Journal of Neuroscience Methods 192 (2010) 49–57 Contents lists available at ScienceDirect Journal of Neuroscience Methods journal homepage: www.elsevier.com/locate/jneumeth A model for ex vivo spinal cord segment culture—A tool for analysis of injury repair strategies Jie Zhang a,∗ , Simon J. O’Carroll b , Ann Wu c , Louise F.B. Nicholson b , Colin R. Green a a Department of Ophthalmology, Faculty of Medical and Health Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand b Department of Anatomy with Radiology and Centre for Brain Research, Faculty of Medical and Health Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand c Neural Injury Research Unit, School of Medical Sciences, University of New South Wales, Sydney, NSW 2052, Australia article info Article history: Received 24 March 2010 Received in revised form 18 June 2010 Accepted 11 July 2010 Keywords: Spinal cord injury Gap junction Connexin43 Glial scar Astrocytes Culture model Regeneration Inflammation abstract Most spinal cord injury research is undertaken using in vivo animal models but the extensive care asso- ciated with spinalised animals, inherent variability between animals, and complex surgeries makes alternative models especially valuable. Here we present a novel ex vivo model that enables culture of intact post-natal spinal cord segments for up to five days and the assessment of peripheral nerve grafting repair, enhanced with connexin43 antisense oligodeoxynucleotides (Cx43 AsODN), in this model. Down- regulating Cx43 expression with Cx43 AsODN in cultured spinal cord segments prevents cell death and inhibits inflammation spreading from the site of injury to neighbouring tissue, hence maintaining culture viability. Reduction in segment swelling and improvement in neuron survival were evident after Cx43 AsODN treatment. Furthermore, the combination of Cx43 AsODN with peripheral nerve graft implants into cultured spinal cords promoted axon sprouting from the spinal cord into the peripheral nerve graft. This ex vivo spinal cord segment culture model provides a valuable addition to tools currently available for spinal cord injury research. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Spinal cord injury (SCI) is a devastating injury with an inci- dence of 40 new cases per million people in the USA (National Spinal Cord Injury Statistical Center, 2008). It is most prevalent in young healthy males, resulting in lifelong paraplegia or tetraple- gia (National Spinal Cord Injury Statistical Center, 2008). A large amount of research on SCI repair treatments has been carried out since the discovery (Richardson et al., 1980) that spinal cord neurons can regenerate when placed into a peripheral nerve envi- ronment. Most of this research is undertaken using in vivo animal models, in which spinal cords are injured by transection, contu- sion or compression. Treatment effects can be studied in a natural physiological environment for as long as required and behavioral improvements measured, but problems exist that call for an alter- native model of SCI repair. Spinal cord injured animals require extensive post injury care including weight management, health management and bladder expression, and there is also inherent variability between animals that can make research data difficult to interpret. In addition, surgeries are relatively complex and exper- iments time consuming. ∗ Corresponding author. Tel.: +64 9 3737599x86386; fax: +64 9 3767173. E-mail address: jie.zhang@auckland.ac.nz (J. Zhang). An ex vivo model is ethically advantageous, does not require post surgical animal care, enables more reproducibility between lesions, and provides a tightly controlled artificial environment that can be reliably compared between studies. Published ex vivo spinal cord models include the culture of several hundred micron thick trans- verse slices maintained for up to three weeks (Krassioukov et al., 2002; Guzmán-Lenis et al., 2008; Cho et al., 2009), unfixed longi- tudinal 16 micron cryostat sections of spinal cord maintained for one week (Pettigrew et al., 2001), and spinal cord segments main- tained for up to 4 h (Saruhashi et al., 2002; Hamann et al., 2008). Here we describe a model that combines all of the advantages of the above-mentioned protocols, with natural tissue architec- ture retained in an intact post-natal spinal cord segment that is 0.5–1.5 cm long over a relatively long culture time that enables analysis of repair strategies. The culture method involving the use of a membrane insert in an air-liquid interface culture was origi- nally developed by Stoppini et al. for culture of hippocampal slices, popularised by its simplicity and maintenance of tissue architec- ture (Stoppini et al., 1991). This novel ex vivo organotypic spinal cord segment culture model can be readily maintained for up to five days through the application of connexin43 specific antisense oligodeoxynucleotides (Cx43 AsODN) that reduce lesion spread and inflammation, and promote neuronal survival. We have already demonstrated an application of this model for testing the efficacy of Cx43 mimetic peptides in reducing damage spread after spinal cord segment culture (O’Carroll et al., 2008). 0165-0270/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jneumeth.2010.07.012