The translation of new therapies for spinal cord injury to clinical trials can be facilitated with large animal models close in morpho-physiological scale to humans. Here, we report functional restoration and morphological reorganization after spinal contusion in pigs, following a combined treatment of locomotor training facilitated with epidural electrical stimulation (EES) and cell-mediated triple gene therapy with umbilical cord blood mononuclear cells overexpressing recombinant vascular endothelial growth factor, glial-derived neurotrophic factor, and neural cell adhesion molecule. Preliminary results obtained on a small sample of pigs 2 months after spinal contusion revealed the difference in post-traumatic spinal cord outcomes in control and treated animals. In treated pigs, motor performance was enabled by EES and the corresponding morpho-functional changes in hind limb skeletal muscles were accompanied by the reorganization of the glial cell, the reaction of stress cell, and synaptic proteins. Our data demonstrate effects of combined EES-facilitated motor training and cell-mediated triple gene therapy after spinal contusion in large animals, informing a background for further animal studies and clinical translation.
The gene therapy has been successful in treatment of spinal cord injury (SCI) in several animal models, although it still remains unavailable for clinical practice. Surprisingly, regardless the fact that multiple reports showed motor recovery with gene therapy, little is known about molecular and cellular changes in the post-traumatic spinal cord following viral vector- or cell-mediated gene therapy. In this study we evaluated the therapeutic efficacy and changes in spinal cord after treatment with the genes encoding vascular endothelial growth factor (VEGF), glial cell-derived neurotrophic factor (GDNF), angiogenin (ANG), and neuronal cell adhesion molecule (NCAM) applied using both approaches. Therapeutic genes were used for viral vector- and cell-mediated gene therapy in two combinations: (1) VEGF+GDNF+NCAM and (2) VEGF+ANG+NCAM. For direct gene therapy adenoviral vectors based on serotype 5 (Ad5) were injected intrathecally and for cell-mediated gene delivery human umbilical cord blood mononuclear cells (UCB-MC) were simultaneously transduced with three Ad5 vectors and injected intrathecally 4 h after the SCI. The efficacy of both treatments was confirmed by improvement in behavioral (BBB) test. Molecular and cellular changes following post-traumatic recovery were evaluated with immunofluorescent staining using antibodies against the functional markers of motorneurons (Hsp27, synaptophysin, PSD95), astrocytes (GFAP, vimentin), oligodendrocytes (Olig2, NG2, Cx47) and microglial cells (Iba1). Our results suggest that both approaches with intrathecal delivery of therapeutic genes may support functional recovery of post-traumatic spinal cord via lowering the stress (down regulation of Hsp25) and enhancing the synaptic plasticity (up regulation of PSD95 and synaptophysin), supporting oligodendrocyte proliferation (up regulation of NG2) and myelination (up regulation of Olig2 and Cx47), modulating astrogliosis by reducing number of astrocytes (down regulation of GFAP and vimetin) and microglial cells (down regulation of Iba1).
Natural brain repair after stroke is extremely limited, and current therapeutic options are even more scarce with no clinical break-through in sight. Despite restricted regeneration in the central nervous system, we have previously proved that human umbilical cord blood mono-nuclear cells (UCB-MC) transduced with adenoviral vectors carrying genes encoding vascular endothelial growth factor (VEGF), glial cell-derived neurotrophic factor (GDNF), and neural cell adhesion molecule (NCAM) successfully rescued neurons in amyotrophic lateral sclerosis and spinal cord injury. This proof-of-principle project was aimed at evaluating the beneficial effects of the same triple-gene approach in stroke. Rats subjected to distal occlusion of the middle cerebral artery were treated intrathecally with a combination of these genes either directly or using our cell-based (UCB-MC) approach. Various techniques and markers were employed to evaluate brain injury and subsequent recovery after treatment. Brain repair was most prominent when therapeutic genes were delivered via adenoviral vector- or UCB-MC-mediated approach. Remodeling of brain cortex in the stroke area was confirmed by reduction of infarct volume and attenuated neural cell death, depletion of astrocytes and microglial cells, and increase in the number of oligodendroglial cells and synaptic proteins expression. These results imply that intrathecal injection of genetically engineered UCB-MC over-expressing therapeutic molecules (VEGF, GDNF, and NCAM) following cerebral blood vessel occlusion might represent a novel avenue for future research into treating stroke.
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