Neurometabolic changes in a rat pup model of type C hepatic encephalopathy depend on age at liver disease onset.

Chronic liver disease (CLD) is a serious condition where various toxins present in the blood affect the brain leading to type C hepatic encephalopathy (HE). Both adults and children are impacted, while children may display unique vulnerabilities depending on the affected window of brain development.We aimed to use the advantages of high field proton Magnetic Resonance Spectroscopy ( <sup>1</sup> H MRS) to study longitudinally the neurometabolic and behavioural effects of Bile Duct Ligation (animal model of CLD-induced type C HE) on rats at post-natal day 15 (p15) to get closer to neonatal onset liver disease. Furthermore, we compared two sets of animals (p15 and p21-previously published) to evaluate whether the brain responds differently to CLD according to age onset.We showed for the first time that when CLD was acquired at p15, the rats presented the typical signs of CLD, i.e. rise in plasma bilirubin and ammonium, and developed the characteristic brain metabolic changes associated with type C HE (e.g. glutamine increase and osmolytes decrease). When compared to rats that acquired CLD at p21, p15 rats did not show any significant difference in plasma biochemistry, but displayed a delayed increase in brain glutamine and decrease in total-choline. The changes in neurotransmitters were milder than in p21 rats. Moreover, p15 rats showed an earlier increase in brain lactate and a different antioxidant response. These findings offer tentative pointers as to which neurodevelopmental processes may be impacted and raise the question of whether similar changes might exist in humans but are missed owing to <sup>1</sup> H MRS methodological limitations in field strength of clinical magnet

A new rat model of creatine transporter deficiency reveals behavioral disorder and altered brain metabolism.

Creatine is an organic compound used as fast phosphate energy buffer to recycle ATP, important in tissues with high energy demand such as muscle or brain. Creatine is taken from the diet or endogenously synthetized by the enzymes AGAT and GAMT, and specifically taken up by the transporter SLC6A8. Deficit in the endogenous synthesis or in the transport leads to Cerebral Creatine Deficiency Syndromes (CCDS). CCDS are characterized by brain creatine deficiency, intellectual disability with severe speech delay, behavioral troubles such as attention deficits and/or autistic features, and epilepsy. Among CCDS, the X-linked creatine transporter deficiency (CTD) is the most prevalent with no efficient treatment so far. Different mouse models of CTD were generated by doing long deletions in the Slc6a8 gene showing reduced brain creatine and cognitive deficiencies or impaired motor function. We present a new knock-in (KI) rat model of CTD holding an identical point mutation found in patients with reported lack of transporter activity. KI males showed brain creatine deficiency, increased urinary creatine/creatinine ratio, cognitive deficits and autistic-like traits. The Slc6a8 <sup>Y389C</sup> KI rat fairly enriches the spectrum of CTD models and provides new data about the pathology, being the first animal model of CTD carrying a point mutation

MP-PCA denoising for diffusion MRS data: promises and pitfalls.

Diffusion-weighted (DW) magnetic resonance spectroscopy (MRS) suffers from a lower signal to noise ratio (SNR) compared to conventional MRS owing to the addition of diffusion attenuation. This technique can therefore strongly benefit from noise reduction strategies. In the present work, Marchenko-Pastur principal component analysis (MP-PCA) denoising is tested on Monte Carlo simulations and on in vivo DW-MRS data acquired at 9.4T in rat brain and at 3T in human brain. We provide a descriptive study of the effects observed following different MP-PCA denoising strategies (denoising the entire matrix versus using a sliding window), in terms of apparent SNR, rank selection, noise correlation within and across b-values and quantification of metabolite concentrations and fitted diffusion coefficients. MP-PCA denoising yielded an increased apparent SNR, a more accurate B <sub>0</sub> drift correction between shots, and similar estimates of metabolite concentrations and diffusivities compared to the raw data. No spectral residuals on individual shots were observed but correlations in the noise level across shells were introduced, an effect which was mitigated using a sliding window, but which should be carefully considered

Central nervous system and systemic oxidative stress interplay with inflammation in a bile duct ligation rat model of type C hepatic encephalopathy.

The role and coexistence of oxidative stress (OS) and inflammation in type C hepatic encephalopathy (C HE) is a subject of intense debate. Under normal conditions the physiological levels of intracellular reactive oxygen species are controlled by the counteracting antioxidant response to maintain redox homeostasis. Our previous in-vivo <sup>1</sup> H-MRS studies revealed the longitudinal impairment of the antioxidant system (ascorbate) in a bile-duct ligation (BDL) rat model of type C HE. Therefore, the aim of this work was to examine the course of central nervous system (CNS) OS and systemic OS, as well as to check for their co-existence with inflammation in the BDL rat model of type C HE. To this end, we implemented a multidisciplinary approach, including ex-vivo and in-vitro electron paramagnetic resonance spectroscopy (EPR) spin-trapping, which was combined with UV-Vis spectroscopy, and histological assessments. We hypothesized that OS and inflammation act synergistically in the pathophysiology of type C HE. Our findings point to an increased CNS- and systemic-OS and inflammation over the course of type C HE progression. In particular, an increase in the CNS OS was observed as early as 2-weeks post-BDL, while the systemic OS became significant at week 6 post-BDL. The CNS EPR measurements were further validated by a substantial accumulation of 8-Oxo-2'-deoxyguanosine (Oxo-8-dG), a marker of oxidative DNA/RNA modifications on immunohistochemistry (IHC). Using IHC, we also detected increased synthesis of antioxidants, glutathione peroxidase 1 (GPX-1) and superoxide dismutases (i.e.Cu/ZnSOD (SOD1) and MnSOD (SOD2)), along with proinflammatory cytokine interleukin-6 (IL-6) in the brains of BDL rats. The presence of systemic inflammation was observed already at 2-weeks post-surgery. Thus, these results suggest that CNS OS is an early event in type C HE rat model, which seems to precede systemic OS. Finally, our results suggest that the increase in CNS OS is due to enhanced formation of intra- and extra-cellular ROS rather than due to reduced antioxidant capacity, and that OS in parallel with inflammation plays a significant role in type C HE

Late post-natal neurometabolic development in healthy male rats using ¹ H and ³¹ P magnetic resonance spectroscopy.

Brain metabolism evolves rapidly during early post-natal development in the rat. While changes in amino acids, energy metabolites, antioxidants or metabolites involved in phospholipid metabolism have been reported in the early stages, neurometabolic changes during the later post-natal period are less well characterized. Therefore, we aimed to assess the neurometabolic changes in male Wistar rats between post-natal days 29 and 77 (p29-p77) using longitudinal magnetic resonance spectroscopy (MRS) in vivo at 9.4 Tesla. <sup>1</sup> H MRS was performed in the hippocampus between p29 and p77 at 1-week intervals (n = 7) and in the cerebellum between p35 and p77 at 2-week intervals (n = 7) using the SPECIAL sequence at ultra-short echo-time. NOE enhanced and <sup>1</sup> H decoupled <sup>31</sup> P MR spectra were acquired at p35, p48 and p63 (n = 7) in a larger voxel covering cortex, hippocampus and part of the striatum. The hippocampus showed a decrease in taurine concentration and an increase in glutamate (with more pronounced changes until p49), seemingly a continuation of their well-described changes in the early post-natal period. A constant increase in myo-inositol and choline-containing compounds in the hippocampus (in particular glycero-phosphocholine as shown by <sup>31</sup> P MRS) was measured throughout the observation period, probably related to membrane metabolism and myelination. The cerebellum showed only a significant increase in myo-inositol between p35 and p77. In conclusion, this study showed important changes in brain metabolites in both the hippocampus and cerebellum in the later post-natal period (p29/p35-p77) of male rats, something previously unreported. Based on these novel data, changes in some neurometabolites beyond p28-35, conventionally accepted as the cut off for adulthood, should be taken into account in both experimental design and data interpretation in this animal model

In vivo macromolecule signals in rat brain 1 H-MR spectra at 9.4T: Parametrization, spline baseline estimation, and T 2 relaxation times

Purpose: Reliable detection and fitting of macromolecules (MM) are crucial for accurate quantification of brain short-echo time (TE) 1 H-MR spectra. An experimentally acquired single MM spectrum is commonly used. Higher spectral resolution at ultra-high field (UHF) led to increased interest in using a parametrized MM spectrum together with flexible spline baselines to address unpredicted spectroscopic components. Herein, we aimed to: (1) implement an advanced methodological approach for post-processing, fitting, and parametrization of 9.4T rat brain MM spectra; (2) assess the concomitant impact of the LCModel baseline and MM model (ie, single vs parametrized); and (3) estimate the apparent T2 relaxation times for seven MM components. Methods: A single inversion recovery sequence combined with advanced AMARES prior knowledge was used to eliminate the metabolite residuals, fit, and parametrize 10 MM components directly from 9.4T rat brain in vivo 1 H-MR spectra at different TEs. Monte Carlo simulations were also used to assess the concomitant influence of parametrized MM and DKNTMN parameter in LCModel. Results: A very stiff baseline (DKNTMN ≥ 1 ppm) in combination with a single MM spectrum led to deviations in metabolite concentrations. For some metabolites the parametrized MM showed deviations from the ground truth for all DKNTMN values. Adding prior knowledge on parametrized MM improved MM and metabolite quantification. The apparent T2 ranged between 12 and 24 ms for seven MM peaks. Conclusion: Moderate flexibility in the spline baseline was required for reliable quantification of real/experimental spectra based on in vivo and Monte Carlo data. Prior knowledge on parametrized MM improved MM and metabolite quantification

PET CMR_glc mapping and ¹H-MRS show altered glucose uptake and neurometabolic profiles in BDL rats.

Type C hepatic encephalopathy (HE) is a complex neuropsychiatric disorder occurring as a consequence of chronic liver disease. Alterations in energy metabolism have been suggested in type C HE, but in vivo studies on this matter remain sparse and have reported conflicting results. Here, we propose a novel preclinical <sup>18</sup> F-FDG PET methodology to compute quantitative 3D maps of the regional cerebral metabolic rate of glucose (CMR <sub>glc</sub> ) from a labelling steady-state PET image of the brain and an image-derived input function. This quantitative approach shows its strength when comparing groups of animals with divergent physiology, such as HE animals. PET CMR <sub>glc</sub> maps were registered to an atlas and the mean CMR <sub>glc</sub> from the hippocampus and the cerebellum were associated to the corresponding localized <sup>1</sup> H MR spectroscopy acquisitions. This study provides for the first time local and quantitative information on both brain glucose uptake and neurometabolic profile alterations in a rat model of type C HE. A 2-fold lower brain glucose uptake, concomitant with an increase in brain glutamine and a decrease in the main osmolytes, was observed in the hippocampus and in the cerebellum. These novel findings are an important step towards new insights into energy metabolism in the pathophysiology of HE

Si/SiGe superlattice I/O finFETs in a vertically-stacked Gate-All-Around horizontal Nanowire Technology

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