Deleterious effects of intestinal ischemia/reperfusion injury in the mouse enteric nervous system are associated with protein nitrosylation

Abstract

Changes in intestinal function, notably impaired transit, following ischemia/reperfusion (I/R) injury are likely to derive, at least in part, from damage to the enteric nervous system. Currently, there is a lack of quantitative data and methods on which to base quantitation of changes that occur in enteric neurons. In the present work, we have investigated quantifiable changes in response to ischemia of the mouse small intestine followed by reperfusion from 1h to 7days. I/R caused distortion of nitric oxide synthase (NOS)-containing neurons, the appearance of a TUNEL reaction in neurons, protein nitrosylation and translocation of Hu protein. Protein nitrosylation was detected after 1h and was detectable in 10% of neurons by 6h in the ischemic region, indicating that reactive peroxynitrites are rapidly produced and can interact with proteins soon after reperfusion. Apoptosis, revealed by TUNEL staining, was apparent at 6h. The profile sizes of NOS neurons were increased by 60% at 2days and neurons were still swollen at 7days, both in the ischemic region and proximal to the ischemia. The distribution of the enteric neuron marker and oligonucleotide binding protein, Hu, was significantly changed in both regions. Hu protein translocation to the nucleus was apparent by 3h and persisted for up to 7days. Particulate Hu immunoreactivity was observed in the ganglia 3h after I/R but was never observed in control. Our observations indicate that effects of I/R injury can be detected after 1h and that neuronal changes persist to at least 7days. Involvement of NO and reactive oxygen species in the changes is indicated by the accumulation of nitrosylated protein aggregates and the swelling and distortion of nitrergic neurons. It is concluded that damage to the enteric nervous system, which is likely to contribute to functional deficits following ischemia and re-oxygenation in the intestine, can be quantified by Hu protein translocation, protein nitrosylation, swelling of nitrergic neurons and apoptosis.