Metabolomics of cerebrospinal fluid reveals change

Authors: Marek J Noga Adrie Dane Shanna Shi Amos Attali Hans van Aken Ernst Suidgeest Tinka Tuinstra Bas Muilwijk Leon Coulier Theo Luider Theo H Reijmers Rob J Vreeken Thomas Hankemeier

Publish Date: 2011/04/16

Volume: 8, Issue: 2, Pages: 253-263

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Abstract

Experimental Autoimmune Encephalomyelitis EAE is the most commonly used animal model for Multiple Sclerosis MScl CSF metabolomics in an acute EAE rat model was investigated using targetted LC–MS and GC–MS Acute EAE in Lewis rats was induced by coinjection of Myelin Basic Protein with Complete Freund’s Adjuvant CSF samples were collected at two time points 10 days after inoculation which was during the onset of the disease and 14 days after inoculation which was during the peak of the disease The obtained metabolite profiles from the two time points of EAE development show profound differences between onset and the peak of the disease suggesting significant changes in CNS metabolism over the course of MBPinduced neuroinflammation Around the onset of EAE the metabolome profile shows significant decreases in arginine alanine and branched amino acid levels relative to controls At the peak of the disease significant increases in concentrations of multiple metabolites are observed including glutamine Ophosphoethanolamine branchedchain amino acids and putrescine Observed changes in metabolite levels suggest profound changes in CNS metabolism over the course of EAE Affected pathways include nitric oxide synthesis altered energy metabolism polyamine synthesis and levels of endogenous antioxidantsMultiple sclerosis MScl is a chronic demyelinating neurodegenerative central nervous system CNS disorder of autoimmune origin affecting over 1 million people worldwide Development of the disease includes emergence of inflammatory lesions in white matter and dysfunction of neural conductivity This leads to impairment of sensory and motor functions Molecular processes associated with the onset and progression of MScl and its etiology are still unknownBecause of obvious ethical and safety constraints access to human brain and spinal cord system samples is very limited Therefore indepth investigation of molecular mechanisms of disorders within CNS is hampered Biochemistry of CNS activity of affected patients can be monitored only indirectly using cerebrospinal fluid CSF for diagnostic purposes Also explorative studies of molecular mechanisms involved in the onset and progression of neurological diseases except for brain tumors are limited to CSF samples The metabolites present in the CSF represent actual metabolism of CNS and the balance between blood and CSF Its analysis can be helpful in the determination of markers for neurological disorders However sampling human CSF is an invasive procedure hence access to such samples is much more limited compared to blood or urine In order to partially avoid these limitations animal models of neurological diseases have been introduced An additional major advantage of animal models is less variation between individual animals compared to variation between individuals in clinical cohorts Therefore the discovery of dominating factors of the onset and progression of the disease is more straightforward On the other hand biomarkers discovered by means of animal models may not be relevant for the human situation and such results need to be validated and confirmed using clinical dataThe most commonly used animal model of MScl is the experimental autoimmune encephalomyelitis EAE The course of EAE resembles the symptoms of MScl and can be used to study some aspects of MScl interconnected with neuroinflammation and neurodegeneration In this model disease is induced by inoculating animal with myelinspecific antigens This paper describes a study investigating changes in metabolites caused by acute EAE an autoimmune reaction was provoked using MBP coinjected with an adjuvant containing Mycobacterium tuberculosis proteins In this model gradual and reversible impairment of motor function is observed however no clear myelin loss is observedMetabolomics the comprehensive analysis of a wide range of metabolites provides a novel perspective for the search of new disease biomarkers and drug targets being an alternative and complementary approach to more established omics techniques such as genomics transcriptomics or proteomics Recent progress in metabolomics resulted in an increasing number of metabolomics applications in neurological research In a recent review Wishart et al 2008 summarized the current status of CSF metabolomics reporting 308 metabolites together with their concentrations in CSF Recent articles investigating CSF metabolome focused on untargeted approaches Crews et al 2009 Myint et al 2009 CarrascoPancorbo et al 2009 Wuolikainen et al 2009 detecting high number of features In this article we used a related approach emphasizing targeted metabolomics We applied two platforms allowing the fully validated analysis of 39 identified metabolites by LC–MS and 64 identified metabolites by GC–MS with an overlap of 18 metabolites between both methodsMale Lewis rats Harlan Laboratories BV the Netherlands kept under normal housing conditions with water and food ad libitum weighing between 175 and 225 g at the start of the experiment were inoculated on day 0 as previously described Hendriks et al 2004 Briefly a 100 μl saline based emulsion containing 50 μl Complete Freund’s Adjuvant H37 RA CFA Difco Laboratories Detroit MI 500 μl Mycobacterium tuberculosis type H37RA Difco and 20 μg guinea pig myelin basic protein MBP was injected subcutaneously in the pad of left hind paw of Isoflurane anaesthetized animals Next to these MBP challenged rats referred to as the EAE group two control groups were included a group of animals receiving the same emulsion without MBP CFA group and a healthy group undergoing anesthesia only Healthy group H Each group consisted of 30 animals Of each group half of the animals were sacrificed to collect plasma and CSF on day 10 day of onset of disease in EAE group resulting in groups further referred as H10 CFA10 and EAE10 and the other half on day 14 peak of disease in EAE group—groups H14 CFA14 and EAE14

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