus (84) Google Scholar Genetic or functional inactivation of P2RX7 can attenuate the development of several neurodegenerative diseases, including Alzheimer's disease31McLarnon J.G. Ryu J.K. Walker D.G. Choi H.B. Upregulated expression of purinergic P2X(7) receptor in Alzheimer disease and amyloid-beta peptide-treated microglia and in peptide-injected rat hippocampus.J Neuropathol Exp Neurol. 2006; 65: 1090-1097Crossref PubMed Scopus (206) Google Scholar and Huntington's disease.32Diaz-Hernandez M. Diez-Zaera M. Sanchez-Nogueiro J. Gomez-Villafuertes R. Canals J.M. Alberch J. Miras-Portugal M.T. Lucas J.J. Altered P2X7-receptor level and function in mouse models of Huntington's disease and therapeutic efficacy of antagonist administration.FASEB J. 2009; 23: 1893-1906Crossref PubMed Scopus (189) Google Scholar Thus, systemic administration of a pharmacologic P2RX7 antagonist, Brilliant Blue G (BBG) can confer neuroprotective effects in models of Alzheimer's, Parkinson's disease, and spinal cord injury.31McLarnon J.G. Ryu J.K. Walker D.G. Choi H.B. Upregulated expression of purinergic P2X(7) receptor in Alzheimer disease and amyloid-beta peptide-treated microglia and in peptide-injected rat hippocampus.J Neuropathol Exp Neurol. 2006; 65: 1090-1097Crossref PubMed Scopus (206) Google Scholar, 32Diaz-Hernandez M. Diez-Zaera M. Sanchez-Nogueiro J. Gomez-Villafuertes R. Canals J.M. Alberch J. Miras-Portugal M.T. Lucas J.J. Altered P2X7-receptor level and function in mouse models of Huntington's disease and therapeutic efficacy of antagonist administration.FASEB J. 2009; 23: 1893-1906Crossref PubMed Scopus (189) Google Scholar, 33Peng W. Cotrina M.L. Han X. Yu H. Bekar L. Blum L. Takano T. Tian G.F. Goldman S.A. Nedergaard M. Systemic administration of an antagonist of the ATP-sensitive receptor P2X7 improves recovery after spinal cord injury.Proc Natl Acad Sci U S A. 2009; 106: 12489-12493Crossref PubMed Scopus (340) Google Scholar BBG is a triphenylmethane dye that has been approved for intraoperative use in ocular surgery, in the context of chromovitrectomy, which involves the use of vital dyes to improve the visualization of intraocular tissues during vitrectomy, thereby improving specific procedures such as internal limiting membrane peeling.34Rodrigues E.B. Meyer C.H. Kroll P. Chromovitrectomy: a new field in vitreoretinal surgery.Graefes Arch Clin Exp Ophthalmol. 2005; 243: 291-293Crossref PubMed Scopus (78) Google Scholar Driven by the recent characterization of BBG as a P2RX7 antagonist,31McLarnon J.G. Ryu J.K. Walker D.G. Choi H.B. Upregulated expression of purinergic P2X(7) receptor in Alzheimer disease and amyloid-beta peptide-treated microglia and in peptide-injected rat hippocampus.J Neuropathol Exp Neurol. 2006; 65: 1090-1097Crossref PubMed Scopus (206) Google Scholar, 32Diaz-Hernandez M. Diez-Zaera M. Sanchez-Nogueiro J. Gomez-Villafuertes R. Canals J.M. Alberch J. Miras-Portugal M.T. Lucas J.J. Altered P2X7-receptor level and function in mouse models of Huntington's disease and therapeutic efficacy of antagonist administration.FASEB J. 2009; 23: 1893-1906Crossref PubMed Scopus (189) Google Scholar, 33Peng W. Cotrina M.L. Han X. Yu H. Bekar L. Blum L. Takano T. Tian G.F. Goldman S.A. Nedergaard M. Systemic administration of an antagonist of the ATP-sensitive receptor P2X7 improves recovery after spinal cord injury.Proc Natl Acad Sci U S A. 2009; 106: 12489-12493Crossref PubMed Scopus (340) Google Scholar we decided to investigate the pathogenic implications of P2RX7 in pathologic photoreceptor loss, as well as the therapeutic utility of BBG in this context. As a result of these investigations, we report here that increased extracellular ATP levels contribute to pathologic conditions of photoreceptor loss and that BBG efficiently avoids photoreceptor cell death. All animal experiments were performed according to the guidelines of the Association for Research in Vision and Ophthalmology on adult (8 weeks of age) male C57BL6JJcl mice (CLEA, Tokyo, Japan) and isogenic P2RX7−/− mice kindly provided from Pfizer Inc. (Groton, CT; now also available from The Jackson Laboratory, Bar Harbor, ME; B6.129P2-P2rx7tm1Gab/J, stock number 005576). Adult primary retinal cell cultures were prepared as previously described with minor modifications.6Hisatomi T. Nakazawa T. Noda K. Almulki L. Miyahara S. Nakao S. Ito Y. She H. Kohno R. Michaud N. Ishibashi T. Hafezi-Moghadam A. Badley A.D. Kroemer G. Miller J.W. HIV protease inhibitors provide neuroprotection through inhibition of mitochondrial apoptosis in mice.J Clin Invest. 2008; 118: 2025-2038PubMed Google Scholar Primary retinal cells were cultured in 4-well chamber (Nunc; part of Thermo Fisher Scientific, Rochester, NY) with Neurobasal-A medium (Invitrogen, Carlsbad, CA) containing B27 supplement without antioxidants (Invitrogen), 1 μg/mL insulin, and 12 μg/mL gentamicin. To determine the number of adherent photoreceptor cells, immunofluorescent staining was performed with a rabbit anti-recoverin antibody (Millipore, Bedford, MA). For nutrient starvation, primary retinal cells were cultured for 3 hours (ATP measurement) or 24 hours (immunocytochemistry and viability assay) with Neurobasal-A medium without B27 supplement as “starvation medium.” P2RX7 agonists [ATP or 2′- and 3′-O-(4-benzoylbenzoyl)-ATP (BzATP); Sigma-Aldrich, St Louis, MO] or P2RX7 antagonists (1-[N, O-bis(5-isoquinolinesulphonyl)-N-methyl-L-tyrosyl]−4-phenylpiperazine (KN-62) or BBG; Sigma-Aldrich) were added to the medium and incubated for 24 hours. To investigate the effect of BBG before incubation, cells were cultured in the presence of BBG for 30 minutes, washed two times with culture medium, and incubated for 24 hours. To examine the secondary effects of death ligands, rat anti-mouse TNF-α neutralizing antibody (1 to 10 ng/mL MP6-XT22; R&D Systems, Minneapolis, MN)35Abrams J.S. Roncarolo M.G. Yssel H. Andersson U. Gleich G.J. Silver J.E. Strategies of anti-cytokine monoclonal antibody development: immunoassay of IL-10 and IL-5 in clinical samples.Immunol Rev. 1992; 127: 5-24Crossref PubMed Scopus (332) Google Scholar or hamster anti-mouse CD95 ligand-neutralizing antibody (1 to 10 μg/mL MFL3; BD Biosciences, San Jose, CA)36Kayagaki N. Yamaguchi N. Nagao F. Matsuo S. Maeda H. Okumura K. Yagita H. Polymorphism of murine Fas ligand that affects the biological activity.Proc Natl Acad Sci U S A. 1997; 94: 3914-3919Crossref PubMed Scopus (178) Google Scholar was added to culture medium, whereas isogenic rat or hamster IgG was added as control. To assess the viability of primary retinal cells, we used calcein AM (2 μmol/L; Invitrogen) or MitoTracker Orange CMTMRos (200 nmol/L, M7510; Invitrogen) that were added for 34 minutes to primary retinal cell cultures. Then, cultured cells were fixed with 4% paraformaldehyde, and photoreceptors were labeled with recoverin. Calcein+ or CMTMRos+ photoreceptors were counted in 10 random fields by blinded observers with the use of ImageJ software version 1.38x (NIH, Bethesda, MD). Values are given as means ± SDs of 10 replicate wells. For evaluation of the toxicity of BzATP and the effect of BBG in vivo, we took advantages of gas compression vitrectomy for uniform diffusion of intravitreally injected solutions. Mouse eyes were vitrectomized with SF6 gas as we and others previously described.37Thresher R.J. Ehrenberg M. Machemer R. Gas-mediated vitreous compression: an experimental alternative to mechanized vitrectomy.Graefes Arch Clin Exp Ophthalmol. 1984; 221: 192-198Crossref PubMed Scopus (80) Google Scholar, 38Irvine W.D. Johnson M.W. Hernandez E. Olsen K.R. Retinal toxicity of human tissue plasminogen activator in vitrectomized rabbit eyes.Arch Ophthalmol. 1991; 109: 718-722Crossref PubMed Scopus (66) Google Scholar, 39Enaida H. Sakamoto T. Hisatomi T. Goto Y. Ishibashi T. Morphological and functional damage of the retina caused by intravitreous indocyanine green in rat eyes.Graefes Arch Clin Exp Ophthalmol. 2002; 240: 209-213Crossref PubMed Scopus (213) Google Scholar, 40Enaida H. Hisatomi T. Goto Y. Hata Y. Ueno A. Miura M. Kubota T. Ishibashi T. Preclinical investigation of internal limiting membrane staining and peeling using intravitreal brilliant blue G.Retina. 2006; 26: 623-630Crossref PubMed Scopus (145) Google Scholar To investigate the effects of gas compression vitrectomy on the viability of retinal cells, we observed ophthalmic examination every day up to 2 weeks and evaluated histochemical changes in H&E staining and TUNEL-positive cells in the retina at 3 and 14 days after gas injections. Two weeks after gas injection, mice were anesthetized with an intraperitoneal injection of pentobarbital, and their pupils were dilated with topical 1% tropicamide and 2.5% phenylephrine hydrochloride. Then, 2 μL of liquid (PBS supplemented with 10 to 20 mmol/L BzATP or BzATP plus 500 μmol/L BBG) was injected into the vitreous cavity with a 32-gauge needle on a Hamilton syringe through posterior to the limbus. Intraocular injections were only performed in the right eye of each animal, and five eyes were examined in each group. The mice were sacrificed 24 hours after treatment, and their eyes were harvested, frozen at nitrogen liquid temperature, and cryosectioned for histochemical or ultrastructural examinations. TUNEL analysis and quantification of TUNEL-positive cells were performed as previously described4Hisatomi T. Sakamoto T. Murata T. Yamanaka I. Oshima Y. Hata Y. Ishibashi T. Inomata H. Susin S.A. Kroemer G. Relocalization of apoptosis-inducing factor in photoreceptor apoptosis induced by retinal detachment in vivo.Am J Pathol. 2001; 158: 1271-1278Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar with the use of the ApopTag Fluorescein In Situ Apoptosis Detection Kit (Millipore). Nuclei were counterstained with propidium iodide or Hoechst 33342. TUNEL-positive cells in the outer nuclear layer (ONL) were counted by two blinded observers, and results were presented as means ± SDs. As previously reported,6Hisatomi T. Nakazawa T. Noda K. Almulki L. Miyahara S. Nakao S. Ito Y. She H. Kohno R. Michaud N. Ishibashi T. Hafezi-Moghadam A. Badley A.D. Kroemer G. Miller J.W. HIV protease inhibitors provide neuroprotection through inhibition of mitochondrial apoptosis in mice.J Clin Invest. 2008; 118: 2025-2038PubMed Google Scholar rabbit anti-AIF (R&D Systems), anti-mouse cleaved caspase-9 (Cell Signaling Technology, Beverly, MA), anti-mouse cleaved caspase-8 (Cell Signaling Technology), and anti-recoverin (Millipore) were used as primary antibodies and incubated at 4°C overnight. Goat anti-rabbit IgG conjugated to Alexa Fluor 546 or 647 (Invitrogen) were used as secondary antibodies and incubated at room temperature for 1 hour. The posterior segments of enucleated eyes were fixed in PBS containing 1% glutaraldehyde and 1% paraformaldehyde, postfixed in veronal acetate buffer osmium tetroxide (2%), dehydrated in ethanol and water, and embedded in Epon. Primary retinal cell cultures were similarly fixed, dehydrated, and embedded in Epon. Ultrathin sections were cut from blocks and mounted on copper grids. The specimens were observed with H-7650 transmission electron microscope (Hitachi, Tokyo, Japan). Primary retinal cells were cultured on 35-mm coverslip dishes (BD Biosciences) in Neurobasal-A medium with B27 supplement. Then the calcium indicator, Fluo-4 AM (5 μmol/L; Invitrogen) was loaded onto primary cultures. The calcium-dependent fluorescence of Fluo-4 was captured by microscopy during 20 minutes of incubation after adding 1 mmol/L BzATP or vehicle PBS. To evaluate the effect of chelation of extracellular Ca2+ or P2RX7 antagonist (BBG), the fluorescence of Fluo-4 was measured in culture medium containing 1 mmol/L EGTA (Sigma-Aldrich) or 10 μmol/L BBG, respectively. For ATP measurements of culture medium, primary retinal cells were starved for 3 hours with Neurobasal-A medium without B27 supplement as starvation cultures, and cultures with Neurobasal-A medium with B27 supplement were prepared as controls. Then 100 μL of culture medium was collected, centrifuged at 4°C, and subjected to ATP measurements as follows. The ATP levels of collected medium (100 μL in 96-well microplates; BD Biosciences) were immediately determined by adding luciferin–luciferase reaction buffer (ATP bioluminescent assay kit, FL-AA; Sigma-Aldrich) and a multimode microplate reader, Flex Station 3 (Molecular Devices, Sunnyvale, CA). The ATP levels in medium of “control cultures” were determined by calibration of standard ATP in Neurobasal-A medium with/without B27 supplement. To define the detection limit, ecto-nucleotidase (Apyrase; 10 U/mL; Sigma-Aldrich) was added to culture medium. In control experiments, β, γ-methylene-ATP, a potent ecto-ATPase inhibitor (300 μmol/L; Sigma-Aldrich), was added to control or starvation cultures. Values are given as mean ± SD of 10 replicate wells. Primary retinal cells were cultured on 35-mm coverslip dishes (BD Biosciences) in Neurobasal A medium with B27 supplement. ATP assay mix (a part of the FL-AA kit; Sigma-Aldrich) was added to culture media (one vial per 1.5 mL of culture medium with/without B27 supplement). Light production from the luciferin–luciferase reaction was captured by an electron multiplier charge-coupled camera (ImagEM; Hamamatsu Photonics, Shizuoka, Japan) with a 40× oil lens (NA 1.35; Olympus, Tokyo, Japan) for 3 hours during 5-minute exposure periods. Statistical differences between two groups were analyzed with the Mann–Whitney U-test. Multiple group comparison was performed by analysis of variance followed by Tukey–Kramer adjustments. Differences were considered significance at P < 0.05 and P < 0.01. All values were expressed as means ± SDs. To investigate the possible implication of P2RX7 in photoreceptor degeneration, we took advantage of primary retinal cell cultures.6Hisatomi T. Nakazawa T. Noda K. Almulki L. Miyahara S. Nakao S. Ito Y. She H. Kohno R. Michaud N. Ishibashi T. Hafezi-Moghadam A. Badley A.D. Kroemer G. Miller J.W. HIV protease inhibitors provide neuroprotection through inhibition of mitochondrial apoptosis in mice.J Clin Invest. 2008; 118: 2025-2038PubMed Google Scholar, 41Nakazawa T. Hisatomi T. Nakazawa C. Noda K. Maruyama K. She H. Matsubara A. Miyahara S. Nakao S. Yin Y. Benowitz L. Hafezi-Moghadam A. Miller J.W. Monocyte chemoattractant protein 1 mediates retinal detachment-induced photoreceptor apoptosis.Proc Natl Acad Sci U S A. 2007; 104: 2425-2430Crossref PubMed Scopus (234) Google Scholar Photoreceptor viability was assessed with two fluorescent sen" @default.
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• W2054724063 date "2011-12-01" @default.
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• W2054724063 title "Critical Involvement of Extracellular ATP Acting on P2RX7 Purinergic Receptors in Photoreceptor Cell Death" @default.
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• W2054724063 doi "https://doi.org/10.1016/j.ajpath.2011.08.035" @default.
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