Abstract
Regeneration in the adult mammalian spinal cord is limited due to intrinsic properties of mature neurons and a hostile environment, mainly provided by central nervous system myelin and reactive astrocytes. Recent results indicate that propriospinal connections are a promising target for intervention to improve functional recovery. To study this functional regeneration in vitro we developed a model consisting of two organotypic spinal cord slices placed adjacently on multi-electrode arrays. The electrodes allow us to record the spontaneously occurring neuronal activity, which is often organized in network bursts. Within a few days in vitro (DIV), these bursts become synchronized between the two slices due to the formation of axonal connections. We cut them with a scalpel at different time points in vitro and record the neuronal activity 3weeks later. The functional recovery ability was assessed by calculating the percentage of synchronized bursts between the two slices. We found that cultures lesioned at a young age (7–9DIV) retained the high regeneration ability of embryonic tissue. However, cultures lesioned at older ages (>19DIV) displayed a distinct reduction of synchronized activity. This reduction was not accompanied by an inability for axons to cross the lesion site. We show that functional regeneration in these old cultures can be improved by increasing the intracellular cAMP level with Rolipram or by placing a young slice next to an old one directly after the lesion. We conclude that co-cultures of two spinal cord slices are an appropriate model to study functional regeneration of intraspinal connections.
Highlights
The adult spinal cord of higher vertebrates fails to regenerate after injury
This has been attributed to an inhibitory environment provided, for example, by myelin associated proteins such as Nogo-A, oligodendrocyte myelin glycoprotein and myelin associated glycoprotein (McKerracher et al, 1994; Mukhopadhyay et al, 1994; Chen et al, 2000; GrandPreet al., 2000; Prinjha et al, 2000; Kottis et al, 2002; Wang et al, 2002) or proteins associated with the extracellular matrix like chondroitin sulfate proteoglycans (CSPGs) (Levine, 1994)
We have previously shown that spontaneous bursting activity appears in organotypic spinal cord cultures, originating from intrinsic spiking and recurrent excitation and reflecting the activation of a neuronal network in the slice (Tscherter et al, 2001; Darbon et al, 2002; Czarnecki et al, 2008)
Summary
The adult spinal cord of higher vertebrates fails to regenerate after injury. This has been attributed to an inhibitory environment provided, for example, by myelin associated proteins such as Nogo-A, oligodendrocyte myelin glycoprotein and myelin associated glycoprotein (McKerracher et al, 1994; Mukhopadhyay et al, 1994; Chen et al, 2000; GrandPreet al., 2000; Prinjha et al, 2000; Kottis et al, 2002; Wang et al, 2002) or proteins associated with the extracellular matrix like chondroitin sulfate proteoglycans (CSPGs) (Levine, 1994). Propriospinal neurons seem to respond better than descending tracts to certain treatments such as application of growth factors or tissue grafting (Houle, 1991; Xu et al, 1995; Blesch et al, 2004). Because of these characteristics, propriospinal fibers are a suitable target for therapeutic interventions to promote functional recovery after SCI. Bonnici and Kapfhammer (2008) used longitudinal spinal cord slices consisting of several segments to study their structural recovery After setting lesions they stained the cultures with an anti-neurofilament SMI-31 antibody and estimated the amount of fibers crossing through the lesion center. The model offers the advantage of circumventing the need for large animal cohorts often required to test the efficacy of strategies to promote regeneration after SCI in vivo
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