Use of human embryonic stem cells with overexpression of FGF2 after ventral root avulsion: neuroprotection and immunomodulation

Author(s)

Marta Rocha Araujo

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2016-10-14

Examining board members

Alexandre Leite Rodrigues de Oliveira; Rosalia Mendez Otero; Angela Cristina Malheiros Luzo; Daniel Martins de Souza; Betania Souza Monteiro

• Advisor: Alexandre Leite Rodrigues de Oliveira

Abstract

Ventral root avulsion (VRA) is a proximal lesion of spinal motoneurons in the central and peripheral nervous system interface. Such injury triggers a cascade of cellular and molecular events leading to retrograde degeneration about of 80% of the axotomized motoneurons two weeks after the injury. Thus, VRA has been a reproducible experimental model of motoneuron degeneration in animal models. Several studies have shown the benefits of cell therapy using stem cells in neuroprotection on the damaged motoneurons and, consequently, the increase of neuronal survival. Other studies have shown that treatment with basic fibroblast growth factors (FGF2) stimulates neurogenesis and synaptic plasticity in models of neurodegenerative diseases. The present study aimed to combine the use of stem cells with the local delivery of FGF2, evaluating their neuroprotective effect on spinal motoneurons in Lewis rats after ventral root avulsion. In addition, we investigated the efficacy of fibrin sealant as a scaffold capable of retaining the grafted cells at the injury site. Thus, was produced, by bioengineering, lineage derived from human embryonic stem cells that overexpress human FGF2-induced manner by doxycycline, containing green fluorescent protein as a marker (hES-FGF2-GFP). The animals were divided into the following groups: Group 1 - ventral root avulsion + fibrin sealant; Group 2 - ventral root avulsion + fibrin sealant + injection of hES-FGF2-GFP + doxycycline; Group 3 - ventral root avulsion + fibrin sealant + doxycycline Group 4 - ventral root avulsion + fibrin sealant + injection of hES-FGF2-GFP; Group 5 - ventral root avulsion + fibrin sealant + FGF2; Group 6 ¿ ventral root avulsion; Group 7- No lesion After 14 days of survival were analyzed (1) the expression of synaptophysin (synapses marker), GFAP (astrocyte marker) and Iba-1 (microglial reactions) by immunohistochemistry; (2) production of neurotrophic factors BDNF and the BDNF by hESC; (3) spinal motoneurons survival by Nissl staining technique and subsequent quantitative analysis by light microscopy; (4) by using qRT-PCR expression markers of the inflammatory response, including TNF-? and interleukins IL1?, IL-6, IL-10, as well as BDNF and GDNF factors. The results showed that treatment with ESC induced to overexpress FGF2 increased neuronal survival by 33%. Immunohistochemical evaluation with synaptophysin showed in groups II and V, treated with doxycycline + hESC and FGF2, respectively, a significant reduction of loss terminals in apposition to the cell body, reflecting a possible decrease in synaptic elimination process. In addition, there was also a significant decrease in anti-GFAP labeling (reactive astrogliosis) in the group treated with hESC + doxycycline compared to the other groups. An important finding was that hESC-GFP overexpressing FGF2, produced BDNF in the injection site of the cells and co-expressed GDNF in perimedullary region. However, cell therapy with hESC and FGF2 did not produce significant differences in the microglial reaction. The FGF2-treated group showed higher expression of ?2? and the group treated with hESC + doxycycline showed lower TNF-? expression and increased IL-10 expression, indicative of anti-inflammatory microenvironment. Taken together, the present results showed a neuroprotective effect of human embryonic stem cells modified to overexpress FGF2 as well as synaptic preservation and reduction of astrogliosis, can these results lead to promising regenerative studies aiming to improve motor recovery after brachial and lumbosacral plexus injuries (AU)

• FAPESP's process: 13/23030-2 - Application of human embryonic stem cells overexpressing FGF2 after ventral root avulsion: neuroprotection and immunomodulation
• Grantee: Marta Rocha Araujo
• Support Opportunities: Scholarships in Brazil - Doctorate