The circuit of recurring inhibition between motor neurons and Renshaw cells in the spinal cord has been known for around 70 years, though no determined function has been outlined as of yet. Renshaw cells are thought to be part of the central pattern generator in the spinal cord establishing them as an important part of the animal’s locomotive properties. In this study we aimed to investigate the role of Renshaw cells in locomotion with the help of optogenetics and electrophysiology. Halorhodopsin was inserted into the genome of mice and driven to expression with Cre recombinase in Renshaw cells. The spinal cord of P3 mice was extracted and by inducing fictive locomotion with appropriate neurotransmitters we could inhibit the Renshaw cells in action with a green laser, opening the halorhodopsin channels for Cl- ions. In previous experiments where the ability of Renshaw cells to release inhibitory neurotransmitters was inactivated, no effect was observed in either behavioral experiments or electrophysiological experiments. In a system where the effect of Renshaw cells was knocked out acutely with optogenetics there was no discernible change in fictive locomotion cycle length, frequency or amplitude. Nor was there an effect on alternation. The access of light to the Renshaw cells area might have been limited during the experiment considering the angle of light delivery and strength of the laser. Furthermore, the maturity of Renshaw cells at P3, the exclusive ability of the marker used to target Renshaw cells and the observed nature of neonatal inhibitory neurons acting as excitatory neurons could all be called into question about whether they contributed to these results or not.

No description available.

The overall aim of this thesis was to establish strategies to minimize secondary damage to the injured spinal cord. Secondary damage that follows spinal cord injury (SCI) involves inflammatory and excitotoxic pathways. Regulation of these pathways using immunomodulatory and neuroprotective substances potentially protects the injured spinal cord from further damage. We also developed and studied resorbable biomaterials to be used as carriers for potential neuroprotectants to the injured spinal cord. We used transversal spinal cord slice cultures (SCSCs) derived from postnatal mice as a model. SCSCs were maintained on different biomaterials and were studied after treatment with immunomodulatory and/or neurotrophic factors. They were further excitotoxically injured and subsequently treated with interleukin-1 receptor antagonist (IL1RA) or by neural crest stem cell (NCSC)-transplantation. The results show that biocompatible and resorbable hydrogels based on hyaluronic acid (HA) preserved neurons in SCSCs to a much higher extent than a conventional collagen-based biomaterial or standard polyethylene terephthalate (PET) membrane inserts. Glial activation was limited in the cultures maintained on HA-based hydrogel. The anti-inflammatory factor IL1RA protected SCSCs from degenerative mechanisms that occur during in vitro incubation, and IL1RA also protected SCSCs from excitotoxic injury induced by N-Methyl-d-Aspartate (NMDA). IL1RA specifically protected neurons that resided in the ventral horn, while other neuronal populations such as dorsal horn neurons and Renshaw cells did not respond to treatment. Finally, transplantation of NCSCs onto excitotoxically injured SCSCs protected from neuronal loss, apoptosis and glial activation, while NCSCs remained undifferentiated. The results presented in this thesis indicate that carriers based on HA seem to be more suitable than conventional collagen-based biomaterials since they enhance neuronal survival per se. The observed neuroprotection is likely due to biomechanical properties of HA. IL1RA protects SCSCs from spontaneous degeneration and from NMDA-induced injury, suggesting that excitotoxic mechanisms can be modulated through anti-inflammatory pathways. Different neuronal populations are affected by IL1RA to various degrees, suggesting that a combination of different neuroprotectants should be used in treatment strategies after SCI. Finally, NCSCs seem to protect SCSCs from excitotoxic injury through paracrine actions, since they remain undifferentiated and do not migrate into the tissue during in vitro incubation. It seems that combinations of neuroprotectants and carrier substances should be considered rather than one single strategy when designing future treatments for SCI. Incorporation of neuroprotectants such as IL1RA combined with stem cells in injectable biocompatible carriers based on HA is the final goal of our group in the treatment of SCI.

No description available.