Pathological implications of iNOS expression in central white matter: an ex vivo study of optic nerves from rats with experimental allergic encephalomyelitis

Abstract

Excessive nitric oxide (NO) production from the inducible isoform of nitric oxide synthase (iNOS) has been invoked as a causative factor in many neurodegenerative disorders, including multiple sclerosis. This hypothesis has been supported by in vitro studies showing that glial iNOS expression results in toxic NO concentrations (near 1 microm). To investigate the relevance of such findings, experiments were carried out ex vivo on optic nerves from rats with exacerbated experimental allergic encephalomyelitis, a model of multiple sclerosis. The nerves displayed characteristic immunopathology and expression of iNOS in macrophages and/or microglia and there was overt axonal damage in localized regions of the optic chiasm. The resulting NO levels in the optic nerve were sufficient to cause activation of guanylyl cyclase-coupled NO receptors, resulting in marked cGMP accumulation in axons throughout the nerve. Nevertheless, calibration of cGMP levels against those evoked by exogenous NO indicated that the nerves were not compromised metabolically and that their ambient NO concentration was only approximately 1 nm. Consistent with this observation, electrophysiological tests indicated that there was no ongoing malfunctioning of the type that can be elicited by high exogenous NO concentrations. It is concluded that, with iNOS expressed in physiological locations and levels, the tissue levels of NO remain at concentrations far lower than those shown to have toxic effects, despite continuous NO synthesis. The fact that NO can rise to much higher levels in dispersed cultures in vitro may be attributable to a deficiency in NO inactivation in such preparations.

What is the real physiological NO concentration in vivo?

“…Only when the iNOS-expressing cell population (microglia) in the slice cultures was expanded further (it was already twice the density found in vivo ) did the prevailing NO become saturating for cGMP generation, signifying a concentration of 10 nM or more [64]. The underlying assumption in this approach is that the tissue NO concentration is fairly uniform, as would be expected theoretically [66] and as was indicated by cGMP immunocytochemistry under similar experimental conditions in the cerebellar cortex [67] and optic nerve [65]. While it cannot be excluded that there existed subregions with higher-than-average NO concentrations, the results suggest that even under potentially pathological conditions, the global NO concentration in brain tissue remains very low, even in vitro when scavenging of NO by circulating red blood cells is lacking.…”

“…Tissue slices of a brain region (cerebellum) consumed NO with an apparent maximal rate of 0.2–2 μM/s and a K m of 10 nM [76,84], meaning that 10 nM NO would decay with a rate constant of 12–120 s −1 , equivalent to a half-life of 6–60 ms. The NO inactivation rate in optic nerve appeared to be similarly high [65]. Hepatocytes consumed NO via a process that depended linearly on both O 2 and NO, such that the parenchymal half-life of NO would be 0.09–2 s, depending on the O 2 concentration [99].…”

“…Here, NO consumption gives rise to substantial NO concentration gradients across the tissue when it is applied in the bathing medium [76], such that high NO concentrations must be used in order to achieve low levels of NO at the centre of the tissue block. In intact brain slices [76] and optic nerve [65], for example, the apparent EC 50 of NO for cGMP accumulation at steady-state is about 1000-fold higher than when NO has direct access to its target cells in isolation. Thus, the EC 50 for NO measured in such tissues reflects the properties of the inactivation mechanism rather than the sensitivity of the constituent cells to NO, much as when glutamate and other transmitters that are subject to avid removal from the extracellular space are administered in the bathing medium [128].…”

Nitric oxide – mediated signalization and nitrosative stress in neuropathology / Azot oksid – posredovana signalizacija i nitrozativni stres u neuropatologiji

“…Animal and cellular models of the disease have confirmed this hypothesis. Thus, in the multiple sclerosis mice experimental model of allergic encepha lo myelitis, NOS2 is profusely expressed in either infiltrating macrophages (14) or in microglia of the white matter (15). The increased NO synthesis is demonstrated by powerful iNOS expression within the CNS at the onset and development of EAE clinical signs, followed by decreased expression in remission (16).…”

Ultrastructure of Renal Tubular Epithelial Cells Of Rat’s Kidneys after Administration of L-Arginine

“…The organism produces NO by endothelial cells, macrophages, hepatocytes, nerve endings, some neurons (10), neutrophils, monocytes, mastocytes, and blood platelets (23). Nitric oxide induces cell apoptosis via free radicals and oxidative stress.…”