EFFECT OF INTEGRIN β<sub>8</sub> ON NEURONAL APOPTOSIS AFTER HYPOXIA ISCHEMIA IN ASTROCYTE/NEURON CO-CULTURE SYSTEM_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

LIJinhui , CHENDapeng , TANGJun , WUJinlin ,  QUYi , MUDezhi

Department of Pediatrics, West China Second Hospital, Key Laboratory of Obstetric & Gynecologic and Pediatric Diseases and Birth Defects of Ministry of Education, Sichuan University, Chengdu Sichuan, 610041, P. R. China;

Corresponding author：

QUYi, Email: quyi712002@163.com

Keywords：

Hypoxic-ischemic encephalopathy; Integrin β₈; Astrocyte; Neuron; Rat

DOI：

10.7507/1002-1892.20140082

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Objective To observe the effect of integrin β₈ on the neuronal apoptosis after hypoxia ischemia (HI) in astrocyte/neuron co-culture system. Methods Astrocytes and neurons were cultured in vitro from cerebral cortex of the P1-3 days Sprague Dawley rats and E16 days fetal rats, respectively. Immunocytochemistry staining was used to identify the purity of cells. Integrin β₈ mRNA expression was qualified in the astrocytes at 12 hours, 1 day, and 2 days after HI and reoxygenation (experimental group) and in normal astrocytes (control group) by RT-PCR. Integrin β₈ small interering RNA (siRNA) system was established to specifically block astrocyte β₈ expression, the efficiency of integrin β₈ inhibition was detected by real-time fluorescent PCR. The astrocytes and neurons were co-cultured to established the astrocyte/neuron co-culture system. The neuronal apoptosis was detected with TUNEL in the normal neurons/astrocytes group (co-cultured HI group), the astrocytes infected by integrin β₈ siRNA for 2 days/normal neurons group (β₈ RNA interference group), and normal neurons in vitro with HI treatment group (HI group) at 1 day after HI and reoxygenation. The normal neurons without treatment as control (control group). Results Glial fibrillary acidic protein and neuronal nuclei staining suggested a purity of more than 90% in cultured cells. HI resulted in an increase of integrin β₈ mRNA expression at 12 hours after reoxygenation in astrocytes, which peaked at 1 day after reoxygenation, then slowly decreased and remained higher at 2 days, showing significant differences between control group and experimental group and among different time points in experimental group (P<0.05). RNA interference efficiency was most significant at 2 days after astrocytes infected with integrin β₈ siRNA (P<0.05). The neuronal apoptosis was significantly increased in HI group, co-cultured HI group, and β₈ RNA interference group when compared with control group (P<0.05). But neuronal apoptosis index (AI) was significantly decreased in co-cultured HI group and β₈ RNA interference group when compared with HI group (P<0.05). The significant difference of AI was found between co-cultured HI group and β₈ RNA interference group (P<0.05). Conclusion Integrin β₈ expression can be induced with hypoxic-ischemic brain damage, leading to decreased AI of neurons and obvious protective effect.

Citation： LIJinhui, CHENDapeng, TANGJun, WUJinlin, QUYi, MUDezhi. EFFECT OF INTEGRIN β₈ ON NEURONAL APOPTOSIS AFTER HYPOXIA ISCHEMIA IN ASTROCYTE/NEURON CO-CULTURE SYSTEM. Chinese Journal of Reparative and Reconstructive Surgery, 2014, 28(3): 366-370. doi: 10.7507/1002-1892.20140082 Copy

1.	Gong SJ, Chen LY, Zhang M, et al. Intermittent hypobaric hypoxia preconditioning induced brain ischemic tolerance by up-regulating glial glutamate transporter-1 in rats. Neurochem Res, 2012, 37(3):527-537.
2.	Kitagawa K. Ischemic tolerance in the brain:endogenous adaptive machinery against ischemic stress. J Neurosci Res, 2012, 90(5):1043-1054.
3.	Ben Achour S, Pascual O. Astrocyte-neuron communication:functional consequences. Neurochem Res, 2012, 37(11):2464-2473.
4.	Mobley AK, McCarty JH. Use of Cre-lox technology to analyze integrin functions in astrocytes. Methods Mol Biol, 2012, 814:555-570.
5.	Baeten KM, Akassoglou K. Extracellular matrix and matrix receptors in blood-brain barrier formation and stroke. Dev Neurobiol, 2011, 71(11):1018-1039.
6.	Li J, Qu Y, Li X, et al. The role of integrin alpha(v)beta(8) in neonatal hypoxic-ischemic brain injury. Neurotox Res, 2010, 17(4):406-417.
7.	Moreno C, Sampieri A, Vivas O, et al. STIM1 and Orai1 mediate thrombin-induced Ca(2+) influx in rat cortical astrocytes. Cell Calcium, 2012, 52(6):457-467.
8.	Geissler M, Faissner A. A new indirect co-culture set up of mouse hippocampal neurons and cortical astrocytes on microelectrode arrays. J Neurosci Methods, 2012, 204(2):262-272.
9.	Fu X, Li Q, Feng Z, et al. The role of aquaporin-4 in brain edema following neonatal hypoxia ischemia and reoxygenation in a cultured rat astrocyte model. Glia, 2007, 55(9):935-941.
10.	Gardiner NJ. Integrins and the extracellular matrix:key mediators of development and regeneration of the sensory nervous system. Dev Neurobiol, 2011, 71(11):1054-1072.
11.	Bridgewater RE, Norman JC, Caswell PT. Integrin trafficking at a glance. J Cell Sci, 2012, 125(Pt16):3695-3701.
12.	Wolfenson H, Lavelin I, Geiger B. Dynamic regulation of the structure and function of integrin adhesion. Dev Cell, 2013, 24(5):447-458.
13.	Mobley AK, McCarty JH. β₈ integrin is essential for neuroblast migration in the rostral migratory stream. Glial, 2011, 59(11):1579-1187.
14.	Tchaicha JH, Reyes SB, Shin J, et al. Glioblastoma angiogenesis and tumor cell invasiveness are differentially regulated by β₈ integrin. Cancer Res, 2011, 71(20):6371-6381.
15.	Zhu J, Motejlek K, Wang D, et al. Beta8 integrins are required for vascular morphogenesis in mouse embryos. Development, 2002, 129(12):2891-2903.
16.	McCarty JH, Lacy-Hulbert A, Charest A, et al. Selective ablation of alphav integrins in the central nervous system leads to cerebral hemorrhage, seizures, axonal degeneration and premature death. Development, 2005, 132(1):165-176.
17.	Cambier S, Gline S, Mu DZ, et al. Integrin alpha(v)beta8-mediated activation of transforming growth factor-beta by perivascular astrocyte. Amer J Pathol, 2005, 166(6):1883-1894.
18.	Beck K, Schachtrup C. Vascular damage in the central nervous system:a multifaceted role for vascular-derived TGF-β. Cell Tissue Res, 2012, 347(1):187-201.
19.	Heinemann U, Kaufer D, Friedman A. Blood-brain barrier dysfunction, TGFβ signaling, and astrocyte dysfunction in epilepsy. Glia, 2012, 60(8):1251-1257.
20.	Rodríquez-Martínez G, Velasco I. Activin and TGF-β effects on brain development and neural stem cells. CNS Neurol Disord Drug Targets, 2012, 11(7):844-855.
21.	Zhu Y, Yang GY, Ahlemeyer B, et al. Transforming growth factor-beta1 increases bad phosphorylation and protects neurons against damage. J Neurosci, 2002, 22(10):3898-3909.
22.	Haas SL, Fitzner B, Jaster R, et al. Transforming growth factor-beta induces nerve growth factor expression in pancreatic stellate cells by activation of the ALK-5 pathway. Growth Fctors, 2009, 27(5):289-299.
23.	König HG, Kögel D, Rami A, et al. TGF-beta1 activates two distinct type Ⅰreceptors in neurons:implications for neuronal NF-κB signaling. J Cell Biol, 2005, 168(7):1077-1086.

1. Gong SJ, Chen LY, Zhang M, et al. Intermittent hypobaric hypoxia preconditioning induced brain ischemic tolerance by up-regulating glial glutamate transporter-1 in rats. Neurochem Res, 2012, 37(3):527-537.
2. Kitagawa K. Ischemic tolerance in the brain:endogenous adaptive machinery against ischemic stress. J Neurosci Res, 2012, 90(5):1043-1054.
3. Ben Achour S, Pascual O. Astrocyte-neuron communication:functional consequences. Neurochem Res, 2012, 37(11):2464-2473.
4. Mobley AK, McCarty JH. Use of Cre-lox technology to analyze integrin functions in astrocytes. Methods Mol Biol, 2012, 814:555-570.
5. Baeten KM, Akassoglou K. Extracellular matrix and matrix receptors in blood-brain barrier formation and stroke. Dev Neurobiol, 2011, 71(11):1018-1039.
6. Li J, Qu Y, Li X, et al. The role of integrin alpha(v)beta(8) in neonatal hypoxic-ischemic brain injury. Neurotox Res, 2010, 17(4):406-417.
7. Moreno C, Sampieri A, Vivas O, et al. STIM1 and Orai1 mediate thrombin-induced Ca(2+) influx in rat cortical astrocytes. Cell Calcium, 2012, 52(6):457-467.
8. Geissler M, Faissner A. A new indirect co-culture set up of mouse hippocampal neurons and cortical astrocytes on microelectrode arrays. J Neurosci Methods, 2012, 204(2):262-272.
9. Fu X, Li Q, Feng Z, et al. The role of aquaporin-4 in brain edema following neonatal hypoxia ischemia and reoxygenation in a cultured rat astrocyte model. Glia, 2007, 55(9):935-941.
10. Gardiner NJ. Integrins and the extracellular matrix:key mediators of development and regeneration of the sensory nervous system. Dev Neurobiol, 2011, 71(11):1054-1072.
11. Bridgewater RE, Norman JC, Caswell PT. Integrin trafficking at a glance. J Cell Sci, 2012, 125(Pt16):3695-3701.
12. Wolfenson H, Lavelin I, Geiger B. Dynamic regulation of the structure and function of integrin adhesion. Dev Cell, 2013, 24(5):447-458.
13. Mobley AK, McCarty JH. β₈ integrin is essential for neuroblast migration in the rostral migratory stream. Glial, 2011, 59(11):1579-1187.
14. Tchaicha JH, Reyes SB, Shin J, et al. Glioblastoma angiogenesis and tumor cell invasiveness are differentially regulated by β₈ integrin. Cancer Res, 2011, 71(20):6371-6381.
15. Zhu J, Motejlek K, Wang D, et al. Beta8 integrins are required for vascular morphogenesis in mouse embryos. Development, 2002, 129(12):2891-2903.
16. McCarty JH, Lacy-Hulbert A, Charest A, et al. Selective ablation of alphav integrins in the central nervous system leads to cerebral hemorrhage, seizures, axonal degeneration and premature death. Development, 2005, 132(1):165-176.
17. Cambier S, Gline S, Mu DZ, et al. Integrin alpha(v)beta8-mediated activation of transforming growth factor-beta by perivascular astrocyte. Amer J Pathol, 2005, 166(6):1883-1894.
18. Beck K, Schachtrup C. Vascular damage in the central nervous system:a multifaceted role for vascular-derived TGF-β. Cell Tissue Res, 2012, 347(1):187-201.
19. Heinemann U, Kaufer D, Friedman A. Blood-brain barrier dysfunction, TGFβ signaling, and astrocyte dysfunction in epilepsy. Glia, 2012, 60(8):1251-1257.
20. Rodríquez-Martínez G, Velasco I. Activin and TGF-β effects on brain development and neural stem cells. CNS Neurol Disord Drug Targets, 2012, 11(7):844-855.
21. Zhu Y, Yang GY, Ahlemeyer B, et al. Transforming growth factor-beta1 increases bad phosphorylation and protects neurons against damage. J Neurosci, 2002, 22(10):3898-3909.
22. Haas SL, Fitzner B, Jaster R, et al. Transforming growth factor-beta induces nerve growth factor expression in pancreatic stellate cells by activation of the ALK-5 pathway. Growth Fctors, 2009, 27(5):289-299.
23. König HG, Kögel D, Rami A, et al. TGF-beta1 activates two distinct type Ⅰreceptors in neurons:implications for neuronal NF-κB signaling. J Cell Biol, 2005, 168(7):1077-1086.

Chinese Journal of Reparative and Reconstructive Surgery

EFFECT OF INTEGRIN β₈ ON NEURONAL APOPTOSIS AFTER HYPOXIA ISCHEMIA IN ASTROCYTE/NEURON CO-CULTURE SYSTEM

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Chinese Journal of Reparative and Reconstructive Surgery

EFFECT OF INTEGRIN β8 ON NEURONAL APOPTOSIS AFTER HYPOXIA ISCHEMIA IN ASTROCYTE/NEURON CO-CULTURE SYSTEM

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EFFECT OF INTEGRIN β₈ ON NEURONAL APOPTOSIS AFTER HYPOXIA ISCHEMIA IN ASTROCYTE/NEURON CO-CULTURE SYSTEM