Abstract
Summary Statement
Spinal cord injury is characterised by an inflammatory cascade that leads to neuronal death by neurotoxicity. In a model of spinal cord damage we successfully preserved the number of ventral horn neurons by treatment with interleukin-1 receptor antagonist (IL1RA) and neurotrophin (NT)-3.
Introduction
Secondary damage after spinal cord injury (SCI) is characterised by activation of microglial cells that release neurotoxic agents. This results in apoptotic death of neurons that survived the initial trauma. Interleukin (IL)-1 is one of the most prominent mediators of neurotoxicity. Organotypic spinal cord slice cultures (OSCSC) are a useful in vitro model of spinal cord injury. We have previously shown that OSCSC degenerate substantially during in vitro incubation under standard conditions. Our aim was to treat OSCSC with the putatively neuroprotective agents IL-1 receptor antagonist (IL1RA) and neurotrophin (NT)-3 and to evaluate neuronal and microglial populations as well as axonal preservation. We hypothesised that treatment with the above substances would enhance neuronal survival and suppress microglial activation.
Materials & Methods
OSCSC were obtained from p9 (p=postnatal) mice and cultured in a 3-D collagen matrix for 0, 2, 4, 6 and 8 days in vitro (div). Neuroprotective substances were added to culture media, resulting in 4 treatment groups: IL1RA, NT3, IL1RA+NT3, and control (only medium). After fixation cultures were stained immunohistochemically for the neuronal marker NeuN and α-neurofilament (NF-L). Microglial cells were marked with isolectin B4 (IB4). Neurons in the ventral and dorsal horns were counted manually. The number of resting and activated microglial cells was calculated within the white and grey matter based on staining intensity and circularity index. Axonal preservation was evaluated qualitatively.
Results
OSCSC under control conditions showed signs of early degeneration after 2 div with decreased number of neurons within both the ventral and dorsal horns. However, significant differences between the groups were noted after 8 div: In the ventral horns, the neurons in all treatment groups were significantly more numerous compared to the control group. In the IL1RA+NT3 group the number of neurons did not differ significantly when compared to directly fixed cultures (div 0), whereas that number was significantly decreased in the other groups. After 8 div the number of activated microglial cells was significantly increased in the control group compared to all treatment groups both in the white and grey matter. Qualitative analysis of axonal preservation after 6 and 8 div revealed axonal sprouting within the white matter in all groups. However, these sprouting axons seemed to expand into the collagen matrix only in the three treatment groups.
Discussion/Conclusion
We demonstrate a neuroprotective effect of IL1RA and NT3 in OSCSC. OSCSC normally degenerate after in vitro incubation, however neurons in the ventral horns are sustained at initial levels in the IL1RA+NT3 group. At the same time microglial activation is suppressed in all treatment groups compared to controls. Finally, treatment of OSCSC with IL1RA and NT3 seem to be associated with axonal sprouting outside the cultures.
