Spinal cord injury (SCI) patients display varying quantities of spinal cord tissue damage with injuries that present as complete, incomplete or compressive. One theory proposed to repair the injured spinal cord and regain motor control is to regenerate axons through the lesion site. This study was designed to quantify the impact of a local injectable in situ forming hydrogel reservoir therapy following rat hemisection SCI. We investigated the effect of hydrogel only treatment following SCI in addition to hydrogels loaded with a neurotrophic factor, Neurotrophin-3 (NT-3), immediately following SCI. Functional recovery, assessed by Basso Beattie Bresnahan (BBB) locomotor test, and local healing mechanisms, including neuronal growth, glial scar formation, inflammation and collagen deposition were investigated one and 6 weeks postsurgery. Delivery of an injectable hydrogel significantly increased functional recovery at four and 6 weeks post injury. In addition, a significant reduction in the inhibitory glial scar and in inflammation was observed at the injury site. Similarly hydrogel + NT-3 delivered directly into the injury site significantly reduced glial scarring and collagen deposition. The hydrogel + NT-3 also resulted in a significant increase in neurons at 6 weeks post injury. This study represents a novel and effective therapy combining growth factor and a biomaterial based therapy following SCI.

Over the past three decades, tremendous progress has been made in elucidating mechanisms underlying regenerative failure after spinal cord injury and in devising therapeutic approaches to promote functional nerve regeneration. Various in vitro assays have been developed using brain and/or spinal cord neuronal cells to study axon growth in conditions that represent the post-injury environment. This review outlines the current models used to dissect, analyze and manipulate specific aspects of spinal cord injury leading to axon growth inhibition.

Background: Following spinal cord injury, a highly inhibitory environment for axonal regeneration develops. One of the main sources of this inhibition is the glial scar that is formed after injury by reactive astrocytes. The inhibitory environment is mainly a result of chondroitin sulphate proteoglycans (CSPGs). Neuroglycan 2 (NG2), one of the main inhibitory CSPGs, is up‐regulated following spinal cord injury.Methods: Small interfering RNA (siRNA) was designed to target NG2 and this short hairpin RNA (shRNA) was cloned into a lentiviral vector (LV). The neurotrophic factor neurotrophin‐3 (NT‐3) promotes the growth and survival of developing neurites and has also been shown to aid regeneration. NT‐3 was also cloned into a LV. In vitro assessment of these vectors using a coculture system of dorsal root ganglia (DRG) neurones and Neu7 astrocytes was carried out. The Neu7 cell line is a rat astrocyte cell line that overexpresses NG2, thereby mimicking the inhibitory environment following spinal cord injury.Results and Discussion: These experiments show that both the knockdown of NG2 via shRNA and over‐expression of NT‐3 can significantly increase neurite growth, although a combination of both vectors did not confer any additional benefit over the vectors used individually. These LVs show promising potential for growth and survival of neurites in injured central nervous system tissue (CNS). Copyright © 2010 John Wiley & Sons, Ltd.

In this study we investigated the effect of neurotrophin-3 (NT-3) and knockdown of NG2, one of the main inhibitory chondroitin sulfate proteoglycans (CSPG), in the glial scar following spinal cord injury (SCI). Short hairpin (sh) RNA were designed to target NG2 and were cloned into a lentiviral vector (LV). A LV was also constructed containing NT-3. LV expressing NT-3, shRNA to NG2 or combinations of both vectors were injected directly into contused rat spinal cords 1 week post-injury. Six weeks post-injection of LV, spinal cords were examined by histology for changes in scar size and by immunohistochemistry for changes in expression of CSPG, NT-3, astrocytes, neurons and microglia/macrophages. Motor function was assessed using the Basso, Beattie and Bresnahan (BBB) locomotor scale. Animals that received the combination treatment of LV shNG2 and LV NT-3 showed reduced scar size. These animals also showed an increase in levels of neurons and NG2, a decrease in levels of astrocytes and a significant functional recovery as assessed using the BBB locomotor scale at 2 weeks post-treatment. The improvement in locomotor recovery and decrease in scar size shows the potential of this gene therapy approach as a therapeutic treatment for SCI.

Background: The development of tissue engineering scaffolds for gene delivery has the potential to enhance gene transfer efficiency and safety via controlled temporal and spatial delivery. Lentiviral delivery can be carried out using the natural biopolymer thermoresponsive gel, chitosan/β‐glycerol phosphate (β‐GP) as a carrier.Methods: Three chitosan/β‐GP scaffolds were prepared with varying concentrations of chitosan and β‐GP to obtain a pH and gelation temperature suitable for in situ delivery. A lentiviral vector expressing either green fluorescent protein (Lenti GFP) or neurotrophin‐3 (Lenti NT‐3) was incorporated into the chitosan/β‐GP scaffolds and also into collagen 0.1% w/v (control). Viral elution medium was removed at various timepoints and added to the culture medium of pre‐seeded HeLa or primary dorsal root ganglia (DRG) cells, respectively. GFP gene expression was quantified using fluorescence‐activated cell sorting analysis. The effect of Lenti NT‐3 was analyzed by measuring DRG neurite outgrowth.Results: Collagen displayed its most significant elution of virus on day 1 and chitosan/β‐GP (with a final concentration of 2.17% chitosan) on day 3.Conclusions: The system shows promise for the in situ, thermoresponsive delivery of lentiviral vectors providing long‐term gene expression for therapeutic factors to treat conditions such as injury to the nervous system. Copyright © 2011 John Wiley & Sons, Ltd.