An improved glial scar model after central nervous system injury in vitro

doi:10.16098/j.issn.0529-1356.2016.03.003

Abstract

Abstract:

Objective To establish a scar model in vitro and observe the morphology and extracellular matrix of the scar. Methods Astrocytes and meningeal fibroblasts were cultured from the cerebral cortex in vitroand identified by glial fibrillary acidic protein (GFAP) and fibronectin(FN) immunofluorescence cell staining respectively. Astrocytes and meningeal fibroblasts were co-cultured and treated by TGF-β1 after 2 days. The group without TGF-β1 treating was used as the control. The morphology of scar was observed by GFAP and FN immunofluorescence cell staining. Immunofluorescence cell staining and Western blotting were used to examine the expression of EphB2, ephrinB2, neurocan and NG2 in the scar. Results The scar-like cell cluster was formed from astrocytes and fibroblasts. In the TGF-β1 group, the expressions of EphB2 and ephrinB2, neurocan and NG2 were significantly increased when compared with the control group (P<0.05). Conclusion A scar model after central nervous system injury has bean established by co-culturing astrocytes and fibroblasts treate by TGF-β1 in vitro.

LI Yi TAN Ling LIN Wei-wei CHEN Xue CHEN Ying PAN Jing-ying WANG Xiao-dong*. An improved glial scar model after central nervous system injury in vitro[J]. AAS, 2016, (3): 303-308.

References

［1］Fawcett JW, Asher RA. The glial scar and central nervous system repair ［J］. Brain Res Bull, 1999, 49(6): 377-391.
［2］Asher RA, Morgenstern DA, Moon LD, et al. Chondroitin sulphate proteoglycans: inhibitory components of the glial scar［J］. Prog Brain Res,2001, 132:611-619.
［3］Moon LD, Asher RA, Rhodes KE, et al. Regeneration of CNS axons back to their target following treatment of adult rat brain with chondroitinase ABC［J］. Nat Neurosci,2001, 4(5):465-466.
［4］Sicotte M, Tsatas O, Jeong SY, et al. Immunization with myelin or recombinant Nogo-66/MAG in alum promotes axon regeneration and sprouting after corticospinal tract lesions in the spinal cord ［J］. Mol Cell Neurosci, 2003, 23(2): 251-263.
［5］Fitch MT, Silver J. CNS injury, glial scars, and inflammation: Inhibitory extracellular matrices and regeneration failure ［J］. Exp Neurol, 2008, 209(2): 294-301.
［6］Kawano H, Kimura-Kuroda J, Komuta Y, et al. Role of the lesion scar in the response to damage and repair of the central nervous system ［J］. Cell Tissue Res, 2012, 349(1): 169-180.
［7］Bundesen LQ, Scheel TA, Bregman BS, et al. Ephrin-B2 and EphB2 regulation of astrocyte-meningeal fibroblast interactions in response to spinal cord lesions in adult rats ［J］. J Neurosci, 2003, 23(21): 7789-7800.
［8］Kimura-Kuroda J, Teng X, Komuta Y, et al. An in vitro model of the inhibition of axon growth in the lesion scar formed after central nervous system injury［J］. Mol Cell Neurosci, 2010,43(2): 177-187.
［9］Rolls A,Shechter R, Schwartz M. The bright side of the glial scar in CNS repair［J］. Nat Rev Neurosci,2009, 10(3):235-241.
［10］Sofroniew MV. Molecular dissection of reactive astrogliosis and glial scar formation［J］. Trends Neurosci,2009, 32(12):638-647.
［11］Yiu G, He Z. Glial inhibition of CNS axon regeneration［J］. Nat Rev Neurosci. 2006, 7(8):617-627.
［12］Faulkner JR, Herrmann JE, Woo MJ, et al. Reactive astrocytes protect tissue and preserve function after spinal cord injury ［J］. J Neurosci, 2004, 24(9): 2143-2155.
［13］Yoshioka N, Kimura-Kuroda J, Saito T,et al. Small molecule inhibitor of type I transforming growth factor-β receptor kinase ameliorates the inhibitory milieu in injured brain and promotes regeneration of nigrostriatal dopaminergic axons［J］.J Neurosci Res, 2011, 89(3):381-393.
［14］Lagord C, Berry M,Logan A. Expression of TGFbeta2 but not TGFbeta1 correlates with the deposition of scar tissue in the lesioned spinal cord ［J］. Mol Cell Neurosci, 2002, 20(1): 69-92.
［15］Goldshmit Y, McLenachan S, Turnley A. Roles of Eph receptors and ephrins in the normal and damaged adult CNS［J］. Brain Res Rev, 2006, 52(2):327-345.
［16］Rhodes KE, Fawcett JW. Chondroitin sulphate proteoglycans: preventing plasticity or protecting the CNS ［J］? J Anat,2004, 204(1):33-48.
［17］Shen LH, Li Y, Gao Q, et al. Down-regulation of neurocan expression in reactive astrocytes promotes axonal regeneration and facilitates the neurorestorative effects of bone marrow stromal cells in the ischemic rat brain ［J］. Glia, 2008, 56(16): 1747-1754.
［18］Naima J, Sari SH. Transforming growth factor β-induced expression of chondroitin sulfate proteoglycans is mediated through non-Smad signaling pathways ［J］. Exp Neurol, 2015,263:372-384.
［19］Davies JE, Tang X, Denning JW, et al. Decorin suppresses neurocan, brevican, phosphacan and NG2 expression and promotes axon growth across adult rat spinal cord injuries［J］. Eur J Neurosci, 2004, 19(5):1226-1242.
［20］Alonso G. NG2 proteoglycan-expressing cells of the adult rat brain: possible involvement in the formation of glial scar astrocytes following stab wound［J］. Glia, 2005,49(3):318-338.
［21］Buss A, Pech K, Kakulas BA, et al. NG2 and phosphacan are present in the astroglial scar after human traumatic spinal cord injury ［J］. BMC Neurol, 2009, 9: 32.
［22］Smith GM, Rutishauser U, Silver J, et al. Maturation of astrocytes in vitro alters the extent and molecular basis of neurite outgrowth ［J］. Dev Biol, 1990, 138(2): 377-390.