臺灣博碩士論文加值系統

活化表皮生長因子受體（epidermal growth factor receptor, EGFR），一個171-kDa的跨膜糖蛋白，並具有酪氨酸激酶(tyrosine kinase, TK)活性，會參與多種信息傳遞，進而調控細胞生長、增殖、細胞的生存、分化和死亡。然而，表皮生長因子受體也會參與神經退化性疾病，包括中風。例如，在中風之腦組織，星狀細胞受到刺激，表皮生長因子受體會將靜止之星狀細胞成為反應性之星狀細胞。因此，我的論文有2個研究主題:第一是表皮生長因子受體在氧糖缺乏處理（oxygen glucose deprivation, OGD）之CTX-TNA2（大鼠星狀細胞株）和大鼠初級培養之星狀細胞參與之角色；第二是afatinib（EGFR-TK抑製劑）對抗星狀細胞之發炎作用。在論文的第一部分，CTX-TNA2（大鼠星狀細胞株）和大鼠初級培養星狀細胞置於細胞培養液不含葡萄糖，培養箱內氣體組成為1％O2/94％N2/5％CO2，37℃，共3、6、12小時。實驗結果顯示，依OGD處理之時間誘導EGFR磷酸化（酪氨酸1068）和其下游之信號傳導途徑，包括AKT和ERK的磷酸化。同時，OGD會增加GFAP (星狀細胞活化之生物標誌物)、COX-II、iNOS (2種促發炎之酵素) 、一氧化氮（NO）的含量，以及活化態之caspase 1（發炎體活化之的生物標誌物）和IL -1β（一促發炎之細胞因子）之的含量。論文的第二部分研究afatinib 對OGD活化之星狀細胞的影響，乃是利用afatinib 對抗12小時OGD 引起的星狀細胞之發炎作用。Western blot分析證實，afatinib會依劑量（1和10 nM）抑制OGD誘導表皮生長因子受體磷酸化及其下游信息傳遞。此外，afatinib會減弱因OGD提高之GFAP的蛋白量，降低EGFR磷酸化和GFAP共同存在之免疫螢光強度，表示afatinib可以抑制OGD誘導表皮生長因子受體磷酸化和星狀細胞活化。afatinib的抗發炎活性之作用如下：首先，afatinib 減弱iNOS和COX-II的蛋白量以及培養液NO含量。另外，afatinib 可以抑制OGD誘導活化之caspase-1 和IL-1β含量。afatinib會減低因 OGD誘導的星狀細胞之遷移和吞噬能力。afatinib會增加OGD誘導之興奮性氨基酸轉運蛋白（EAAT）-1，但不影響EAAT2之表現。總之，afatinib可以抑制OGD誘導星狀細胞之EGFR活化，神經發炎和遷移。此外，afatinib會抑制細胞吞噬。因此，表皮生長因子受體抑制劑具有潛力被用來抑制腦部缺血引起之神經發炎，並減緩中樞神經退化疾病。

Activation of epidermal growth factor receptor (EGFR), a 171-kDa transmembrane glycoprotein with tyrosine kinase (TK) activity is known to initiate multiple signaling cascades that generate intracellular mediators to regulate cell growth and proliferation as well as regulate cell survival, migration, differentiation, and death. However, EGFR has been proposed to be involved in the pathophysiology of neurodegenerative diseases, including stroke. In response to injuries, EGFR reportedly triggers quiescent astrocytes to become reactive astrocytes. In my thesis, two aims were included, one was the effect of oxygen glucose deprivation (OGD) on astrocytes, CTX-TNA2 (a rat astrocyte cell line) and primary cultured rat astrocytes. The other was the anti-inflammatory effect of afatinib (an EGFR-TK inhibitor) on OGD-treated astrocytes. In the first part of the thesis, both CTX-TNA2 and primary cultured rat astrocytes were incubated in a cultured medium deprived of glucose in a chamber with 1% O2/ 94% N2/ 5% CO2 at 37°C for 3, 6, and 12 h. Western blot assay showed that OGD induced EGFR phosphorylation (tyrosine 1068) and activated subsequent signaling pathways, including phosphorylation of AKT and extracellular signal-regulated kinases (ERK) in time dependent manner. Besides, OGD elevated the level of activated glial fibrillary acidic protein (GFAP, a biomarker of activated astrocytes), cyclooxygenase (COX)-II, inducible nitric oxide synthase (iNOS), nitric oxide (NO), caspase 1 (a biomarker of inflammasome activation), and IL-1β (a pro-inflammatory cytokine). In the second part of the thesis, the effect of afatinib on OGD-induced activation of astrocytes was investigated by incubating afatinib with OGD (12h)-treated astrocytes. Western blot assay demonstrated that afatinib (1 and 10 nM) inhibited OGD (12h)-induced EGFR phosphorylation and its signaling pathways in dose dependent manner. Furthermore, afatinib attenuated OGD-elevated GFAP expression and reduced the colocalized immunofluorescent intensities of phosphorylated EGFR and GFAP, indicating that afatinib inhibited OGD-induced EGFR phosphorylation and astrocyte reactivation. The anti-inflammatory activity of afatinib was demonstrated as follows. First, afatinib reduced OGD-induced elevation in iNOS and COX-II levels as well as NO levels in the cultured medium. Moreover, afatinib inhibited OGD-induced activation of caspase 1 and IL-1β elevation. In addition, afatinib decreased the migration ability and engulfment of microspheres of activated astrocytes. afatinib was capable of potentiating OGD-induced elevation in excitatory amino acid transporter (EAAT)-1 but not EAAT2. In conclusion, afatinib may exert its neuroprotective effect by inhibiting OGD-induced EGFR activation, neuroinflammation and migration. Furthermore, afatinib appears to attenuate phagocytosis in treated astrocytes. In conclusion, afatinib is capable of inhibiting EGFR activation, neuroinflammation, and migration. Translationally, EGFR-TKIs may be neuroprotective by exerting its anti-inflammatory action and thus attenuating brain damages.

第一章、中文摘要 I
第二章、英文摘要 III
縮寫表 V
目錄 VII
第三章、緒論 1
1. 腦中風 1
1.1 腦中風的分類 1
1.2 腦中風的治療 1
2. EGFR 2
2.1 EGFR activation 2
2.2 Regulation of signaling pathways 3
2.3 Distribution of EGFR in brain 4
2.4 EGFR ligands 5
3. EGFR在中樞神經退化性疾病中扮演之生理與病理的角色 6
3.1 EGFR的生理角色 6
3.2 EGFR在腦部缺氧中扮演的角色 7
4. EGFR在星狀細胞中的定位：EGFR 與星狀細胞之雙重角色 7
4.1 星狀細胞的生理特性 7
4.2 星狀細胞的病理特性（Pathophysiology of Astrocyte） 9
5. 抑制EGFR之治療策略 14
5.1 EGFR-TKI 的機轉 14
5.2 EGFR-TKI的臨床應用 15
5.3 EGFR-TKIs 之老藥新用：作為神經保護製劑（EGFR-TKIs repurposing
as neuroprotective agents） 16
第四章、研究目的 17
第五章、實驗材料與方法 18
1. 實驗材料 18
1.1 細胞培養/繼代/計數 18
1.2 藥品與溶劑 19
1.3 細胞生長測定 20
1.4 西方點墨法 20
1.5 流式細胞儀測定 20
1.6 ELISA kit：Rat IL-1β (R&D #RLB00, U.S.A) ; TGF-α (Cloud-CloneCorp. #SEA123Ra , U.S.A)。 21
1.7 一級抗體 (Primary antibody) 21
1.8 二級抗體 (Secondary antibody) 23
1.9儀器耗材 23
2. 實驗方法 24
2.1 大鼠初代星狀細胞培養（Primary cultured astrocyte culture） 24
2.2 細胞培養（Cell culture） 25
2.3 氧糖缺乏處理（OGD condition and hypoxia condition） 25
2.4 藥物配製 26
2.5細胞生長測定 (Sulforhodamine B assay; SRB assay) 26
2.6 乳酸脫氫酶活性測試 (Lactate dehydrogenase release assay, LDH release assay) 27
2.7 細胞蛋白質均質液 27
2.8 細胞蛋白質濃度測定 (BCATM Protein Assay Kit) 28
2.9 西方墨點法 (Western blotting) 28
2.10 免疫螢光染色（Immunocytochemical staining） 29
2.11 酵素免疫分析法(ELISA, Enzyme-linked immunosorbent assay) 29
2.12 細胞移動能力試驗 (Migration assay) 30
2.13 流式細胞儀（Flow cytometry） 31
2.14 數據統計分析 (Statistics analysis) 31
第六章、實驗結果 33
1. OGD (氧糖缺乏處理)活化EGFR以及其下游ERK與AKT訊息傳遞路徑 33
2. afatinib 對於CTX-TNA2與初代培養星狀細胞之細胞存活率影響 33
3. afatinib可抑制OGD誘發之EGFR的活化 34
4. OGD處理會活化星狀細胞 35
5. afatinib可抑制OGD誘發之神經發炎反應 35
6. afatinib透過抑制caspase 1活化而降低IL-1β之釋出 36
7. afatinib可抑制低氧情況下CTX-TNA2之遷移能力 37
8. afatinib對於OGD處理之初代培養星狀細胞之EAAT1與EAAT2的影響 37
9. afatinib對於細胞增生之影響 38
10. afatinib與AZD3759會抑制低氧環境誘發CTX-TNA2細胞株之吞噬能力 39
11. OGD處理與afatinib不會影響TGF-α的表現 39
第七章、討論 40
1. OGD(氧糖缺乏處理)之離體模式 40
1.1 OGD活化EGFR及其下游ERK與AKT訊息傳遞路徑 40
1.2 OGD活化星狀細胞，促進細胞增生和細胞遷移 40
1.3 OGD引起神經發炎 41
2. afatinib的抗神經發炎作用 42
2.1 afatinib抑制EGFR活化及其下游訊息傳遞路徑 42
2.2 afatinib抑制OGD引起星狀細胞之活化、細胞增生和遷移 42
2.3 afatinib可抑制OGD誘發之神經發炎反應 43
2.4 afatinib對於OGD處理下CTX-TNA2 與初代培養星狀細胞之EAAT1與 EAAT2的影響 43
2.5 afatinib與AZD3759會抑制低氧環境誘發CTX-TNA2細胞株之吞噬能力 44
2.6 OGD處理與afatinib不會影響TGF-α的表現 44
第八章、結論 46
第九章、參考文獻 47
第十一章、附圖 72

1. Tissue plasminogen activator for acute ischemic stroke. The National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group. N Engl J Med, 1995. 333(24): p. 1581-7.
2. Wardlaw, J.M., et al., Recombinant tissue plasminogen activator for acute ischaemic stroke: an updated systematic review and meta-analysis. Lancet, 2012. 379(9834): p. 2364-72.
3. Albers, G.W., et al., Intravenous tissue-type plasminogen activator for treatment of acute stroke: the Standard Treatment with Alteplase to Reverse Stroke (STARS) study. Jama, 2000. 283(9): p. 1145-50.
4. Prenzel, N., et al., The epidermal growth factor receptor family as a central element for cellular signal transduction and diversification. Endocr Relat Cancer, 2001. 8(1): p. 11-31.
5. Bishayee, S., Role of conformational alteration in the epidermal growth factor receptor (EGFR) function. Biochem Pharmacol, 2000. 60(8): p. 1217-23.
6. Li, Q., et al., Effect of neuregulin on apoptosis and expressions of STAT3 and GFAP in rats following cerebral ischemic reperfusion. J Mol Neurosci, 2009. 37(1): p. 67-73.
7. Yano, S., et al., Distribution and function of EGFR in human tissue and the effect of EGFR tyrosine kinase inhibition. Anticancer Res, 2003. 23(5a): p. 3639-50.
8. Gomez-Pinilla, F., D.J. Knauer, and M. Nieto-Sampedro, Epidermal growth factor receptor immunoreactivity in rat brain. Development and cellular localization. Brain Res, 1988. 438(1-2): p. 385-90.
9. Erschbamer, M., K. Pernold, and L. Olson, Inhibiting epidermal growth factor receptor improves structural, locomotor, sensory, and bladder recovery from experimental spinal cord injury. J Neurosci, 2007. 27(24): p. 6428-35.
10. Wilson, K.J., et al., EGFR ligands exhibit functional differences in models of paracrine and autocrine signaling. Growth Factors, 2012. 30(2): p. 107-16.
11. Lazar, L.M. and M. Blum, Regional distribution and developmental expression of epidermal growth factor and transforming growth factor-alpha mRNA in mouse brain by a quantitative nuclease protection assay. J Neurosci, 1992. 12(5): p. 1688-97.
12. Kaser, M.R., J. Lakshmanan, and D.A. Fisher, Comparison between epidermal growth factor, transforming growth factor-alpha and EGF receptor levels in regions of adult rat brain. Brain Res Mol Brain Res, 1992. 16(3-4): p. 316-22.
13. Fallon, J., et al., In vivo induction of massive proliferation, directed migration, and differentiation of neural cells in the adult mammalian brain. Proc Natl Acad Sci U S A, 2000. 97(26): p. 14686-91.
14. Koprivica, V., et al., EGFR activation mediates inhibition of axon regeneration by myelin and chondroitin sulfate proteoglycans. Science, 2005. 310(5745): p. 106-10.
15. Smith, G.M. and C. Strunz, Growth factor and cytokine regulation of chondroitin sulfate proteoglycans by astrocytes. Glia, 2005. 52(3): p. 209-18.
16. Ayuso-Sacido, A., et al., Activated EGFR signaling increases proliferation, survival, and migration and blocks neuronal differentiation in post-natal neural stem cells. J Neurooncol, 2010. 97(3): p. 323-37.
17. Gallo, V. and B. Deneen, Glial development: the crossroads of regeneration and repair in the CNS. Neuron, 2014. 83(2): p. 283-308.
18. Liu, B. and A.H. Neufeld, Activation of epidermal growth factor receptor causes astrocytes to form cribriform structures. Glia, 2004. 46(2): p. 153-68.
19. Zhang, S., et al., Inhibition of Epidermal Growth Factor Receptor Improves Myelination and Attenuates Tissue Damage of Spinal Cord Injury. Cell Mol Neurobiol, 2016.
20. Justicia, C. and A.M. Planas, Transforming growth factor-alpha acting at the epidermal growth factor receptor reduces infarct volume after permanent middle cerebral artery occlusion in rats. J Cereb Blood Flow Metab, 1999. 19(2): p. 128-32.
21. Stoll, G., S. Jander, and M. Schroeter, Neuroglia: definition, classification, evolution, numbers, development. . glial physiology and pathophysiology, 2013: p. 73-104.
22. Nedergaard, M., B. Ransom, and S.A. Goldman, New roles for astrocytes: redefining the functional architecture of the brain. Trends Neurosci, 2003. 26(10): p. 523-30.
23. Seifert, G., K. Schilling, and C. Steinhauser, Astrocyte dysfunction in neurological disorders: a molecular perspective. Nat Rev Neurosci, 2006. 7(3): p. 194-206.
24. Pellerin, L., et al., Activity-dependent regulation of energy metabolism by astrocytes: an update. Glia, 2007. 55(12): p. 1251-62.
25. Barres, B.A., The mystery and magic of glia: a perspective on their roles in health and disease. Neuron, 2008. 60(3): p. 430-40.
26. Cataldo, A.M. and R.D. Broadwell, Cytochemical identification of cerebral glycogen and glucose-6-phosphatase activity under normal and experimental conditions. II. Choroid plexus and ependymal epithelia, endothelia and pericytes. J Neurocytol, 1986. 15(4): p. 511-24.
27. Brown, A.M. and B.R. Ransom, Astrocyte glycogen and brain energy metabolism. Glia, 2007. 55(12): p. 1263-71.
28. Alvarez, J.I., T. Katayama, and A. Prat, Glial influence on the blood brain barrier. Glia, 2013. 61(12): p. 1939-58.
29. Daneman, R. and A. Prat, The blood-brain barrier. Cold Spring Harb Perspect Biol, 2015. 7(1): p. a020412.
30. Janzer, R.C. and M.C. Raff, Astrocytes induce blood-brain barrier properties in endothelial cells. Nature, 1987. 325(6101): p. 253-7.
31. Abbott, N.J., L. Ronnback, and E. Hansson, Astrocyte-endothelial interactions at the blood-brain barrier. Nat Rev Neurosci, 2006. 7(1): p. 41-53.
32. Broux, B., E. Gowing, and A. Prat, Glial regulation of the blood-brain barrier in health and disease. Semin Immunopathol, 2015. 37(6): p. 577-90.
33. Savchenko, V.L., et al., Microglia and astrocytes in the adult rat brain: comparative immunocytochemical analysis demonstrates the efficacy of lipocortin 1 immunoreactivity. Neuroscience, 2000. 96(1): p. 195-203.
34. Li, C., et al., Astrocytes: implications for neuroinflammatory pathogenesis of Alzheimer's disease. Curr Alzheimer Res, 2011. 8(1): p. 67-80.
35. Gendelman, H.E., Neural immunity: Friend or foe? J Neurovirol, 2002. 8(6): p. 474-9.
36. Pekny, M. and M. Nilsson, Astrocyte activation and reactive gliosis. Glia, 2005. 50(4): p. 427-34.
37. Sofroniew, M.V., Reactive astrocytes in neural repair and protection. Neuroscientist, 2005. 11(5): p. 400-7.
38. Correa-Cerro, L.S. and J.W. Mandell, Molecular mechanisms of astrogliosis: new approaches with mouse genetics. J Neuropathol Exp Neurol, 2007. 66(3): p. 169-76.
39. Jones, L.L., R.U. Margolis, and M.H. Tuszynski, The chondroitin sulfate proteoglycans neurocan, brevican, phosphacan, and versican are differentially regulated following spinal cord injury. Exp Neurol, 2003. 182(2): p. 399-411.
40. Silver, J., M.E. Schwab, and P.G. Popovich, Central nervous system regenerative failure: role of oligodendrocytes, astrocytes, and microglia. Cold Spring Harb Perspect Biol, 2015. 7(3): p. a020602.
41. Stichel, C.C. and H.W. Muller, The CNS lesion scar: new vistas on an old regeneration barrier. Cell Tissue Res, 1998. 294(1): p. 1-9.
42. Anderson, M.A., Y. Ao, and M.V. Sofroniew, Heterogeneity of reactive astrocytes. Neurosci Lett, 2014. 565: p. 23-9.
43. Brosius Lutz, A. and B.A. Barres, Contrasting the glial response to axon injury in the central and peripheral nervous systems. Dev Cell, 2014. 28(1): p. 7-17.
44. Davies, S.J., et al., Regeneration of adult axons in white matter tracts of the central nervous system. Nature, 1997. 390(6661): p. 680-3.
45. Griffith, O.W. and D.J. Stuehr, Nitric oxide synthases: properties and catalytic mechanism. Annu Rev Physiol, 1995. 57: p. 707-36.
46. Zhang, R.L., Z.G. Zhang, and M. Chopp, Targeting nitric oxide in the subacute restorative treatment of ischemic stroke. Expert Opin Investig Drugs, 2013. 22(7): p. 843-51.
47. Amantea, D., et al., Rational modulation of the innate immune system for neuroprotection in ischemic stroke. Front Neurosci, 2015. 9: p. 147.
48. Arend, W.P., et al., Interleukin-1 receptor antagonist: role in biology. Annu Rev Immunol, 1998. 16: p. 27-55.
49. Bhat, R.V., et al., Increased expression of IL-1beta converting enzyme in hippocampus after ischemia: selective localization in microglia. J Neurosci, 1996. 16(13): p. 4146-54.
50. Orzylowska, O., et al., Prolonged and concomitant induction of astroglial immunoreactivity of interleukin-1beta and interleukin-6 in the rat hippocampus after transient global ischemia. Neurosci Lett, 1999. 263(1): p. 72-6.
51. Hara, H., et al., Attenuation of transient focal cerebral ischemic injury in transgenic mice expressing a mutant ICE inhibitory protein. J Cereb Blood Flow Metab, 1997. 17(4): p. 370-5.
52. Schielke, G.P., et al., Reduced ischemic brain injury in interleukin-1 beta converting enzyme-deficient mice. J Cereb Blood Flow Metab, 1998. 18(2): p. 180-5.
53. Fairman, W.A., et al., An excitatory amino-acid transporter with properties of a ligand-gated chloride channel. Nature, 1995. 375(6532): p. 599-603.
54. Lehre, K.P., et al., Differential expression of two glial glutamate transporters in the rat brain: quantitative and immunocytochemical observations. J Neurosci, 1995. 15(3 Pt 1): p. 1835-53.
55. Lehre, K.P. and N.C. Danbolt, The number of glutamate transporter subtype molecules at glutamatergic synapses: chemical and stereological quantification in young adult rat brain. J Neurosci, 1998. 18(21): p. 8751-7.
56. Furness, D.N. and K.P. Lehre, Immunocytochemical localization of a high-affinity glutamate-aspartate transporter, GLAST, in the rat and guinea-pig cochlea. Eur J Neurosci, 1997. 9(9): p. 1961-9.
57. Lehre, K.P., S. Davanger, and N.C. Danbolt, Localization of the glutamate transporter protein GLAST in rat retina. Brain Res, 1997. 744(1): p. 129-37.
58. Haugeto, O., et al., Brain glutamate transporter proteins form homomultimers. J Biol Chem, 1996. 271(44): p. 27715-22.
59. Tanaka, K., et al., Epilepsy and exacerbation of brain injury in mice lacking the glutamate transporter GLT-1. Science, 1997. 276(5319): p. 1699-702.
60. Furness, D.N., et al., A quantitative assessment of glutamate uptake into hippocampal synaptic terminals and astrocytes: new insights into a neuronal role for excitatory amino acid transporter 2 (EAAT2). Neuroscience, 2008. 157(1): p. 80-94.
61. Sutherland, M.L., T.A. Delaney, and J.L. Noebels, Glutamate transporter mRNA expression in proliferative zones of the developing and adult murine CNS. J Neurosci, 1996. 16(7): p. 2191-207.
62. Shashidharan, P., et al., Immunohistochemical localization of the neuron-specific glutamate transporter EAAC1 (EAAT3) in rat brain and spinal cord revealed by a novel monoclonal antibody. Brain Res, 1997. 773(1-2): p. 139-48.
63. Holmseth, S., et al., The density of EAAC1 (EAAT3) glutamate transporters expressed by neurons in the mammalian CNS. J Neurosci, 2012. 32(17): p. 6000-13.
64. Dehnes, Y., et al., The glutamate transporter EAAT4 in rat cerebellar Purkinje cells: a glutamate-gated chloride channel concentrated near the synapse in parts of the dendritic membrane facing astroglia. J Neurosci, 1998. 18(10): p. 3606-19.
65. Rothstein, J.D., et al., Localization of neuronal and glial glutamate transporters. Neuron, 1994. 13(3): p. 713-25.
66. Arriza, J.L., et al., Excitatory amino acid transporter 5, a retinal glutamate transporter coupled to a chloride conductance. Proc Natl Acad Sci U S A, 1997. 94(8): p. 4155-60.
67. Allen, N.J., R. Karadottir, and D. Attwell, Reversal or reduction of glutamate and GABA transport in CNS pathology and therapy. Pflugers Arch, 2004. 449(2): p. 132-42.
68. Yun, C.H., et al., Structures of lung cancer-derived EGFR mutants and inhibitor complexes: mechanism of activation and insights into differential inhibitor sensitivity. Cancer Cell, 2007. 11(3): p. 217-27.
69. Zhou, W., et al., Novel mutant-selective EGFR kinase inhibitors against EGFR T790M. Nature, 2009. 462(7276): p. 1070-4.
70. Yang, Q., et al., Blocking epidermal growth factor receptor attenuates reactive astrogliosis through inhibiting cell cycle progression and protects against ischemic brain injury in rats. J Neurochem, 2011. 119(3): p. 644-53.
71. Solca, F., et al., Target binding properties and cellular activity of afatinib (BIBW 2992), an irreversible ErbB family blocker. J Pharmacol Exp Ther, 2012. 343(2): p. 342-50.
72. Li, Z.W., et al., Epidermal growth factor receptor inhibitor ameliorates excessive astrogliosis and improves the regeneration microenvironment and functional recovery in adult rats following spinal cord injury. J Neuroinflammation, 2014. 11: p. 71.
73. Planas, A.M., et al., Epidermal growth factor receptor in proliferating reactive glia following transient focal ischemia in the rat brain. Glia, 1998. 23(2): p. 120-9.
74. Hoffknecht, P., et al., Efficacy of the irreversible ErbB family blocker afatinib in epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI)-pretreated non-small-cell lung cancer patients with brain metastases or leptomeningeal disease. J Thorac Oncol, 2015. 10(1): p. 156-63.
75. Zeng, Q., et al., Discovery and Evaluation of Clinical Candidate AZD3759, a Potent, Oral Active, Central Nervous System-Penetrant, Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor. J Med Chem, 2015. 58(20): p. 8200-15.
76. Zhang, C., et al., Role of astrocyte activation in fine particulate matter-enhancement of existing ischemic stroke in Sprague-Dawley male rats. J Toxicol Environ Health A, 2016. 79(9-10): p. 393-401.
77. Chen, C.H., et al., The role of the PI3K/Akt/mTOR pathway in glial scar formation following spinal cord injury. Exp Neurol, 2016. 278: p. 27-41.
78. Huang, W., et al., Paracrine Factors Secreted by MSCs Promote Astrocyte Survival Associated With GFAP Downregulation After Ischemic Stroke via p38 MAPK and JNK. J Cell Physiol, 2015. 230(10): p. 2461-75.
79. Beck, H., et al., Egr-1 regulates expression of the glial scar component phosphacan in astrocytes after experimental stroke. Am J Pathol, 2008. 173(1): p. 77-92.
80. Allaman, I., M. Belanger, and P.J. Magistretti, Astrocyte-neuron metabolic relationships: for better and for worse. Trends Neurosci, 2011. 34(2): p. 76-87.
81. Wang, R., et al., Oxygen-glucose deprivation induced glial scar-like change in astrocytes. PLoS One, 2012. 7(5): p. e37574.
82. Roy Choudhury, G., et al., Involvement of p38 MAPK in reactive astrogliosis induced by ischemic stroke. Brain Res, 2014. 1551: p. 45-58.
83. Sofroniew, M.V., Molecular dissection of reactive astrogliosis and glial scar formation. Trends Neurosci, 2009. 32(12): p. 638-47.
84. Prim, N., M. Fore, and B. Mennecier, [Afatinib (BIBW 2992)]. Rev Pneumol Clin, 2014. 70(5): p. 279-85.
85. Mukai, H., et al., Phase I trial of afatinib plus vinorelbine in Japanese patients with advanced solid tumors, including breast cancer. Cancer Chemother Pharmacol, 2015. 76(4): p. 739-50.
86. Engle, J.A. and J.M. Kolesar, Afatinib: A first-line treatment for selected patients with metastatic non-small-cell lung cancer. Am J Health Syst Pharm, 2014. 71(22): p. 1933-8.
87. Mander, P., et al., Nitric oxide from inflammatory-activated glia synergizes with hypoxia to induce neuronal death. J Neurosci Res, 2005. 79(1-2): p. 208-15.
88. Rao, V.L., K.K. Bowen, and R.J. Dempsey, Transient focal cerebral ischemia down-regulates glutamate transporters GLT-1 and EAAC1 expression in rat brain. Neurochem Res, 2001. 26(5): p. 497-502.
89. Ketheeswaranathan, P., et al., Changes in glutamate transporter expression in mouse forebrain areas following focal ischemia. Brain Res, 2011. 1418: p. 93-103.
90. Kang, T.C., et al., Chronological changes of N-methyl-D-aspartate receptors and excitatory amino acid carrier 1 immunoreactivities in CA1 area and subiculum after transient forebrain ischemia. J Neurocytol, 2001. 30(12): p. 945-55.
91. Gottlieb, M., M. Domercq, and C. Matute, Altered expression of the glutamate transporter EAAC1 in neurons and immature oligodendrocytes after transient forebrain ischemia. J Cereb Blood Flow Metab, 2000. 20(4): p. 678-87.
92. Arranz, A.M., et al., Increased expression of glutamate transporters in subcortical white matter after transient focal cerebral ischemia. Neurobiol Dis, 2010. 37(1): p. 156-65.
93. Rao, V.L., et al., Antisense knockdown of the glial glutamate transporter GLT-1, but not the neuronal glutamate transporter EAAC1, exacerbates transient focal cerebral ischemia-induced neuronal damage in rat brain. J Neurosci, 2001. 21(6): p. 1876-83.
94. Hughes, E.G., et al., Loss of glial fibrillary acidic protein results in decreased glutamate transport and inhibition of PKA-induced EAAT2 cell surface trafficking. Brain Res Mol Brain Res, 2004. 124(2): p. 114-23.
95. Eugenin, J., et al., Expression Pattern of Scavenger Receptors and Amyloid-beta Phagocytosis of Astrocytes and Microglia in Culture are Modified by Acidosis: Implications for Alzheimer's Disease. J Alzheimers Dis, 2016.
96. Tasdemir-Yilmaz, O.E. and M.R. Freeman, Astrocytes engage unique molecular programs to engulf pruned neuronal debris from distinct subsets of neurons. Genes Dev, 2014. 28(1): p. 20-33.
97. Tansey, F.A. and W. Cammer, Differential uptake of dextran beads by astrocytes, macrophages and oligodendrocytes in mixed glial-cell cultures from brains of neonatal rats. Neurosci Lett, 1998. 248(3): p. 159-62.