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 (1H 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 1H MRS methodological limitations in field strength of clinical magnet.

AQP1 in the Gastrointestinal Tract of Mice: Expression Pattern and Impact of AQP1 Knockout on Colonic Function.

Aquaporin 1 (AQP1) is one of thirteen known mammalian aquaporins. Its main function is the transport of water across cell membranes. Lately, a role of AQP has been attributed to other physiological and pathological functions including cell migration and peripheral pain perception. AQP1 has been found in several parts of the enteric nervous system, e.g., in the rat ileum and in the ovine duodenum. Its function in the intestine appears to be multifaceted and is still not completely understood. The aim of the study was to analyze the distribution and localization of AQP1 in the entire intestinal tract of mice. AQP1 expression was correlated with the hypoxic expression profile of the various intestinal segments, intestinal wall thickness and edema, as well as other aspects of colon function including the ability of mice to concentrate stools and their microbiome composition. AQP1 was found in a specific pattern in the serosa, the mucosa, and the enteric nervous system throughout the gastrointestinal tract. The highest amount of AQP1 in the gastrointestinal tract was found in the small intestine. AQP1 expression correlated with the expression profiles of hypoxia-dependent proteins such as HIF-1α and PGK1. Loss of AQP1 through knockout of AQP1 in these mice led to a reduced amount of bacteroidetes and firmicutes but an increased amount of the rest of the phyla, especially deferribacteres, proteobacteria, and verrucomicrobia. Although AQP-KO mice retained gastrointestinal function, distinct changes regarding the anatomy of the intestinal wall including intestinal wall thickness and edema were observed. Loss of AQP1 might interfere with the ability of the mice to concentrate their stool and it is associated with a significantly different composition of the of the bacterial stool microbiome.

Current and potential new treatment strategies for creatine deficiency syndromes.

Creatine deficiency syndromes (CDS) are inherited metabolic disorders caused by mutations in GATM, GAMT and SLC6A8 and mainly affect central nervous system (CNS). AGAT- and GAMT-deficient patients lack the functional brain endogenous creatine (Cr) synthesis pathway but express the Cr transporter SLC6A8 at blood-brain barrier (BBB), and can thus be treated by oral supplementation of high doses of Cr. For Cr transporter deficiency (SLC6A8 deficiency or CTD), current treatment strategies benefit one-third of patients. However, as their phenotype is not completely reversed, and for the other two-thirds of CTD patients, the development of novel more effective therapies is needed. This article aims to review the current knowledge on Cr metabolism and CDS clinical aspects, highlighting their current treatment possibilities and the most recent research perspectives on CDS potential therapeutics designed, in particular, to bring new options for the treatment of CTD.

PET CMRglc 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 18F-FDG PET methodology to compute quantitative 3D maps of the regional cerebral metabolic rate of glucose (CMRglc) 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 CMRglc maps were registered to an atlas and the mean CMRglc from the hippocampus and the cerebellum were associated to the corresponding localized 1H 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.

Creatine transporter-deficient rat model shows motor dysfunction, cerebellar alterations, and muscle creatine deficiency without muscle atrophy.

Creatine (Cr) is a nitrogenous organic acid and plays roles such as fast phosphate energy buffer to replenish ATP, osmolyte, antioxidant, neuromodulator, and as a compound with anabolic and ergogenic properties in muscle. Cr is taken from the diet or endogenously synthetized by the enzymes arginine:glycine amidinotransferase and guanidinoacetate methyltransferase, and specifically taken up by the transporter SLC6A8. Loss-of-function mutations in the genes encoding for the enzymes or the transporter cause creatine deficiency syndromes (CDS). CDS are characterized by brain Cr deficiency, intellectual disability with severe speech delay, behavioral troubles, epilepsy, and motor dysfunction. Among CDS, the X-linked Cr transporter deficiency (CTD) is the most prevalent with no efficient treatment so far. Different animal models of CTD show reduced brain Cr levels, cognitive deficiencies, and together they cover other traits similar to those of patients. However, motor function was poorly explored in CTD models, and some controversies in the phenotype exist in comparison with CTD patients. Our recently described Slc6a8Y389C knock-in rat model of CTD showed mild impaired motor function, morphological alterations in cerebellum, reduced muscular mass, Cr deficiency, and increased guanidinoacetate content in muscle, although no consistent signs of muscle atrophy. Our results indicate that such motor dysfunction co-occurred with both nervous and muscle dysfunctions, suggesting that muscle strength and performance as well as neuronal connectivity might be affected by this Cr deficiency in muscle and brain.

Cinnamaldehyde as antimicrobial in cellulose-based dental appliances.

AIMS: In the context of minor orthodontic intervention using clear aligner technologies, we determined antimicrobial properties of a cellulose-based material loaded with essential oils such as cinnamaldehyde. METHODS AND RESULTS: Isothermal microcalorimetry was used to assess the growth of bacterial biofilms at the interface between the tested material and the solid growth medium. The calorimetric data were analyzed using conventional growth models (Gompertz and Richards), and inhibition at 12 and 24 h was calculated. CONCLUSIONS: The tested material showed antimicrobial properties against Staphylococcus epidermidis as well as Streptococcus mutans and Streptococcus mitis clinical isolates. The inhibition was more pronounced against S. epidermidis, for which growth rate was reduced by 70% and lag phase was extended by 12 h. For S. mutans and S. mitis, the decrease in growth rate was 20% and 10%, and the lag phase increased by 2 and 6 h, respectively. SIGNIFICANCE AND IMPACT: Clear aligners for minor teeth alignment are becoming very popular. As they must be worn for at least 22 h per day for up to 40 weeks, it is important that they remain clean and do not promote caries formation or other oral infections. Therefore, introducing material with antimicrobial properties is expected to maintain oral hygiene during the aligner therapy. Here, we demonstrate the use of cinnamaldehyde for reducing microbial growth and biofilm formation on cellulose-based dental clear aligners.

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. 1 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 1 H decoupled 31 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 31 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.

A knock-in rat model unravels acute and chronic renal toxicity in glutaric aciduria type I.

Glutaric aciduria type I (GA-I, OMIM # 231670) is an autosomal recessive inborn error of metabolism caused by deficiency of the mitochondrial enzyme glutaryl-CoA dehydrogenase (GCDH). The principal clinical manifestation in GA-I patients is striatal injury most often triggered by catabolic stress. Early diagnosis by newborn screening programs improved survival and reduced striatal damage in GA-I patients. However, the clinical phenotype is still evolving in the aging patient population. Evaluation of long-term outcome in GA-I patients recently identified glomerular filtration rate (GFR) decline with increasing age. We recently created the first knock-in rat model for GA-I harboring the mutation p.R411W (c.1231 C>T), corresponding to the most frequent GCDH human mutation p.R402W. In this study, we evaluated the effect of an acute metabolic stress in form of high lysine diet (HLD) on young Gcdhki/ki rats. We further studied the chronic effect of GCDH deficiency on kidney function in a longitudinal study on a cohort of Gcdhki/ki rats by repetitive 68Ga-EDTA positron emission tomography (PET) renography, biochemical and histological analyses. In young Gcdhki/ki rats exposed to HLD, we observed a GFR decline and biochemical signs of a tubulopathy. Histological analyses revealed lipophilic vacuoles, thinning of apical brush border membranes and increased numbers of mitochondria in proximal tubular (PT) cells. HLD also altered OXPHOS activities and proteome in kidneys of Gcdhki/ki rats. In the longitudinal cohort, we showed a progressive GFR decline in Gcdhki/ki rats starting at young adult age and a decline of renal clearance. Histopathological analyses in aged Gcdhki/ki rats revealed tubular dilatation, protein accumulation in PT cells and mononuclear infiltrations. These observations confirm that GA-I leads to acute and chronic renal damage. This raises questions on indication for follow-up on kidney function in GA-I patients and possible therapeutic interventions to avoid renal damage.

The first knock-in rat model for glutaric aciduria type I allows further insights into pathophysiology in brain and periphery.

Glutaric aciduria type I (GA-I, OMIM # 231670) is an inborn error of metabolism caused by a deficiency of glutaryl-CoA dehydrogenase (GCDH). Patients develop acute encephalopathic crises (AEC) with striatal injury most often triggered by catabolic stress. The pathophysiology of GA-I, particularly in brain, is still not fully understood. We generated the first knock-in rat model for GA-I by introduction of the mutation p.R411W, the rat sequence homologue of the most common Caucasian mutation p.R402W, into the Gcdh gene of Sprague Dawley rats by CRISPR/CAS9 technology. Homozygous Gcdhki/ki rats revealed a high excretor phenotype, but did not present any signs of AEC under normal diet (ND). Exposure to a high lysine diet (HLD, 4.7%) after weaning resulted in clinical and biochemical signs of AEC. A significant increase of plasmatic ammonium concentrations was found in Gcdhki/ki rats under HLD, accompanied by a decrease of urea concentrations and a concomitant increase of arginine excretion. This might indicate an inhibition of the urea cycle. Gcdhki/ki rats exposed to HLD showed highly diminished food intake resulting in severely decreased weight gain and moderate reduction of body mass index (BMI). This constellation suggests a loss of appetite. Under HLD, pipecolic acid increased significantly in cerebral and extra-cerebral liquids and tissues of Gcdhki/ki rats, but not in WT rats. It seems that Gcdhki/ki rats under HLD activate the pipecolate pathway for lysine degradation. Gcdhki/ki rat brains revealed depletion of free carnitine, microglial activation, astroglyosis, astrocytic death by apoptosis, increased vacuole numbers, impaired OXPHOS activities and neuronal damage. Under HLD, Gcdhki/ki rats showed imbalance of intra- and extracellular creatine concentrations and indirect signs of an intracerebral ammonium accumulation. We successfully created the first rat model for GA-I. Characterization of this Gcdhki/ki strain confirmed that it is a suitable model not only for the study of pathophysiological processes, but also for the development of new therapeutic interventions. We further brought up interesting new insights into the pathophysiology of GA-I in brain and periphery.

Effect of divalent ions on cariogenic biofilm formation.

BACKGROUND: Divalent cations are able to interact with exopolysaccharides (EPS) and thus are capable to modify the structure and composition of dental biofilm. At the moment, little is known about the adsorption of metals by cariogenic EPS; thus, the aim of the present study was to evaluate the effect of divalent ions (calcium, magnesium, and zinc) on the growth and biofilm formation of mutans streptococci and on the dissolution of hydroxyapatite as well as to investigate their binding to the bacterial EPS. RESULTS: S. mutans strains used in this study show the highest tolerance towards calcium of the ions tested. Growth parameters showed no differences to control condition for both strains up to 100 mM; revealing natural tolerance to higher concentration of calcium in the surroundings. Although excessive levels of calcium did not impair the growth parameters, it also did not have a positive effect on biofilm formation or its binding affinity to EPS. Magnesium-saturated environment proved to be counterproductive as strains were able to dissolve more Ca2+ from the tooth surface in the presence of magnesium, therefore releasing excessive amounts of Ca2+ in the environment and leading to the progression of the disease. Thus, this supports the idea of self-regulation, when more Ca2+ is released, more calcium is bound to the biofilm strengthening its structure and however, also less is left for remineralization. Zinc inhibited bacterial adhesion already at low concentrations and had a strong antibacterial effect on the strains as well as on calcium dissolution; leading to less biofilm and less EPS. Additionally, Zn2+ had almost always the lowest affinity to all EPS; thus, the unbound zinc could also still remain in the surrounding environment and keep its antimicrobial properties. CONCLUSION: It is important to maintain a stable relationship between calcium, magnesium and zinc as excessive concentrations of one can easily destroy the balance between the three in cariogenic environment and lead to progression of the disease.

Magnetic resonance spectroscopy in the rodent brain: Experts' consensus recommendations.

In vivo MRS is a non-invasive measurement technique used not only in humans, but also in animal models using high-field magnets. MRS enables the measurement of metabolite concentrations as well as metabolic rates and their modifications in healthy animals and disease models. Such data open the way to a deeper understanding of the underlying biochemistry, related disturbances and mechanisms taking place during or prior to symptoms and tissue changes. In this work, we focus on the main preclinical 1H, 31P and 13C MRS approaches to study brain metabolism in rodent models, with the aim of providing general experts' consensus recommendations (animal models, anesthesia, data acquisition protocols). An overview of the main practical differences in preclinical compared with clinical MRS studies is presented, as well as the additional biochemical information that can be obtained in animal models in terms of metabolite concentrations and metabolic flux measurements. The properties of high-field preclinical MRS and the technical limitations are also described.

Longitudinal neurometabolic changes in the hippocampus of a rat model of chronic hepatic encephalopathy.

BACKGROUND & AIMS: The sequence of events in hepatic encephalopathy (HE) remains unclear. Using the advantages of in vivo 1H-MRS (9.4T) we aimed to analyse the time-course of disease in an established model of type C HE by analysing the longitudinal changes in a large number of brain metabolites together with biochemical, histological and behavioural assessment. We hypothesized that neurometabolic changes are detectable very early, and that these early changes will offer insight into the primary events underpinning HE. METHODS: Wistar rats underwent bile-duct ligation (BDL) and were studied before BDL and at post-operative weeks 2, 4, 6 and 8 (n = 26). In vivo short echo-time 1H-MRS (9.4T) of the hippocampus was performed in a longitudinal manner, as were biochemical (plasma), histological and behavioural tests. RESULTS: Plasma ammonium increased early after BDL and remained high during the study. Brain glutamine increased (+47%) as early as 2-4 weeks post-BDL while creatine (-8%) and ascorbate (-12%) decreased. Brain glutamine and ascorbate correlated closely with rising plasma ammonium, while brain creatine correlated with brain glutamine. The increases in brain glutamine and plasma ammonium were correlated, while plasma ammonium correlated negatively with distance moved. Changes in astrocyte morphology were observed at 4 weeks. These early changes were further accentuated at 6-8 weeks post-BDL, concurrently with the known decreases in brain organic osmolytes. CONCLUSION: Using a multimodal, in vivo and longitudinal approach we have shown that neurometabolic changes are already noticeable 2 weeks after BDL. These early changes are suggestive of osmotic/oxidative stress and are likely the premise of some later changes. Early decreases in cerebral creatine and ascorbate are novel findings offering new avenues to explore neuroprotective strategies for HE treatment. LAY SUMMARY: The sequence of events in chronic hepatic encephalopathy (HE) remains unclear, therefore using the advantages of in vivo proton magnetic resonance spectroscopy at 9.4T we aimed to test the hypothesis that neurometabolic changes are detectable very early in an established model of type C HE, offering insight into the primary events underpinning HE, before advanced liver disease confounds the findings. These early, previously unreported neurometabolic changes occurred as early as 2 to 4 weeks after bile-duct ligation, namely an increase in plasma ammonium and brain glutamine, a decrease in brain creatine and ascorbate together with behavioural and astrocyte morphology changes, and continued to progress throughout the 8-week course of the disease.

Ammonium accumulation and chemokine decrease in culture media of Gcdh-/- 3D reaggregated brain cell cultures.

Glutaric Aciduria type I (GA-I) is caused by mutations in the GCDH gene. Its deficiency results in accumulation of the key metabolites glutaric acid (GA) and 3-hydroxyglutaric acid (3-OHGA) in body tissues and fluids. Present knowledge on the neuropathogenesis of GA-I suggests that GA and 3-OHGA have toxic properties on the developing brain. We analyzed morphological and biochemical features of 3D brain cell aggregates issued from Gcdh-/- mice at two different developmental stages, day-in-vitro (DIV) 8 and 14, corresponding to the neonatal period and early childhood. We also induced a metabolic stress by exposing the aggregates to 10 mM l-lysine (Lys). Significant amounts of GA and 3-OHGA were detected in Gcdh-/- aggregates and their culture media. Ammonium was significantly increased in culture media of Gcdh-/- aggregates at the early developmental stage. Concentrations of GA, 3-OHGA and ammonium increased significantly after exposure to Lys. Gcdh-/- aggregates manifested morphological alterations of all brain cell types at DIV 8 while at DIV 14 they were only visible after exposure to Lys. Several chemokine levels were significantly decreased in culture media of Gcdh-/- aggregates at DIV 14 and after exposure to Lys at DIV 8. This new in vitro model for brain damage in GA-I mimics well in vivo conditions. As seen previously in WT aggregates exposed to 3-OHGA, we confirmed a significant ammonium production by immature Gcdh-/- brain cells. We described for the first time a decrease of chemokines in Gcdh-/- culture media which might contribute to brain cell injury in GA-I.

Argininosuccinate neurotoxicity and prevention by creatine in argininosuccinate lyase deficiency: An in vitro study in rat three-dimensional organotypic brain cell cultures.

The urea cycle disorder (UCD) argininosuccinate lyase (ASL) deficiency, caused by a defective ASL enzyme, exhibits a wide range of phenotypes, from life-threatening neonatal hyperammonemia to asymptomatic patients, with only the biochemical marker argininosuccinic acid (ASA) elevated in body fluids. Remarkably, even without ever suffering from hyperammonemia, patients often develop severe cognitive impairment and seizures. The goal of this study was to understand the effect on the known toxic metabolite ASA and the assumed toxic metabolite guanidinosuccinic acid (GSA) on developing brain cells, and to evaluate the potential role of creatine (Cr) supplementation, as it was described protective for brain cells exposed to ammonia. We used an in vitro model, in which we exposed three-dimensional (3D) organotypic rat brain cell cultures in aggregates to different combinations of the metabolites of interest at two time points (representing two different developmental stages). After harvest and cryopreservation of the cell cultures, the samples were analyzed mainly by metabolite analysis, immunohistochemistry, and western blotting. ASA and GSA were found toxic for astrocytes and neurons. This toxicity could be reverted in vitro by Cr. As well, an antiapoptotic effect of ASA was revealed, which could contribute to the neurotoxicity in ASL deficiency. Further studies in human ASL deficiency will be required to understand the biochemical situation in the brain of affected patients, and to investigate the impact of high or low arginine doses on brain Cr availability. In addition, clinical trials to evaluate the beneficial effect of Cr supplementation in ASL deficiency would be valuable.

Personalized Treatment Response Assessment for Rare Childhood Tumors Using Microcalorimetry-Exemplified by Use of Carbonic Anhydrase IX and Aquaporin 1 Inhibitors.

We present a novel approach to a personalized therapeutic concept for solid tumors. We illustrate this on a rare childhood tumor for which only a generalized treatment concept exists using carbonic anhydrase IX and aquaporin 1 inhibitors. The use of microcalorimetry as a refined in vitro method for evaluation of drug susceptibility in organotypic slice culture has not previously been established. Rapid microcalorimetric drug response assessment can refine a general treatment concept when it is applied in cases in which tumors do not respond to conventional chemo-radiation treatment. For solid tumors, which do not respond to classical treatment, and especially for rare tumors without an established protocol rapid microcalorimetric drug response testing presents an elegant novel approach to test alternative therapeutic approaches. While improved treatment concepts have led to improved outcome over the past decades, the prognosis of high risk disease is still poor and rethinking of clinical trial design is necessary. A small patient population combined with the necessity to assess experimental therapies for rare solid tumors rather at the time of diagnosis than in relapsed or refractory patients provides great challenges. The possibility to rapidly compare established protocols with innovative therapeutics presents an elegant novel approach to refine and personalize treatment.

New in vitro model derived from brain-specific Mut-/- mice confirms cerebral ammonium accumulation in methylmalonic aciduria.

BACKGROUND: Methylmalonic aciduria (MMAuria) is an inborn error of metabolism leading to neurological deterioration. In this study, we used 3D organotypic brain cell cultures derived from embryos of a brain-specific Mut-/- (brain KO) mouse to investigate mechanisms leading to brain damage. We challenged our in vitro model by a catabolic stress (temperature shift). RESULTS: Typical metabolites for MMAuria as well as a massive NH4+ increase were found in the media of brain KO cultures. We investigated different pathways of intracerebral NH4+ production and found increased expression of glutaminase 2 and diminished expression of GDH1 in Mut-/- aggregates. While all brain cell types appeared affected in their morphological development in Mut-/- aggregates, the most pronounced effects were observed on astrocytes showing swollen fibers and cell bodies. Inhibited axonal elongation and delayed myelination of oligodendrocytes were also noted. Most effects were even more pronounced after 48 h at 39 °C. Microglia activation and an increased apoptosis rate suggested degeneration of Mut-/- brain cells. NH4+ accumulation might be the trigger for all observed alterations. We also found a generalized increase of chemokine concentrations in Mut-/- culture media at an early developmental stage followed by a decrease at a later stage. CONCLUSION: We proved for the first time that Mut-/- brain cells are indeed able to produce the characteristic metabolites of MMAuria. We confirmed significant NH4+ accumulation in culture media of Mut-/- aggregates, suggesting that intracellular NH4+ concentrations might even be higher, gave first clues on the mechanisms leading to NH4+ accumulation in Mut-/- brain cells, and showed the involvement of neuroinflammatory processes in the neuropathophysiology of MMAuria.

MRS studies of neuroenergetics and glutamate/glutamine exchange in rats: Extensions to hyperammonemic models.

In vivo Magnetic Resonance Spectroscopy is a useful tool to characterize brain biochemistry as well as its alteration in a large number of major central nervous system diseases. The present review will focus on the study of the glutamate-glutamine cycle, an important biochemical pathway in excitatory neurotransmission, analyzed using in vivo MRS of different accessible nuclei: 1H, 13C, 15N and 31P. The different methodological aspects of data acquisition, processing and absolute quantification of the MRS data for each nucleus will be presented, as well as the description of the mathematical modeling approach to interpret the MRS measurements in terms of biochemical kinetics. The unique advantages of MRS, especially its non-invasive nature enabling longitudinal monitoring of brain disease progression and/or effect of treatment is illustrated in the particular context of hyperammonemic disorders with a specific focus on animal models. We review the current possibilities given by in vivo MRS to investigate some of the molecular mechanisms involved in hyperammonemic disorders and to give a better understanding of the process of development of hepatic encephalopathy, a severe neuropsychiatric disorder that frequently accompanies liver disease.

Creatine in the central nervous system: From magnetic resonance spectroscopy to creatine deficiencies.

Creatine (Cr) is an important organic compound acting as intracellular high-energy phosphate shuttle and in energy storage. While located in most cells where it plays its main roles in energy metabolism and cytoprotection, Cr is highly concentrated in muscle and brain tissues, in which Cr also appears to act in osmoregulation and neurotransmission. This review discusses the basis of Cr metabolism, synthesis and transport within brain cells. The importance of Cr in brain function and the consequences of its impaired metabolism in primary and secondary Cr deficiencies are also discussed. Cr and phosphocreatine (PCr) in living systems can be well characterized using in vivo magnetic resonance spectroscopy (MRS). This review describes how 1H MRS allows the measurement of Cr and PCr, and how 31P MRS makes it possible to estimate the creatine kinase (CK) rate constant and so detect dynamic changes in the Cr/PCr/CK system. Absolute quantification by MRS using creatine as internal reference is also debated. The use of in vivo MRS to study brain Cr in a non-invasive way is presented, as well as its use in clinical and preclinical studies, including diagnosis and treatment follow-up in patients.

Determinants of biofilm formation and cleanability of titanium surfaces.

OBJECTIVE: The aim of the present study was to analyze biofilm formation on four different titanium-based surfaces (machined titanium zirconium (TiZr) alloy, M; machined, acid-etched TiZr alloy, modMA; machined, sandblasted, acid-etched TiZr alloy, modSLA; and micro-grooved titanium aluminum vanadium alloy, TAV MG) in an experimental human model. MATERIAL AND METHODS: Custom-made discs were mounted in individual intraoral splint housings and worn by 16 volunteers for 24 h. The safranin staining assay, isothermal microcalorimetry (IMC), and SEM were applied before and after surface cleaning. RESULTS: The hydrophilic surfaces modMA and modSLA with greater surface micro-roughness exhibited significantly more biofilm than the hydrophobic surfaces TAV MG and M. The standardized cleaning procedure substantially reduced the biofilm mass on all surfaces. After cleaning, the IMC analyses demonstrated a longer lag time of the growth curve on TAV MG compared to modSLA. Inter- and intraindividual variations in biofilm formation on the titanium discs were evident throughout the study. CONCLUSIONS: Surface hydrophilicity and roughness enhanced biofilm formation in vivo, whereas surface topography was the most influential factor that determined surface cleanability. While the grooved surface retained larger amounts of initial biofilm, the machined surface was easier to clean, but proliferation indicated by increased metabolic activity (growth rate) in IMC occurred despite mechanical biofilm removal.

Ammonium accumulation is a primary effect of 2-methylcitrate exposure in an in vitro model for brain damage in methylmalonic aciduria.

Using 3D organotypic rat brain cell cultures in aggregates we recently identified 2-methylcitrate (2-MCA) as the main toxic metabolite for developing brain cells in methylmalonic aciduria. Exposure to 2-MCA triggered morphological changes and apoptosis of brain cells. This was accompanied by increased ammonium and decreased glutamine levels. However, the sequence and causal relationship between these phenomena remained unclear. To understand the sequence and time course of pathogenic events, we exposed 3D rat brain cell aggregates to different concentrations of 2-MCA (0.1, 0.33 and 1.0mM) from day in vitro (DIV) 11 to 14. Aggregates were harvested at different time points from DIV 12 to 19. We compared the effects of a single dose of 1mM 2-MCA administered on DIV 11 to the effects of repeated doses of 1mM 2-MCA. Pan-caspase inhibitors Z-VAD FMK or Q-VD-OPh were used to block apoptosis. Ammonium accumulation in the culture medium started within few hours after the first 2-MCA exposure. Morphological changes of the developing brain cells were already visible after 17h. The highest rate of cleaved caspase-3 was observed after 72h. A dose-response relationship was observed for all effects. Surprisingly, a single dose of 1mM 2-MCA was sufficient to induce all of the biochemical and morphological changes in this model. 2-MCA-induced ammonium accumulation and morphological changes were not prevented by concomitant treatment of the cultures with pan-caspase inhibitors Z-VAD FMK or Q-VD-OPh: ammonium increased rapidly after a single 1mM 2-MCA administration even after apoptosis blockade. We conclude that following exposure to 2-MCA, ammonium production in brain cell cultures is an early phenomenon, preceding cell degeneration and apoptosis, and may actually be the cause of the other changes observed. The fact that a single dose of 1mM 2-MCA is sufficient to induce deleterious effects over several days highlights the potential damaging effects of even short-lasting metabolic decompensations in children affected by methylmalonic aciduria.