East, E.

Golding, J. P.

Phillips, J. B.

English

Traumatic injuries to the spinal cord and dorsal root entry zone (DREZ) are debilitating and often lead to paralysis and loss of sensation for the patient. At the cellular and molecular level, the repair site microenvironment is inhibitory for axon growth due to formation of a glial scar. A common finding of current strategies aimed at bridging CNS lesions, in particular recent tissue engineering approaches using fibronectin (Phillips et al., Biomaterials 2004), is that whilst axons readily enter and traverse the bridging graft, they are less likely to exit the graft and reconnect with their targets. The aim of this work was to develop a cell culture model to investigate reactive gliosis following damage to the spinal cord.

Astrocytes in the CNS under physiological conditions express low levels of GFAP, but following trauma exhibit a reactive phenotype characterised by GFAP up-regulation. Primary glial cell cultures were analysed in 2D monolayers and 3D collagen gels for GFAP expression. In 2D cultures approximately twice the number of cells were positive for GFAP compared to those cultured in 3D collagen gels. As 3D astrocyte cultures more closely modelled the in vivo situation, we used this model to investigate the response of astrocytes to cells found at the inhibitory interface following damage. The preliminary model involved placing dorsal root ganglia (DRG) explants into the centre of astrocyte gels. Classification of astrocyte morphology revealed significantly more ramified cells in DRG-adjacent regions (6 fold higher), than in control areas away from the DRG. A more advanced model was then developed in which dissociated DRGs were labelled with CellTrackerTM, seeded onto astrocyte-populated collagen gels and maintained in culture for 5 days. Astrocytes near the DRG interface showed marked GFAP up-regulation and adopted a reactive morphology which was observed up to 1mm away. Intensity of GFAP fluorescence at this interface was 3 fold higher than that seen away from the interface or in controls (astrocyte only gels).

Astrocytes in 3D culture exhibit a lower basal level of reactivity than cells grown in monolayer. This model provides a useful tool for investigating triggers of reactive gliosis, as demonstrated by the response observed to cells found at the inhibitory interfaces formed following damage to the spinal cord and could be used as a way to improve existing therapies and develop new strategies aimed at CNS repair

Golding, J.P.

Phillips, J.B.

Open Research Online

Modelling of the injured spinal cord using 3-dimensional cell cultures; strategies for improving tissue engineered repair

Open Research OnlineThe Open University’s repository of research publicationsand other research outputsModelling the injured spinal cord using 3-dimensionalcell cultures; strategies for improving tissue engineeredrepairConference or Workshop ItemHow to cite:East, E.; Golding, J. P. and Phillips, J. B. (2008). Modelling the injured spinal cord using 3-dimensional cellcultures; strategies for improving tissue engineered repair. In: European Cells and Materials, 16(Supple) p. 51.For guidance on citations see FAQs.c© 2008 AO FoundationVersion: Version of RecordLink(s) to article on publisher’s website:http://www.ecmjournal.org/journal/supplements/vol016supp03/pdf/v016supp03a051.pdfCopyright and Moral Rights for the articles on this site are retained by the individual authors and/or other copyrightowners. For more information on Open Research Online’s data policy on reuse of materials please consult the policiespage.oro.open.ac.ukEuropean Cells and Materials Vol. 16. Suppl. 3, 2008 (page 51)                                            ISSN 1473-2262  Modelling the injured spinal cord using 3-dimensional cell cultures; strategies for improving tissue engineered repair E East, J P Golding & J B Phillips Department of Life Science, The Open University, Walton Hall, Milton Keynes, UK.  INTRODUCTION: Traumatic injuries to the spinal cord are debilitating and often lead to paralysis and loss of sensation.  CNS injury results in the formation of a glial scar that is largely composed of reactive, hypertrophic astrocytes which upregulate various markers including glial fibrillary acidic protein (GFAP) and sulphated protoglycans.  The glial scar microenvironment is inhibitory for axon growth and regeneration A common finding of strategies aimed at bridging CNS lesions [1], in particular recent tissue engineered approaches using fibronectin [2], is that whilst axons readily enter and traverse the bridging graft they generally fail to exit the graft and re-enter the parenchyma due to the inhibitory glial scar at the graft-CNS interface.  The aim of this work was to create a 3D cell culture model of CNS reactive gliosis, in which improved tissue-engineered repair strategies can be developed.  METHODS:  Primary rat astrocytes were seeded onto collagen coated coverslips (2D) or into 3D collagen gels and the expression of markers associated with glial scar formation were analysed by immunocytochemistry (GFAP, chondroitin sulphate proteoglycan (CSPG), vimentin, aquaporin 4 (AqP4) and S100β).  To stimulate reactive gliosis, 3D astrocyte cultures were treated with TGFβ1 (10ng/ml) every other day for 15 days [3].  Cultures without TGF β1 were used as controls.  Expression of glial scar markers was assessed and measured using Volocity image analysis software (Improvision).  Cell perimeter was measured as an indicator of astrocyte activation. Area of immunoreactivity was measured for CSPGs released from astrocytes.  RESULTS:  Astrocytes in 3D cultures had significantly less immunoreactivity for GFAP, CSPG, vimentin, AqP4 and S100β, than astrocytes in 2D cultures indicating a lower level of activation in 3D (fig 1).  However immunoreactivity for β-actin did not differ in 2D or 3D.  Following treatment with TGFβ1, astrocytes in 3D became hypertrophic and reactive, with changes in the expression of gliosis markers over time relative to control cultures (fig 2).               Fig. 1: Comparison of astrocyte activation markers in 2D and 3D cultures in type I collagen.  Bars represent mean ± SD, ***P<0.001 2D vs 3D.              Fig 2.  Increased astrocyte activation following TGFβ1 treatment of 3D cultures.  DISCUSSION & CONCLUSIONS:  Astrocytes in 3D are less reactive than their 2D counterparts, thus resembling the physiological in vivo situation and providing a useful system in which activation can be investigated.  TGFβ1 treated astrocytes over 15 days take on characteristics of the mature glial scar, including their morphology and protein expression profile.  This model will provide a useful test-bed for tissue engineered strategies to improve axonal growth through the glial scar environment.  REFERENCES: 1 Geller HM & Fawcett JW (2002) Exp Neurol 172:125-36. 2 Phillips JB et al, (2004) Biomaterials 25:2769-79. 3 DK Cullen, CM Simon & MC LaPlaca (2007) Brain Res 1158:103-115. ACKNOWLEDGEMENTS: This research was funded by the Wellcome Trust.   GFAPGFAPCSPGCSPGVimentinVimentinAqP4 AqP4 βS100 βS100-Actinβ -Actinβ02550751003D collagen2D collagen************ ***% positive cells/field0 5 10 15123456789AqP4VimentinGFAPCSPG   (area mm2)(perimeter mm)Time (days of TGFβ1 treatment)Ratio TGF β1/control(cell perimeter (mm) or area of staining (mm2 ))2D CSPG 3D CSPG Control 15d - GFAP TGFβ1 15d - GFAP  

Modelling the injured spinal cord using 3-dimensional cell cultures; strategies for improving tissue engineered repair

http://oro.open.ac.uk/11407/1/EmmaTCES2008abstract.pdf

Modelling the injured spinal cord using 3-dimensional cell cultures; strategies for improving tissue engineered repair

Abstract

Similar works

Full text

Available Versions

Open Research Online

Open Research Online