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PROTECTIVE EFFECT OF CYTOKINES AGAINST OXIDATIVE DAMAGE TO ENTERIC NEURONS IN VITRO
S Lourenssen, P-Y Gougeon, MG Blennerhassett
GIDRU, Queen’s University, Kingston, Ontario
BACKGROUND: The enteric nervous system is a sensitive target in intestinal inflammation. For example, there is a 50% reduction in neuron number by Day 2 of TNBS-induced colitis, as well as a transient decrease in axon density. This is followed by axonal proliferation which restores the innervation density to control levels by day 6 post-TNBS. To study the mechanism of axonal plasticity, we have used an enteric co-culture model to determine the effects of the TH1 cytokines TNFalpha and IL-1beta on neuron survival and axonal proliferation, both alone and in the presence of the oxidizing molecule hydrogen peroxide (H2O2).
METHODS: Enteric co-cultures were established from neonatal rat intestine and maintained in DMEM before addition of TNFalpha (50 ng/ml) or IL-1beta (25 ng/ml). Neuron and axon structure were analyzed using immunocytochemistry and Western blot analysis. Cytokines were added 3 hr prior to addition of 50 µM H2O2, a concentration previously shown to cause significant damage to enteric neuron structure and function (Lourenssen et. al, CDDW, 2006).
RESULTS: Addition of TNFalpha or IL-1beta to enteric co-cultures resulted in a significant increase in axon number to 183±19% and 155±10% of control (n=6), respectively, with no significant change in neuron number. Western blot analysis showed an increase in the expression of the synaptic vesicle protein SNAP-25 in cytokine-treated co-cultures relative to control. While addition of 50 µM H2O2 to co-cultures caused a significant loss of both neurons and axons to 74±9% and 72±6% of control (n=4), respectively, prior addition of TNFalpha diminished neuron loss (88±11% control) and completely prevented axon damage (126±16% control). In contrast, IL-1beta did not significantly affect the H2O2-induced loss in neuron or axon number.
CONCLUSIONS: Both TNFalpha and IL-1beta promote axonal outgrowth from enteric neurons, but there is a differential effect of these cytokines in the presence of an oxidative damaging compound, since TNFalpha but not IL-1beta was able to diminish the loss of neurons and completely prevent axonal loss. This suggests that TNFalpha may support axon outgrowth following damage in intestinal inflammation.
Supported by the CCFC