One-time intrathecal triamcinolone acetonide application alters the redox potential in cerebrospinal fluid of progressive multiple sclerosis patients: a pilot study

Introduction

Multiple sclerosis (MS), as a chronic inflammatory disease of the central nervous system, is characterized by glial lesions that are monitored by MRI. Most patients finally suffer from a smouldering, chronic inflammatory, progressive MS process in the long term. Concomitantly, an upregulated free-radical synthesis may occur in the central nervous system [Ljubisavljevic et al. 2013; Mitosek-Szewczyk et al. 2010] that may be influenced by steroids [Seven et al. 2013]. Free radicals play a role in a variety of normal regulatory systems. Their dysregulation is an important component of chronic inflammation. Free radicals have the capacity to mediate tissue destruction, either alone, or in concert with proteases [Ljubisavljevic et al. 2013; Mitosek-Szewczyk et al. 2010]. Disturbances in free-radical-regulated second-messenger systems may contribute to the inflammatory process [Golde et al. 2003; Sellebjerg et al. 2002]. Cells and tissues have an abundance of antioxidant systems to scavenge or otherwise eliminate reactive oxygen species (ROS). Under normal circumstances, there is a well-managed balance between formation and neutralization of ROS [Aiken et al. 2011]. A potent mode of direct oxidative attack on a protein derives from site-specific metal-catalysed oxidation, that is, via protein-bound transition metals like copper (Cu (II)). These processes generate active oxidative stress-related intermediates, such as superoxide anion, hydrogen peroxide or hydroxyl radicals. They oxidise, for instance, the chelating amino acids in proteins [Aiken et al. 2011; Mitosek-Szewczyk et al. 2010]. In the end, various oxidative mechanisms may modify the structure of proteins and cause loss of function, like in the case of oxidation of the plasma protein, fibrinogen, by treatment with an iron or ascorbate radical-generating system with its component C. This water-soluble vitamin reacts with several radical species, producing semidehydroascorbic acid or ascorbyl radicals. Here, the enzyme NADH-semidehydroascorbate reductase reduces ascorbyl radicals back to ascorbate, whilst oxidizing glutathione to its dimer, GSSG [Spasojevic et al. 2010]. In a healthy population, ascorbyl radicals were not found in cerebrospinal fluid (CSF), whereas they were elevated in amyotrophic lateral sclerosis, probably as a feature of the chronic neurodegenerative process [Spasojevic et al. 2010]. Generally, cells also possess oxidation–reduction–dependent repair pathways, which are triggered by oxidation of redox proteins. If cellular defending systems and repair processes fail, oxidatively damaged proteins can undergo direct chemical fragmentation or form large aggregates that may accumulate and disrupt important cellular processes. Therefore, timely removal of these lesioned proteins is important for maintenance of cellular homeostasis and viability [Aiken et al. 2011]. Failure of homeostasis ultimately causes apoptotic or necrotic cell death, both of which represent features of chronic progression in MS. From this point of view, it is interesting that intravenous methylprednisolone application beneficially altered substrates of upregulated free-radical peroxidation processes related to the pathophysiology of MS relapses [Mooradian, 1993]. Therefore, investigations of CSF and proteins that mediate the antioxidative potential in MS patients may provide some insight into the interplay between disease progression, chronic inflammation and response to treatment, such as intrathecal retarded-release steroid applications with triamcinolone (TCA) [Ljubisavljevic et al. 2013; Mitosek-Szewczyk et al. 2010; Müller, 2009; Sloka and Stefanelli, 2005]. The objective was to demonstrate the effect of one intrathecal TCA injection on the redox potential in the cerebrospinal fluid of chronic progressive MS patients in this observational pilot study.

Methods

Results

The second CSF assessment following the initial TCA administration showed an increase of Cu (II) ion absorption, which reflects an elevated content of reduced proteins in the cerebrospinal fluid (p < 0.0005) (Figure 1). At the second determination, there was no significant decline of the ascorbyl radical levels (Figure 2). There were no relevant changes of protein content, cells and other concomitantly assessed routine parameters in CSF. The patients did not significantly improve after this one TCA application only (results not shown).

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Figure 1.

Increase of Cu (II) ion absorption determined before the first triamcinolone (TCA) administration and at the moment of the second cerebrospinal fluid sampling before the second TCA application.

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Figure 2.

Ascorbyl radical levels before and after triamcinolone (TCA) application determined before the first TCA administration and at the moment of the second cerebrospinal fluid sampling before the second TCA application.

Discussion

Generally, intrathecal application of retarded-release steroids is under debate; not only in MS but also in other indications, that is, post-herpetic neuralgia or lumbar disk herniation [Kneissel, 1976; Kotani et al. 2000; Langmayr et al. 1995; Marinangeli et al. 2002]. To date, trials were mostly only observational and not placebo controlled due to ethical concerns. A certain spasticity-reducing and MS-process-improving effect is well known due to the observed, mean threefold increase of walking distance in progressive MS patients [Müller, 2009].

Our experimental results suggest a potential anti-inflammatory effect due to a decreased free radical synthesis. Thus our findings support the beneficial role of steroids on oxidative stress, which is a feature of the acute and chronic smouldering inflammation in MS [Melcangi et al. 2000; Sellebjerg et al. 2002]. Decline of ROS is the prerequisite for the observed upregulated production of reduced proteins [Ljubisavljevic et al. 2013; Mitosek-Szewczyk et al. 2010]. Generally, it is known that free radicals play an important role in acute and chronic inflammation of the central nervous system in MS [Ljubisavljevic et al. 2013; Mitosek-Szewczyk et al. 2010]. The observed levels of ascorbyl radical concentrations in CSF seem to confirm this, since these radicals were not detectable in control CSF [Spasojevic et al. 2010]. However, this comparison with data from the literature must be viewed critically. There might be differences in terms of conditions of lumbar puncture and techniques of CSF analysis, etc. Our current outcomes do not allow any conclusion on the induction of putative regenerative mechanisms, which would be in line with the observed clinical benefit after repeat TCA therapy over an interval of at least 1 year [Müller, 2009]. But one should consider that oxidation–reduction–dependent repair pathways triggered by oxidation of redox proteins play an important role in proteasome metabolism, which is closely related to the improvement of cell viability, cell recovery, as well as the attenuation of oxidation-induced cytotoxicity associated with neuronal death in chronic neurological disorders [Aiken et al. 2011; Shacter, 2000]. Hypothetically, our findings suggest that an initial TCA application may modify via a free-radical-mediated signal-transduction pathway the function and regulation of regenerative proteins and growth factors. There are hints that TCA application may induce fall of soluble repulsive guidance molecule a (RGMa) fragments. This decline generally precedes neuronal recovery. One may even hypothesize that changes of free radical metabolism may trigger the observed descent of RGMa concentrations in patients with an enhancement of MS symptoms during TCA treatment [Müller et al. 2014, 2015]. However, this hypothesis warrants further investigation in combination with repeat clinical evaluation of MS patients, and assessment of further metabolic CSF substrates that are responsible for regenerative processes according to a similar signal-transduction pathway as in the case of fibrinogen generation [Aiken et al. 2011].

This observational pilot study has limitations. We did not perform our investigations in controls or in saline, respectively placebo-treated MS patients. Moreover, it is not ethical to obtain CSF from normal healthy controls for comparison of the free radical status with the healthy condition. However, there is a need for further investigation; for instance, during acute relapse in larger cohorts of MS patients.

In conclusion, we show that intrathecal steroid application alters the redox potential in CSF.