Modelling and overcoming the biological interfaces that prevent nerve regeneration

Supervisors – Dr. James Phillips and Dr. Jon Golding

Spinal cord injury

Traumatic injuries to the spinal cord and the dorsal root are severely debilitating and result in permanent paralysis and loss of sensation for patients. These injuries directly affect around 2 million people worldwide (ISRT 2002). In contrast to the PNS situation, neuronal regeneration in the central nervous system (CNS) is generally unsuccessful. Lack of repair in the injured CNS is widely attributed to the inhibitory environment of the glial scar surrounding the lesion site, largely composed of reactive astrocytes, which forms a physical and physiological barrier to axon regeneration.


Figure 1A. Astrocytes stained for GFAP in 2D culture (x 40)		Figure 1B. Astrocytes in 3D cultures upregulate GFAP following stimulation (x 40)

Current project

A common finding of strategies aimed at bridging CNS spinal cord lesions, in particular tissue engineered approaches, is that whilst axons readily enter and traverse the bridging graft, they are less likely to exit the graft and reconnect with their targets. This is largely due to the inhibitory interface that forms between cells within the graft and cells of the glial scar surrounding the graft. This interface is much like that seen at the dorsal root entry zone i.e. the CNS-PNS boundary. The aim of this work is to develop a powerful in vitro 3-dimensional cell culture model to study these biological interfaces and develop ways of overcoming them.

Recent work has focused on the comparison of astrocytes (Figure 1) and neurons (Figure 2) grown in conventional 2D monolayers and cells cultured in 3D collagen gels. Furthermore we have developed and established an in vitro 3D model of astrogliosis in which astrocyte gels are treated with TGF-beta1 for 15 days (Figure 3B) and compared to untreated control gels (3A) (East et al. 2009)


Figure 2. DRG neurites growing over satellite cells in 2D culture (x 20)	Figure 3A. Control 3D astrocyte gels after 15 days in culture, stained for GFAP (x 10).	Figure 3B. 3D astrocyte gels after 15 days of treatment with TGF-beta1, stained for GFAP (x 10).

More recently we have developed an astrocyte / satellite glia interface model, which recreates aspects of the graft-host interface that occurs following implantation of repair devices in the injured spinal cord. Our integrated 3D model allows detailed analysis of astrocyte reactivity in response to cells of the peripheral nervous system (Figure 4) and allows us to monitor the behaviour of axons at the interface (Figure 5). Using this model we are investigating strategies to improve axon growth through the interface. One such approach involves aligning astrocytes (Figure 6), creating a more permissive environment, enhancing and guiding neuronal growth (Figure 7).

Figure 4. Astrocytes become reactive at the interface with satellite glial cells in 3D culture. Figure 5. Monitoring neurons at the astrocyte interface. Figure 6. Aligned astrocytes in 3D culture stained for GFAP. Figure 7. Neurites (red) growing in an environment of aligned astrocytes (green).

This work has been disseminated at TCES 2008, 2009, Glial Cells in Health and Disease 2007, 2009, TERMIS 2007 and at the Society for Neuroscience 2007, 2008. Our recent work was presented at TERMIS in Galway 2010 and at TCES in Manchester 2010.

Poster presentation at SFN, 2007.

Funded by the Wellcome Trust

Publications

East E., Blum de Oliveira D., Golding J.P. and Phillips J.B. (2010). Alignment of astrocytes increases neuronal growth in 3D collagen gels and is maintained following plastic compression to form a spinal cord repair conduit. Tissue Eng., 16(10); 3173-3184.

East E., Georgiou M., Loughlin A.J., Golding J.P. and Phillips J.B. (2010). Plastic compression of aligned cellular collagen gels for nervous system repair. In press European cells and Materials.

East E., Golding J.P. and Phillips J.B. (2010). Development of an integrated collagen gel system for studying cellular interfaces following spinal cord injury. In press Tissue Engineering.

East E., Golding J.P. and Phillips J.B. (2009). A versatile 3D culture model facilitates monitoring of astrocytes undergoing reactive gliosis. J. Tissue. Eng. Regen. Med., 3; 634-646.

East E., Blum de Oliveira D., Golding J. and Phillips J. (2009). Astrocyte alignment increases neurite outgrowth in a 3D cell culture model. Glia, 57(S13), S159.

Loughlin J., East E., Golding J. and Phillips J. (2009). Developing a 3D culture model of CNS myelination. Glia, 57(S13), S119.

Piers T.M., East E. and Pocock J.M. (2009). Fibrin and fibrinogen cause neuron non-cell autonomous degeneration. Glia, 57(S13), S61.

East E. and Phillips J.B. (2008). Tissue engineered cell culture models for nervous system research. In “Tissue engineering research”. Editor Greco, published by Nova Science Publishers. ISBN 978-1-60456-264-4.

East E., Gveric D., Baker D., Pryce G., Lijnen H.R. and Cuzner M.L (2008). Chronic relapsing EAE in plasminogen activator inhibitor-1 knockout mice; the effect of fibrinolysis during neuroinflammation. Neuropathol. App. Neurobiol., 34(2); 216-230.

East E., Golding J.P. and Phillips J.B. (2007) Modelling of the injured spinal cord using 3-dimensional cell cultures; strategies for improving tissue engineered repair. 600.7/BB3 Society for Neuroscience. San Diego.

East E., Golding J.P. and Phillips J.B. (2007). Development of a 3-dimensional in vitro model to study reactive gliosis following nervous system injury. Tissue Engineering; 13(7), 1668.

East E., Golding J. and Phillips J. (2007). Increased GFAP immunoreactivity by astrocytes in response to contact with dorsal root ganglia cells in a 3D culture model. Neuron Glia Biology; 3(S1), S119.

Sevastou I.G., Pinteaux-Jones F., Hooper C., East E., Fry V.A.H. and Pocock J.M. (2007). Microglial Rock and Rho in Multiple Sclerosis. 804.10/U11 Society for Neuroscience. San Diego.

Pocock J.M., Hooper C., East E. and Jones F. (2006). Cell death pathways and the immune response. In “Glia and inflammation in neurodegenerative disease”. Editors Yenari and Giffard, published by Nova Science Publishers.

East E., Baker D., Pryce G., Lijnen H. R., Cuzner M. L. and Gveric D. (2005). A role for the plasminogen activator system in inflammation and neurodegeneration in the CNS during experimental allergic encephalomyelitis. Am. J. Pathol., 167, 545 – 554.

Pryce G., O’Neill J. K., Croxford J. L., Amor S., Hankey D. R. J., East E., Giovannoni G. and Baker D. (2005). Autoimmune tolerance eliminates relapses but fails to halt progression in a model of multiple sclerosis. J. Neuroimmunology, 165; 41 - 52.

East E., Baker D., Pryce G., Cuzner M. L. and Gveric D. (2004). The role of plasminogen activators in experimental allergic encephalomyelitis: inflammation and axonal pathology. J. Neuroimmunology, 154; 167.

East E., Baker D., Pryce G. M., Cuzner M. L. and Gveric D. (2003). The role of plasminogen activators in experimental allergic encephalomyelitis. Glia, Suppl 2; 40

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Figure 4. Astrocytes become reactive at the interface with satellite glial cells in 3D culture.	Figure 5. Monitoring neurons at the astrocyte interface.	Figure 6. Aligned astrocytes in 3D culture stained for GFAP.	Figure 7. Neurites (red) growing in an environment of aligned astrocytes (green).