Proton Magnetic Resonance Spectroscopy in Chronic HE

In vivo localized proton magnetic resonance spectroscopy (¹H MRS) is complementary to MRI and is a powerful technique to investigate brain metabolism of rodents and humans non-invasively and in a longitudinal manner.^{67, 68} It provides a spectrum as a readout, consisting of peaks at different resonant frequencies. In single voxel MRS, spectra are acquired from a well-defined volume, positioned in a specific brain region, using a combination of band-selective radiofrequency pulses and magnetic field gradients.^{33, 69 1}H MRS is one of the most sensitive techniques, and nearly all brain metabolites contain hydrogen nuclei. An important number of biologically relevant metabolites can be observed and quantified in vivo within minutes. This technique can detect low molecular weight metabolites at concentrations as low as 0.5 mM.

Reliable quantification of the concentration of known metabolites and the extension of the number of quantifiable metabolites represent the main goal of in vivo ¹H MRS.^{70, 71, 72, 73, 74} Accurate and precise quantification of brain metabolites is challenging and depends on hardware performance, pulse sequence design and adjustment, data processing, and quantification strategies. The choice of data processing software is very important, since many algorithms depend on user input, which might lead to inaccuracies. Moreover, published recommendations encourage the usage of quantification algorithms where metabolite concentrations are determined by fitting the measured in vivo ¹H MRS spectrum to a linear combination of spectra of individual metabolites (the metabolite basis set).⁶⁷ In clinical settings, metabolite concentration ratios are often used (mainly ratios to total creatine [tCr]); however, absolute metabolite concentrations are more valuable especially when tCr might change.

¹H MRS was among the first techniques which provided indications of the presence of low-grade cerebral edema in chronic HE by reporting changes in brain organic osmolytes (an increase in glutamine [Gln] concentration, together with a decrease in myo-inositol [mIns] that partially compensates for increased intracellular osmotic pressure).^{30, 75} The glial localization of these osmolytes suggests a disturbance of astrocyte volume homeostasis.^{30, 75, 76} However, the information provided by ¹H MRS is an indirect evidence of astrocyte swelling.

A detailed description of the main findings using ¹H MRS in chronic HE in human patients can be found in Table 3. In clinical settings, the MRS acquisitions were performed at magnetic fields of 1.5T-3T and echo times (TEs) ≥20 ms, leading to the quantification of few metabolites (e.g. the sum of glutamine and glutamate [Glx], tCr [sometimes also simply called creatine {Cr}], total choline [tCho] and myo-inositol or inositol [mIns or Ins]). It is interesting to note that the stronger changes in brain metabolites (Glx/Cr, mIns/Cr, and tCho/Cr) were observed in overt HE, while in minimal HE, the decrease in mIns/Cr was observed more often than an increase in Glx/Cr. Finally, in functionally well-compensated liver cirrhosis, no significant changes were measured. This raises the question as to whether few metabolite changes occur in well-compensated liver disease patients, or if these changes are very small, and thus they are not detected at lower magnetic fields. Therefore, nowadays the availability of high magnetic fields (≥7T), together with MRS acquisitions at shorter TEs (<10–20 ms) might offer opportunities to better quantify and understand brain metabolites changes in chronic HE. Using this methodology, both in animal models and humans, about 19 brain metabolites can be quantified in the brain: glutamate, Gln, aspartate, γ-aminobutyrate, and glycine (neurotransmitters and associated metabolites); glucose, lactate (Lac), Cr, phosphocreatine, and alanine (markers of energy metabolism); taurine and mIns (markers of osmoregulation); phosphocholine, glycerophosphocholine, phosphoethanolamine, N-acetylaspartate, and N-acetylaspartylglutamate (markers of myelination/cell proliferation); and ascorbate and glutathione (antioxidants).^{67, 70, 71, 73, 77, 78} Table 3 also presents some interesting correlations between MRS changes and other MRI or blood parameters. In addition, some brain regional differences were observed in brain metabolites, but this observation requires further validation.

To date, brain water mapping³⁴ is the direct method for absolute quantification of water content in vivo in humans. In animal models, a multimodal approach is desired combining in vivo and longitudinal measurements with an ex vivo technique assessing the absolute brain water content. This combination provides additional information on the temporal resolution of the onset of brain edema by monitoring the progression of the syndrome longitudinally. None of these techniques provides information on the type of the edema or which cell is involved. Therefore, using parallel electron microscopy or a similar technique would be very useful in animal models.