The evidence presented in this paper demonstrates that NAC treatment reduced the inflammatory monocyte/macrophage cells in the CNS of Lewis rats with acute monophasic EAE. This in turn results in protection both in terms of clinical and histopathological changes. These conclusions are based on the following observations. 1) NAC treatment of EAE rats reduced the severity of EAE clinical symptoms, 2) attenuated the infiltration of mononuclear cells into the CNS of EAE rats, 3) blocked the induction of proinflammatory cytokines, iNOS and nitrotyrosine in the CNS, and 4) decreased proinflammatory Th1 cytokine responses (IFN-γ) from ex vivo splenocytes while increasing anti-inflammatory cytokine production (IL-10), and decreasing NO production in LPS-stimulated splenocytes.

The infiltration of activated mononuclear cells into the CNS of EAE is a critical event in the progression of the disease [26]. We have shown both qualitatively and quantitatively that ED1 positive leukocytes, namely macrophage/monocytes, were significantly decreased in animals treated with NAC as compared to the EAE animals. This decrease also correlated with the amelioration of clinical disease in female Lewis rats. As compared to our previous studies with lovastatin, NAC was not as effective in blocking the transmigration of inflammatory cells (NAC reduced by an average of 46%, while lovastatin reduced by 85%) and hence did not delay the onset of disease as was achieved with lovastatin treatment (EAE, EAE + NAC onset day 8 versus EAE + lovastatin onset day 11). However, NAC reduced the clinical scores to the same levels as those obtained with lovastatin (both had clinical scores maximum of 3). Other studies have also shown a correlation between macrophage infiltration and EAE clinical disease [27]. Inflammatory cytokine expression (IFN-γ, IL-1β, and TNF-α) was also inhibited in the CNS of EAE animals treated with NAC. As a consequence, inhibition of IFN-γ expression in NAC treated animals could in turn result in the reduced expression of MHC II molecules thereby inhibiting the proliferation of T-lymphocytes as has been shown with statins [28, 29], copolymer 1 [30] and IFN-β [31].

Evidence indicates that iNOS while not a crucial factor for induction of EAE, plays a major role in the progression of the disease. The critical factors is the amount of NO produced that tips the balance in favor or against the pathogenesis of EAE [32]. The peroxynitrite (ONOO-) produced by reaction of NO and O 2 - can damage membranes and cells by nitrosylation of lipids, proteins and nucleic acids. The induction of IL-1β and activation of NFκB were shown to precede the induction of iNOS in ED1+ cells [33]. Here we report that NAC blocked the induction of IL-1β in the CNS of EAE animals. Ex vivo studies using splenocytes isolated from control, EAE and EAE+NAC treated animals showed that NAC inhibited IFN-γ production while increasing IL-10 production. These changes coincided with a decreased NO production in the cultured splenocytes. NAC treatment was not as effective as lovastatin in altering cytokine production, but the reduction in nitrite was identical. NAC treatment reduced IFN-γ production by splenocytes (NAC by 59% and 40%, LOV by 76% and 60% for PHA and MBP respectively) and up-regulated IL-10 production by EAE splenocytes (NAC by 31% and 34%, LOV by 350% and 490% for PHA and MBP respectively). NAC treatment also inhibited the production of nitrite by LPS-stimulated splenocytes by 71% as compared to splenocytes from EAE animals. These studies indicate that NAC treatment inhibited a proinflammatory Th1 biased cytokine response (IFN-γ) while promoting an increase in IL-10, an anti-inflammatory cytokine. Similar shifts from Th1 cytokine profile to Th2 have been correlated with disease recovery or improvement in both EAE and MS [16, 18, 19, 34–37].

The brain is particularly vulnerable to oxidative stress due to its high consumption of oxygen and glucose, enrichment in unsaturated fatty acids that are subject to oxidation, and presence of regions enriched in iron and ascorbate that are potent pro-oxidants for brain membranes. Moreover, higher levels of glucose upregulate the neuroinflammatory process measured as induction of iNOS and NO production [38]. Coupled with the relatively reduced antioxidant defenses in the brain, exposure of brain cells to reactive oxygen or nitrogen species can be detrimental and is thought to contribute to the pathogenesis of many brain disorders [39]. Oxidative stress is important in the etiology of EAE and is thought to contribute directly to CNS damage [7, 40]. N-acetyl-L-cysteine (NAC) as cysteine, a precursor of glutathione, is a potent anti-oxidant. By scavenging superoxide radicals, metallothionein and other antioxidants such as cysteine, N-acetyl-cysteine and glutathione offer neuroprotection [41]. In vivo NAC enhances hippocampal neuronal survival after transient forebrain ischemia in rats [42]. Partial protection of neurons from the dopaminergic neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine was achieved by four different antioxidants including NAC in the mouse [43]. NAC also has a protective effect in pneumococcal meningitis in the rat [44]. In vitro, NAC promotes oligodendrocyte survival in the presence of toxic stimuli or due to withdrawal of growth factors [45] and maturation of oligodendrocytes [46]. NAC inhibits Theiler's virus-induced NO and TNF-α production by murine SJL/J astrocyte cultures [47]. NAC treatment prevented cytokine-induced decrease in GSH and degradation of sphingomyelin to ceramide, also blocked cytokine-mediated ceramide production in rat primary oligodendrocytes, microglia, and C6 glial cells, thereby preventing cell death. These results suggest that intracellular levels of GSH may play a critical role in the regulation of cytokine-induced generation of ceramide leading to apoptosis of brain cells in demyelinating diseases. [48]

In summary, the ability of NAC to inhibit the induction of proinflammatory cytokines and inhibit the transmigration of inflammatory cells into the CNS of EAE-induced rats identifies it as a potential drug for the treatment of neuroinflammatory diseases and possibly other Th1-mediated autoimmune diseases. In addition, in vitro studies suggest that NAC may also promote survival of neurons and oligodendrocytes and thereby potentially facilitating remyelination. MS is a multifactorial disease and the etiology of the disease in unknown. Consequently, the targets for the prevention of the disease are currently unknown. However the disease signs and causes of these are known. For example an increase in pro-inflammatory cytokines and iNOS activity has been linked increase in clinical sign. As evidenced in the manuscript, NAC can inhibit the production of inflammatory cytokines and nitrotyrosine in the CNS during EAE pathogenesis. Thus, NAC holds out to be a promising therapeutic agent for the amelioration of MS/EAE.