Capillary Electrophoresis-Laser Induced Fluorescence Method Development and Validation for Quantification of Nine Gangliosides-Application to Analysis of Cell Lines of CNS Origin.

Gangliosides are sialic acid-containing glycosphingolipids that play an essential role in many biological and pathophysiological processes. They are present in high amounts in the central nervous system and their abnormal metabolism or expression has been observed in many diseases. We have developed and validated a sensitive capillary electrophoresis laser-induced fluorescence (CE-LIF) method for the separation and quantification of oligosaccharides digested from nine gangliosides of high biological relevance. APTS was used for the labeling of the glycans. Reverse polarity CE was performed for the separation of the labeled glycans bearing negative charges. The optimized background electrolyte is a 15 mM lithium acetate buffer with pH of 5 containing 5% w/v linear polyacrylamide, which allows for the separation of all nine gangliosides. Validation parameters including linearity, precision, and accuracy were evaluated. LOQ and LOD were in the nM range, comparable to those of LC-MS techniques. The method was used to identify and quantify the ganglioside pattern of glioblastoma and neuroblastoma cell lines. The presented method is a valuable tool for further investigations aiming at understanding the role of gangliosides in various neurological diseases or CNS tumors.

Differential Ganglioside and Cholesterol Depletion by Various Cyclodextrin Derivatives and Their Effect on Synaptosomal Glutamate Release.

Gangliosides are glycosphingolipids of the plasma membrane and are highly enriched in the nervous system where they play a vital role in normal cell functions. Furthermore, several studies suggest their potential involvement in the pathogenesis of neurological conditions. Since cyclodextrins (CDs) can form inclusion complexes with various lipids, methylated beta-CDs are widely used in biomedical research to extract cholesterol from the membrane and study its cellular role. Despite CDs being known to interact with other membrane lipid components, their effect on gangliosides is poorly characterized. The aim of this research was to investigate the effect of dimethyl-beta-cyclodextrin (DIMEB), hydroxypropyl-beta-cyclodextrin (HPBCD), randomly methylated-alpha-cyclodextrin (RAMEA), and hydroxypropyl-alpha-cyclodextrin (HPACD) on ganglioside and cholesterol levels in rat brain synaptosomes. Their effect on membrane integrity and viability was also assessed. We examined the role of lipid depletion by CDs on the release of the major excitatory neurotransmitter, glutamate. Selective concentration range for cholesterol depletion was only found with HPBCD, but not with DIMEB. Selective depletion of gangliosides was achieved by both RAMEA and HPACD. The inhibition of stimulated glutamate release upon ganglioside depletion was found, suggesting their potential role in neurotransmission. Our study highlights the importance of the characterization of the lipid depleting capability of different CDs.

The Acute Antiallodynic Effect of Tolperisone in Rat Neuropathic Pain and Evaluation of Its Mechanism of Action.

Current treatment approaches to manage neuropathic pain have a slow onset and their use is largely hampered by side-effects, thus there is a significant need for finding new medications. Tolperisone, a centrally acting muscle relaxant with a favorable side effect profile, has been reported to affect ion channels, which are targets for current first-line medications in neuropathic pain. Our aim was to explore its antinociceptive potency in rats developing neuropathic pain evoked by partial sciatic nerve ligation and the mechanisms involved. Acute oral tolperisone restores both the decreased paw pressure threshold and the elevated glutamate level in cerebrospinal fluid in neuropathic rats. These effects were comparable to those of pregabalin, a first-line medication in neuropathy. Tolperisone also inhibits release of glutamate from rat brain synaptosomes primarily by blockade of voltage-dependent sodium channels, although inhibition of calcium channels may also be involved at higher concentrations. However, pregabalin fails to affect glutamate release under our present conditions, indicating a different mechanism of action. These results lay the foundation of the avenue for repurposing tolperisone as an analgesic drug to relieve neuropathic pain.

Pregabalin-Tolperisone Combination to Treat Neuropathic Pain: Improved Analgesia and Reduced Side Effects in Rats.

The current treatment of neuropathic pain (NP) is unsatisfactory; therefore, effective novel agents or combination-based analgesic therapies are needed. Herein, oral tolperisone, pregabalin, and duloxetine were tested for their antinociceptive effect against rat partial sciatic nerve ligation (pSNL)-induced tactile allodynia described by a decrease in the paw withdrawal threshold (PWT) measured by a dynamic plantar aesthesiometer. On day 7 after the operation, PWTs were assessed at 60, 120, and 180 min post-treatment. Chronic treatment was continued for 2 weeks, and again, PWTs were measured on day 14 and 21. None of the test compounds produced an acute antiallodynic effect. In contrast, after chronic treatment, tolperisone and pregabalin alleviated allodynia. In other experiments, on day 14, the acute antiallodynic effect of the tolperisone/pregabalin or duloxetine combination was measured. As a novel finding, a single dose of the tolperisone/pregabalin combination could remarkably alleviate allodynia acutely. It also restored the neuropathy-induced elevated CSF glutamate content. Furthermore, the combination is devoid of adverse effects related to motor and gastrointestinal transit functions. Tolperisone and pregabalin target voltage-gated sodium and calcium channels, respectively. The dual blockade effect of the combination might explain its advantageous acute analgesic effect in the present work.

Development and validation of capillary electrophoresis method for quantification of gangliosides in brain synaptosomes.

Gangliosides are sialic acid containing glycosphingolipids of the plasma membrane with diverse biological functions. They are most abundant in neural tissues where their dysregulation has been suggested to be involved in various pathological conditions. Due to their importance, efficient analytical methods are needed to determine individual gangliosides in biological samples. Here we report a capillary electrophoresis method, optimized and validated for the simultaneous quantification of major neural gangliosides GM1, GD1a, GD1b, GT1b and GQ1b in their underivatized form. The most abundant extraneural monosialogangloside, GM3 can also be separated by this method. Micelles of the highly amphiphilic gangliosides were disrupted with cyclodextrins (CyDs) in the aqueous separation buffer. Among the tested CyDs, the best resolution was observed using 20 mM randomly methylated alpha-CyD in alkaline sodium borate buffer enabling the separation of all studied gangliosides. The method was applied for the quantification of gangliosides in rat cerebral and cerebellar synaptosomes.