Use of fibrin sealant combined with mesenchymal stem cells for anterior funiculus injury repair: effects on inflammation, axonal regeneration and neuroprotection

Author(s)

Aline Barroso Spejo

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2017-08-30

Examining board members

Alexandre Leite Rodrigues de Oliveira; Marcondes Cavalcante Franca Junior; Andre Schwambach Vieira; Rui Seabra Ferreira Junior; Taize Machado Augusto

• Advisor: Alexandre Leite Rodrigues de Oliveira

Abstract

The intense neuronal death, inflammation, and formation of an inhibitory microenvironment represent a great challenge for spinal cord injury repair. In this sense, the use of mesenchymal stem cells (MSC) has been considered as an alternative for the treatment of trauma to the spinal cord. Thus, to evaluate neuroprotection, modulation of the inflammatory response and formation of the glial scar, MSC treatment was performed after intramedullary axotomy (IA), through a perforating lesion at the anterior funiculus, at the spinal levels L4-6, in female Lewis rats. To retain the MSC at the site of the lesion, a fibrin sealant (FS) scaffold was used. After 7 days of post-injury survival, real-time quantitative PCR was performed to evaluate the levels of VEGF and BDNF gene transcripts; INOS2 and Arginase-1 (M1 and M2 macrophage markers, respectively); proinflammatory cytokines TNF-alpha, IL-6 and IL-1beta and anti-inflammatory cytokines IL-4, IL-10, IL-13 and TGF-beta. The FS-treated group had higher expression of the iNOS2 and arginase-1 genes, proinflammatory cytokines TNF-alpha and IL-1beta and anti-inflammatory cytokines IL-10, IL-4 and IL-13. Thus, FS promoted greater inflammatory response, as well as an anticipation of its resolution. With survival of 14 days after injury, spinal alpha motor neurons (MN), evidenced by Nissl staining were counted. For the analysis of astrogliosis in spinal lamina IX and synaptic detachment around lesioned MN (GAP-43 positive), anti-GFAP and anti-synaptophysin immunohistochemistry were performed, respectively. Cell death of about 70% MN was observed in the untreated groups. The groups treated with FS and/or MSC presented higher neuronal survival compared to the other groups, revealing the neuroprotective effect of these treatments. Astroglial reactivity was less intense in the MSC-treated groups when compared to the other groups, showing that MSC caused reduced astrogliosis in this period. The analysis of the synaptic detachment showed greater synaptic preservation around MN cell bodies in the MSC-treated group, reflecting a possible role for the cells on the preservation of synaptic circuits. With a 28-day survival after injury, the glial scar on the anterior funiculus was studied by anti-GFAP (astrocyte marker) and anti-CS-56 (chondroitin sulfate proteoglycan marker) immunohistochemistry. Throughout this period, the gait of the animals was evaluated by the walking track test (Catwalk). The study of glial scar revealed similar quantity of astrocytes and chondroitin sulfate in all experimental groups. In the evaluation of the motor recovery, the animals treated with MSC presented better performance. FS alone was shown to be neuroprotective and promoter of pro-regenerative inflammation, as well as acceleration of its resolution. The combination of FS and MSC promoted neuroprotection, decreased astrogliosis and synaptic preservation. However, such a combined treatment generated a different inflammation pattern compared to the FS-treated group and did not show a similar improvement in motor function observed in the MSC-treated group. Thus, animals treated only with MSC showed a better regenerative performance. In this group, it was observed neuroprotection, reduced astrogliosis, preserved spinal circuits and better motor function. Therefore, MSC therapy can be successfully performed, even without a co-administration of a substrate for the retention at the lesion site. Possibly these cells are actively able to remain within the injured microenvironment, through molecular signals emanated by neurons/glia present in lesion site (AU)

• FAPESP's process: 12/22750-9 - Use of fibrin sealant combined with mesenchymal stem cells for anterior funiculus injury repair: effects on inflammation, axonal regeneration and neuroprotection.
• Grantee: Aline Barroso Spejo
• Support Opportunities: Scholarships in Brazil - Doctorate