Sensorimotor recovery after spinal dorsal roots transecction and repair with platelet-rich plasma (PRP) associated with human embryonic stem cells (hESC)

Author(s)

Mateus Vidigal Castro

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2020-02-28

Examining board members

Alexandre Leite Rodrigues de Oliveira; Fábio Rogério; André Luis Bombeiro; Rogério Martins Amorim; Victor Túlio Ribeiro de Resende

• Advisor: Alexandre Leite Rodrigues de Oliveira

Abstract

Motor coordination involves specific neural processes ranging from the perception of stimuli to the motor response, being dependent on the sensorimotor integration, which is particularly important in the spinal cord. In this sense, spinal cord injuries can result in sensory and motor losses depending on the affected root, becoming an important medical problem. The repair of such lesions is challenging, because of the risk of additional damage to the spinal cord and surrounding structures, as well as the possibility of generating neuropathic pain. Thus, surgical procedures for restoration of root lesions prioritize the repair of the ventral roots (motor component) over the sensory component (dorsal roots). In this context, we propose a new treatment using human platelet-rich plasma (PRP) gel for the repair of injured dorsal roots. Further, in view of stem cell therapy as a promising tool to improve regeneration, the present work investigated the benefits of human embryonic stem cells (hESC) in association with PRP following root lesion. Such cells have been bioengineered for inducible overexpression fibroblast growth factor-2 (FGF2). We also propose the use of poly L-lactic acid (PLLA) nanofibers to provide mechanical support for repaired dorsal roots, as a negative control. Therefore, to investigate the efficacy of PRP gel as an adhesive and inductive element of axonal regeneration, as well as the benefits of modified hESC, 8-week-old female Lewis rats underwent unilateral dorsal rhizotomy (L4-L6), where the roots were repaired using PRP, associated or not with cell therapy (N = 5; per experimental group). PRP was obtained from human blood and characterized regarding platelet concentration, integrity, and viability. The human cells were characterized, phenotyped by flow cytometry and processed to evaluate the expression of gene transcripts for BDNF, GDNF, FGF-2, IGF and VEGFA, by qRT-PCR. The reflex arc recovery was evaluated weekly through the electronic von-Frey method, for eight weeks. The spinal cords were processed to evaluate the in vivo expression of gene transcripts for TNFa, TGFb, BDNF, GDNF, FGF2, VEGF, NGF, IL4, IL6 and IL13 by qRT-PCR, 1-week post-lesion. Also, changes in the glutamatergic synaptic circuits (anti-VGLUT1) and glial reactions (anti-GFAP e anti-Iba-1) were evaluated by immunofluorescence, 1-week (acute phase) and 8-weeks (chronic phase) post-lesion. Taken together, the results of the present study indicate that the activated platelet rich plasma (PRP) used to repair the dorsal spinal roots was efficient, allowing the physical reconnection of the sectioned root to the spinal surface and the sensory reinnervation in the spinal environment. Thus, the surgical approach used herein contributed to axon regeneration, as seen by the partial restoration of the glutamatergic synaptic network. This happened without exacerbation of glial reactivity, producing behavioral improvement overtime. In addition, root repair with PRP showed immunomodulatory effects. Also, PRP served as a scaffold for human embryonic stem cells (hESC). Cell therapy did not bring additional improvement in functional terms, despite the significant expression of trophic factors in vitro. In conclusion, the present study indicates the feasibility of repairing dorsal spinal roots, partially restoring the integration of sensory and motor inputs, with important functional gain (AU)

• FAPESP's process: 15/26206-0 - Sensorimotor recovery after transection and spinal dorsal roots repair with platelet rich plasma (PRP) and human embryonic stem cells treatment (hESC).
• Grantee: Mateus Vidigal de Castro
• Support Opportunities: Scholarships in Brazil - Doctorate