Embryonic stem cells overexpressing high molecular weight FGF2 isoform enhance recovery of pre-ganglionic spinal root lesion in combination with fibrin biopolymer mediated root repair.

BACKGROUND: Spinal ventral root avulsion results in massive motoneuron degeneration with poor prognosis and high costs. In this study, we compared different isoforms of basic fibroblast growth factor 2 (FGF2), overexpressed in stably transfected Human embryonic stem cells (hESCs), following motor root avulsion and repair with a heterologous fibrin biopolymer (HFB). METHODS: In the present work, hESCs bioengineered to overexpress 18, 23, and 31 kD isoforms of FGF2, were used in combination with reimplantation of the avulsed roots using HFB. Statistical analysis was conducted using GraphPad Prism software with one-way or two-way ANOVA, followed by Tukey's or Dunnett's multiple comparison tests. Significance was set at *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. RESULTS: For the first set of experiments, rats underwent avulsion of the ventral roots with local administration of HFB and engraftment of hESCs expressing the above-mentioned FGF2 isoforms. Analysis of motoneuron survival, glial reaction, and synaptic coverage, two weeks after the lesion, indicated that therapy with hESCs overexpressing 31 kD FGF2 was the most effective. Consequently, the second set of experiments was performed with that isoform, so that ventral root avulsion was followed by direct spinal cord reimplantation. Motoneuron survival, glial reaction, synaptic coverage, and gene expression were analyzed 2 weeks post-lesion; while the functional recovery was evaluated by the walking track test and von Frey test for 12 weeks. We showed that engraftment of hESCs led to significant neuroprotection, coupled with immunomodulation, attenuation of astrogliosis, and preservation of inputs to the rescued motoneurons. Behaviorally, the 31 kD FGF2 - hESC therapy enhanced both motor and sensory recovery. CONCLUSION: Transgenic hESCs were an effective delivery platform for neurotrophic factors, rescuing axotomized motoneurons and modulating glial response after proximal spinal cord root injury, while the 31 kD isoform of FGF2 showed superior regenerative properties over other isoforms in addition to the significant functional recovery.

Interferon (IFN) beta treatment induces major histocompatibility complex (MHC) class I expression in the spinal cord and enhances axonal growth and motor function recovery following sciatic nerve crush in mice.

AIMS: Major histocompatibility complex (MHC) class I expression by neurones and glia constitutes an important pathway that regulates synaptic plasticity. The upregulation of MHC class I after treatment with interferon beta (IFN beta) accelerates the response to injury. Therefore the present work studied the regenerative outcome after peripheral nerve lesion and treatment with IFN beta, aiming at increasing MHC class I upregulation in the spinal cord. METHODS: C57BL/6J mice were subjected to unilateral sciatic nerve crush and treatment with IFN beta. The lumbar spinal cords were processed for immunohistochemistry, in situ hybridization, Western blotting and RT-PCR, while the sciatic nerves were submitted for immunohistochemistry, morphometry and counting of regenerated axons. Motor function recovery was monitored using the walking track test. RESULTS: Increased MHC class I expression in the motor nucleus of IFN beta-treated animals was detected. In the peripheral nerve, IFN beta-treated animals showed increased S100, GAP-43 and p75NTR labelling coupled with a significantly greater number of regenerated axons. No significant differences were found in neurofilament or laminin labelling. The morphological findings, indicating improvements in the regenerative process after IFN treatment were in line with the motor behaviour test applied to the animals during the recovery process. CONCLUSIONS: The present data reinforce the role of MHC class I upregulation in the response to injury, and suggest that IFN treatment may be beneficial to motor recovery after axotomy.