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79 AXONAL PLASTICITY OF ENTERIC NEURONS IN VITRO SR Lourenssen, RW Wells, MG Blennerhassett BACKGROUND: TNBS-induced colitis causes significant death of myenteric neurons by day 2 of inflammation. This is associated with a transient decrease of intestinal smooth muscle innervation, followed by axonal proliferation which restores the innervation density to control levels by day 6 post-TNBS. Since the mechanism of axonal plasticity remains unknown, a coculture model was developed, and neuronal damage was studied using the neurotoxins acrylamide (ACR) and hydrogen peroxide (H2O2).
GIDRU, Queen's University, Kingston, Ontario
METHODS: Enteric co-cultures were established from neonatal rat intestine. ACR or H2O2 was added for 6 h, 24 h or 96 h, and changes to neuron structure and function were analyzed using immunocytochemistry, Western blotting, and measurement of stimulated release of acetylcholine (3H-ACh).
RESULTS: Addition of ACR at doses of 0.5 mM to 2 mM to co-cultures caused damage to axon structure without decrease in neuron number, with axon number decreasing to 71%±4% of control (0.5 mM ACR) at 24 h. Western blot analysis showed decreased expression of the synaptic vesicle protein SNAP-25 relative to the pan-neuronal marker PGP 9.5 (73%±3% of control; 0.5 mM). In addition, the stimulated release of 3H-ACh was decreased to 64%±6% of control (0.5 mM), without change in uptake of the ACh precursor 3H-choline. By 96 h post-ACR, both axon number and SNAP-25 expression were again similar to time-matched control values, although 3H-ACh release remained impaired. This showed that axonal regeneration led to restoration of structure without parallel increase in function.
By 6 h post-ACR, no axon damage was evident, but SNAP-25 expression was markedly decreased relative to PGP 9.5 (66%±13% relative to control; 1 mM), further supporting a distinct time course for changes to neuronal function vs structure.
In contrast, exposure to H2O2 caused neuronal damage characterized by the parallel loss of axons and cell bodies with doses of 1 µM to 200 µM. These structural changes were also associated with decreased expression of SNAP-25 and syntaxin relative to PGP 9.5. The uptake of the ACh precursor 3H-choline was decreased after addition of 50 µm H2O2, and absent at higher doses, suggesting a high sensitivity of cholinergic metabolism to this neurotoxic agent.
CONCLUSIONS: The actions of ACR and H2O2 on enteric neurons in vitro cause selective impairment of function in the apparent absence of structural damage, although there are distinct mechanisms of response to specific challenges. Both models show aspects of neuronal damage similar to those observed in intestinal inflammation, where we reported the selective decrease in expression of the calcium-sensitive synaptic vesicle protein NCS-1. These models can be used to better understand axonal plasticity in vivo.
Supported by CCFC