Peripherally delivered Adeno-associated viral vectors for spinal cord injury repair.

Via the peripheral and autonomic nervous systems, the spinal cord directly or indirectly connects reciprocally with many body systems (muscular, intengumentary, respiratory, immune, digestive, excretory, reproductive, cardiovascular, etc). Accordingly, spinal cord injury (SCI) can result in catastrophe for multiple body systems including muscle paralysis affecting movement and loss of normal sensation, as well as neuropathic pain, spasticity, reduced fertility and autonomic dysreflexia. Treatments and cure for an injured spinal cord will likely require access of therapeutic agents across the blood-CNS (central nervous system) barrier. However, some types of repair within the CNS may be possible by targeting treatment to peripherally located cells or by delivering Adeno-Associated Viral vectors (AAVs) by peripheral routes (e.g., intrathecal, intravenous). This review will consider some future possibilities for SCI repair generated by therapeutic peripheral gene delivery. There are now six gene therapies approved worldwide as safe and effective medicines of which three were created by modification of the apparently nonpathogenic Adeno-Associated Virus. One of these AAVs, Zolgensma, is injected intrathecally for treatment of spinal muscular atrophy in children. One day, delivery of AAVs into peripheral tissues might improve recovery after spinal cord injury in humans; we discuss experiments by us and others delivering transgenes into nerves or muscles for sensorimotor recovery in animal models of SCI or of stroke including human Neurotrophin-3. We also describe ongoing efforts to develop AAVs that are delivered to particular targets within and without the CNS after peripheral administration using capsids with improved tropisms, promoters that are selective for particular cell types, and methods for controlling the dose and duration of expression of a transgene. In conclusion, in the future, minimally invasive administration of AAVs may improve recovery after SCI with minimal side effects.

Toll-like receptor 4 (TLR4) influences the glial reaction in the spinal cord and the neural response to injury following peripheral nerve crush.

After peripheral axotomy, there is a selective retraction of synaptic terminals in contact with injured motoneurons. This process, which actively involves glial cells, is influenced by the expression of immune-related molecules. Since toll-like receptors (TLRs) are upregulated by astrocytes and microglia following lesions, they might be involved in synaptic plasticity processes. Therefore, we administered lipopolysaccharide (LPS) to enhance TLR4 expression in mice and studied retrograde changes in the spinal cord ventral horn following sciatic nerve crush. To this end, adult C57BL/6J male mice were subjected to unilateral sciatic nerve crush at the mid-thigh level and, after a survival time of seven and forty days (acute and chronic phases, respectively), the spinal cords were paraformaldehyde-fixed and dissected out for immunolabeling for synaptophysin, glial fibrillary acidic protein (GFAP) and ionized calcium-binding adapter molecule 1 (Iba1). The results show that TLR4 upregulation leads to synaptophysin downregulation close to spinal motoneuron cell bodies, indicating increased synaptic elimination. LPS exposure also further increases astrogliosis and microglial reactions in the both ventral and dorsal horns, especially ipsilateral to nerve axotomy, compared to those in untreated mice. Notably, LPS administration to TLR4-/- mice produces results similar to those observed in untreated wild-type counterparts, reinforcing the role of this receptor in the glial response to injury. Therefore, our results suggest that the overexpression of the TLR4 receptor results in augmented astrogliosis/microglial reactions and the excessive loss of synapses postinjury, which may, in turn, affect the motoneuronal regenerative response and functionality. Additionally, treatment with LPS increases the expression of ß2-microglobulin, a subcomponent of MHC I. Importantly, the absence of TLR4 results in imbalanced axonal regeneration, inducing subsequent improvements and setbacks. In conclusion, our results show the involvement of TLR4 in the process of synaptic remodeling, indicating a new target for future research aimed at developing therapies for CNS and PNS repair.

Mesenchymal stem cells engrafted in a fibrin scaffold stimulate Schwann cell reactivity and axonal regeneration following sciatic nerve tubulization.

The present study investigated the effectiveness of mesenchymal stem cells (MSCs) associated with a fibrin scaffold (FS) for the peripheral regenerative process after nerve tubulization. Adult female Lewis rats received a unilateral sciatic nerve transection followed by repair with a polycaprolactone (PCL)-based tubular prosthesis. Sixty days after injury, the regenerated nerves were studied by immunohistochemistry. Anti-p75NTR immunostaining was used to investigate the reactivity of the MSCs. Basal labeling, which was upregulated during the regenerative process, was detected in uninjured nerves and was significantly greater in the MSC-treated group. The presence of GFP-positive MSCs was detected in the nerves, indicating the long term survival of such cells. Moreover, there was co-localization between MSCs and BNDF immunoreactivity, showing a possible mechanism by which MSCs improve the reactivity of SCs. Myelinated axon counting and morphometric analyses showed that MSC engrafting led to a higher degree of fiber compaction combined with a trend of increased myelin sheath thickness, when compared with other groups. The functional result of MSC engrafting was that the animals showed higher motor function recovery at the seventh and eighth week after lesion. The findings herein show that MSC+FS therapy improves the nerve regeneration process by positively modulating the reactivity of SCs.

Synaptic plasticity and sensory-motor improvement following fibrin sealant dorsal root reimplantation and mononuclear cell therapy.

Root lesions may affect both dorsal and ventral roots. However, due to the possibility of generating further inflammation and neuropathic pain, surgical procedures do not prioritize the repair of the afferent component. The loss of such sensorial input directly disturbs the spinal circuits thus affecting the functionality of the injuried limb. The present study evaluated the motor and sensory improvement following dorsal root reimplantation with fibrin sealant (FS) plus bone marrow mononuclear cells (MC) after dorsal rhizotomy. MC were used to enhance the repair process. We also analyzed changes in the glial response and synaptic circuits within the spinal cord. Female Lewis rats (6-8 weeks old) were divided in three groups: rhizotomy (RZ group), rhizotomy repaired with FS (RZ+FS group) and rhizotomy repaired with FS and MC (RZ+FS+MC group). The behavioral tests electronic von-Frey and Walking track test were carried out. For immunohistochemistry we used markers to detect different synapse profiles as well as glial reaction. The behavioral results showed a significant decrease in sensory and motor function after lesion. The reimplantation decreased glial reaction and improved synaptic plasticity of afferent inputs. Cell therapy further enhanced the rewiring process. In addition, both reimplanted groups presented twice as much motor control compared to the non-treated group. In conclusion, the reimplantation with FS and MC is efficient and may be considered an approach to improve sensory-motor recovery following dorsal rhizotomy.