Abstract
Alterations of the enteric glutamatergic transmission may underlay changes in the function of myenteric neurons following intestinal ischemia and reperfusion (I/R) contributing to impairment of gastrointestinal motility occurring in these pathological conditions. The aim of the present study was to evaluate whether glutamate receptors of the NMDA and AMPA/kainate type are involved in myenteric neuron cell damage induced by I/R. Primary cultured rat myenteric ganglia were exposed to sodium azide and glucose deprivation (in vitro chemical ischemia). After 6 days of culture, immunoreactivity for NMDA, AMPA and kainate receptors subunits, GluN1 and GluA1–4, GluK1–3 respectively, was found in myenteric neurons. In myenteric cultured ganglia, in normal metabolic conditions, -AP5, an NMDA antagonist, decreased myenteric neuron number and viability, determined by calcein AM/ethidium homodimer-1 assay, and increased reactive oxygen species (ROS) levels, measured with hydroxyphenyl fluorescein. CNQX, an AMPA/kainate antagonist exerted an opposite action on the same parameters. The total number and viability of myenteric neurons significantly decreased after I/R. In these conditions, the number of neurons staining for GluN1 and GluA1–4 subunits remained unchanged, while, the number of GluK1–3-immunopositive neurons increased. After I/R, -AP5 and CNQX, concentration-dependently increased myenteric neuron number and significantly increased the number of living neurons. Both -AP5 and CNQX (100–500 µM) decreased I/R-induced increase of ROS levels in myenteric ganglia. On the whole, the present data provide evidence that, under normal metabolic conditions, the enteric glutamatergic system exerts a dualistic effect on cultured myenteric ganglia, either by improving or reducing neuron survival via NMDA or AMPA/kainate receptor activation, respectively. However, blockade of both receptor pathways may exert a protective role on myenteric neurons following and I/R damage. The neuroprotective effect may depend, at least in part, on the ability of both receptors to increase intraneuronal ROS production.
Highlights
The intestine is one of the most sensitive organs to ischemia/reperfusion (I/R) injury which may occur as a consequence of embolism, arterial or venous thrombosis, shock [1], intestinal transplantation, necrotising enterocolitis in the human premature newborn or chronic inflammatory diseases [2,3]
To further investigate the mechanisms underlying glutamate-mediated neurotoxicity in myenteric neurons following an I/R insult, we evaluated whether ionotropic glutamate receptors of the NMDA and AMPA/kainate type are involved in myenteric neuron cell damage induced by I/R
There are, evidences suggesting that overactivation of ionotropic receptors of the NMDA and AMPA/kainate type, may exert a neurotoxic effect on myenteric neurons, as observed in the central nervous system (CNS) [14]
Summary
The intestine is one of the most sensitive organs to ischemia/reperfusion (I/R) injury which may occur as a consequence of embolism, arterial or venous thrombosis, shock [1], intestinal transplantation, necrotising enterocolitis in the human premature newborn or chronic inflammatory diseases [2,3]. Increasing evidence is, available to suggest that intrinsic neuronal circuitries may be damaged following I/R: some neurons may die, while others may undergo changes lasting for many weeks after the injury [5,6,7]. Since both intestinal motility and secretion are highly dependent upon the activity of intrinsic enteric circuitries, such damage may be at the basis of intestinal dysfunctions associated with an ischemic injury in the gut [8]. Accumulation of nitrosylated protein aggregates resulting from the reaction between nitric oxide (NO) and ROS has been suggested to participate to degeneration of nitrergic neurons following an in vivo I/R damage in the mouse gut [7]
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