Age-Related 1H NMR Characterization of Cerebrospinal Fluid in Newborn and Young Healthy Piglets

Abstract

When it comes to neuroscience, pigs represent an important animal model due to their resemblance with humans' brains for several patterns including anatomy and developmental stages. Cerebrospinal fluid (CSF) is a relatively easy-to-collect specimen that can provide important information about neurological health and function, proving its importance as both a diagnostic and biomedical monitoring tool. Consequently, it would be of high scientific interest and value to obtain more standard physiological information regarding its composition and dynamics for both swine pathology and the refinement of experimental protocols. Recently, proton nuclear magnetic resonance (1H NMR) spectroscopy has been applied in order to analyze the metabolomic profile of this biological fluid, and results showed the technique to be highly reproducible and reliable. The aim of the present study was to investigate in both qualitative and quantitative manner the composition of Cerebrospinal Fluid harvested form healthy newborn (5 days old-P5) and young (30-P30 and 50-P50 days old) piglets using 1H NMR Spectroscopy, and to analyze any possible difference in metabolites concentration between age groups, related to age and Blood-Brain-Barrier maturation. On each of the analyzed samples, 30 molecules could be observed above their limit of quantification, accounting for 95-98% of the total area of the spectra. The concentrations of adenine, tyrosine, leucine, valine, 3-hydroxyvalerate, 3-methyl-2-oxovalerate were found to decrease between P05 and P50, while the concentrations of glutamine, creatinine, methanol, trimethylamine and myo-inositol were found to increase. The P05-P30 comparison was also significant for glutamine, creatinine, adenine, tyrosine, leucine, valine, 3-hydroxyisovalerate, 3-methyl-2-oxovalerate, while for the P30-P50 comparison we found significant differences for glutamine, myo-inositol, leucine and trimethylamine. None of these molecules showed at P30 concentrations outside the P05 -P50 range.

Fig 1. A 1D-NMR spectrum of CSF from a 30d pig, representative of all the registered spectra.

Fig 2. Scoreplot of a PCA model built on the concentrations of 29 molecules listed in Table 1.

For each group, segments are drawn from each sample position to the median of the group. “Expl.Var.” stands for explained variance of the original data.

Fig 3

Scoreplot (A) and correlation plot (B) of one of the PLS-DA models built. The base of data was represented by the centered and scaled concentrations of the 11 molecules showing statistically significant differences between P05 and P50. The inner and outer circles of the correlation plots represent correlations of 0.5 and 1 respectively. P05, P30 and P50 samples are represented with filled circles, empty squares and empty tringles respectively.