Influence of Delivery Method on Neuroprotection by Bone Marrow Mononuclear Cell Therapy following Ventral Root Reimplantation with Fibrin Sealant

Oliveira ALR (2014) Influence of Delivery Method on Neuroprotection by Bone Marrow Mononuclear Cell Therapy following Ventral Root Reimplantation with Fibrin Sealant. PLoS ONE 9(8): e105712. doi:10.1371/journal.pone.0105712 Influence of Delivery Method on Neuroprotection by Bone Marrow Mononuclear Cell Therapy following Ventral Root Reimplantation with Fibrin Sealant Roberta Barbizan 0 Mateus V. Castro 0 Benedito Barraviera 0 Rui S. Ferreira Jr. 0 Alexandre L. R. Oliveira 0 Graca Almeida-Porada, Wake Forest Institute for Regenerative Medicine, United States of America 0 1 Laboratory of Nerve Regeneration, Department of Structural and Functional Biology, University of Campinas - UNICAMP , Campinas, Sa o Paulo , Brazil , 2 Center for the Study of Venoms and Venomous Animals (CEVAP), Sa o Paulo State University (UNESP - Univ Estadual Paulista) , Botucatu, Sa o Paulo , Brazil The present work compared the local injection of mononuclear cells to the spinal cord lateral funiculus with the alternative approach of local delivery with fibrin sealant after ventral root avulsion (VRA) and reimplantation. For that, female adult Lewis rats were divided into the following groups: avulsion only, reimplantation with fibrin sealant; root repair with fibrin sealant associated with mononuclear cells; and repair with fibrin sealant and injected mononuclear cells. Cell therapy resulted in greater survival of spinal motoneurons up to four weeks post-surgery, especially when mononuclear cells were added to the fibrin glue. Injection of mononuclear cells to the lateral funiculus yield similar results to the reimplantation alone. Additionally, mononuclear cells added to the fibrin glue increased neurotrophic factor gene transcript levels in the spinal cord ventral horn. Regarding the motor recovery, evaluated by the functional peroneal index, as well as the paw print pressure, cell treated rats performed equally well as compared to reimplanted only animals, and significantly better than the avulsion only subjects. The results herein demonstrate that mononuclear cells therapy is neuroprotective by increasing levels of brain derived neurotrophic factor (BDNF) and glial derived neurotrophic factor (GDNF). Moreover, the use of fibrin sealant mononuclear cells delivery approach gave the best and more long lasting results. - Funding: The present work was supported by a grant from Fundacao de Amparo a Pesquisa do Estado de Sao Paulo FAPESP, Brazil (2010/0986-5). Barbizan R. was supported by Fundacao de Amparo a Pesquisa do Estado de Sao Paulo FAPESP (process number: 2010/00729-2). This work was funded by grants from Coordenacao de Aperfeicoamento de Pessoal de Nvel Superior (CAPES), Conselho Nacional de Desenvolvimento Cientifico e Tecnolo gico (CNPq) and Fundacao de Amparo a` Pesquisa do Estado de Sao Paulo (FAPESP). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. In order to enhance the success of adult stem cell (SC) translational medicine efforts, the source as well as the most effective delivery method has to be considered. The bone marrow contains endothelial progenitor cells and mononuclear cells (MC). The MC fraction corresponds to the totality of hematopoietic and mesenchymal stem cells. MC present clinical advantages over other stem cells, based on the minimally invasive harvesting procedures, which are fast and cost-effective. Also, the possibility of autografting avoids the use of immunosuppressants, present low oncogenic potential and does not raise ethical issues [1] as compared to other SC. Moreover, MCs have similar potential therapeutic outcome for nerve regeneration in comparison to mesenchymal cells [2]. The peripheral nerve regeneration after MC has been connected to the local production of neurotrophic factors [1,3,4]. Relevantly, stem cell therapy may also present an immunomodulatory effect, reducing pro-inflammatory events as well as glial reaction following lesion. Ventral root avulsion in rats has been used as a model for brachial plexus lesion (BPL). BPL is frequently caused by motorbike accidents in young adults as well as following complicated child-birth delivery [5]. It causes paralysis in the corresponding muscle groups and loss of sensory functions [6]. The degenerative impact on motoneurons is well characterized and is potentiated by pulling out the ventral roots from the CNS/ PNS interface at the spinal cord surface [6]. Similarly to BPL, VRA results in extensive loss of neurons in the first weeks after injury [7,8]. Reimplantation of avulsed roots can rescue motoneurons from degeneration, increasing the regenerative capacity of axonal regrowth [9,10]. As a result, anatomical and functional reinnervation of denervated muscles can be obtained [1113]. As seen in a previous work [10], a snake venom derived fibrin sealant allowed successful and stable ventral root implantation. Nevertheless, additional therapeutic approaches need to be developed, since root reimplantation alone, although neuroprotective, results in insufficient functional sensory-motor recovery [12,1416]. In order to improve the outcome following VRA, regarding neuronal survival, several attempts have been made to provide neurotrophic molecules at the site of injury. In this regard, the association of the root reimplantation with BDNF and CNTF resulted in rescue of injured motoneurons after avulsion in rabbits [17]. Therefore, the use of neurotrophic factors in combination with root reimplantation is a potential therapy to be used in patients. The use of recombinant neurotrophic factors, however, present important drawbacks. One of them is the need of relatively large amounts of the purified substance, to reach the target lesioned area. Due to the short biological activity window of such substances, there is also need of constant perfusion, what may contribute to infection and further lesion of the affected spinal cord area. Additionally, it is improbable that a single neurothrophic molecule will be sufficient to provide the necessary conditions for optimal regeneration. Based on such facts, the advent of stem cell technology brought new insights on cell therapy and local delivery of trophic substances. To date, however, there is not sufficient data on the delivery method to the nervous system, especially following VRA. So far, it is known that mesenchymal stem cells synthesize and possibly release BDNF and GDNF, when grafted to the VRA lesion area [18]. No data, however, indicates that MC exhibit the same properties. Therefore, the present study investigated two delivery strategies of MC, comparing the local injection to the spinal cord with the possibility of mixing MC with fibrin sealant on the interface of the CNS/PNS. Local production of BDNF and GDNF were evaluated in both situations. The results herein demonstrate that MC (...truncated)