Progressive brain metabolic changes under deep brain stimulation of subthalamic nucleus in parkinsonian rats.

Deep brain stimulation (DBS) of the subthalamic nucleus (STN) is an efficient neurosurgical treatment for advanced Parkinson's disease. Non-invasive metabolic neuroimaging during the course of DBS in animal models may contribute to our understanding of its action mechanisms. Here, DBS was adapted to in vivo proton magnetic resonance spectroscopy at 11.7 T in the rat to follow metabolic changes in main basal ganglia structures, the striatum, and the substantia nigra pars reticulata (SNr). Measurements were repeated OFF and ON acute and subchronic (7 days) STN-DBS in control and parkinsonian (6-hydroxydopamine lesion) conditions. Acute DBS reversed the increases in glutamate, glutamine, and GABA levels induced by the dopamine lesion in the striatum but not in the SNr. Subchronic DBS normalized GABA in both the striatum and SNr, and glutamate in the striatum. Taurine levels were markedly decreased under subchronic DBS in the striatum and SNr in both lesioned and unlesioned rats. Microdialysis in the striatum further showed that extracellular taurine was increased. These data reveal that STN-DBS has duration-dependent metabolic effects in the basal ganglia, consistent with development of adaptive mechanisms. In addition to counteracting defects induced by the dopamine lesion, prolonged DBS has proper effects independent of the pathological condition. Non-invasive metabolic neuroimaging might be useful to understand the physiological mechanisms of deep brain stimulation (DBS). Here, we demonstrate the feasibility of repeated high-field proton magnetic resonance spectroscopy of basal ganglia structures under subthalamic nucleus DBS in control and parkinsonian rats. Results show that DBS has both rapid and delayed effects either dependent or independent of disease state.

Does MPTP intoxication in mice induce metabolite changes in the nucleus accumbens? A ¹H nuclear MRS study.

Using in vivo ¹H NMR spectroscopy in a mouse model of Parkinson's disease, we previously showed that glutamate concentrations in the dorsal striatum were highest after dopamine denervation associated with an increase in gamma-aminobutyric acid (GABA) and (Gln) glutamine levels. The aim of this study was to determine whether the changes previously observed in the motor part of the striatum were reproduced in a ventral part of the striatum, the nucleus accumbens (NAc). This study was carried out on controls and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated mice. In vivo spectra were acquired for a voxel (8 µL) in the dorsal striatum, and in the NAc (1.56 µL). NMR acquisitions were first performed 10 days after the last MPTP injection in a basal condition [after saline intraperitoneal (i.p.) injection] and then in the same animal the week after basal NMR acquisitions, after acute levodopa administration (200 mg kg⁻¹, i.p.). Immunohistochemistry was used to determine the levels of (Glu) glutamate, glutamine synthetase (GS) and glutamic acid decarboxylase (GAD) isoform 67 in these two structures. The Glu, Gln and GABA concentrations obtained in the basal state were higher in the NAc of MPTP-intoxicated mice which have the higher dopamine denervation in the ventral tegmental area (VTA) and in the dorsal striatum. Levodopa decreased the levels of these metabolites in MPTP-intoxicated mice to levels similar to those in controls. In parallel, immunohistochemical staining showed that glutamate, GS and GAD67 immunoreactivity increased in the dorsal striatum of MPTP-intoxicated mice and in the NAc for animals with a severe dopamine denervation in VTA. These findings strongly supported a hyperactivity of the glutamatergic cortico-striatal pathway and changes in glial activity when the dopaminergic denervation in the VTA and substantia nigra pars compacta (SNc) was severe.

The human OPA1delTTAG mutation induces premature age-related systemic neurodegeneration in mouse.

Dominant optic atrophy is a rare inherited optic nerve degeneration caused by mutations in the mitochondrial fusion gene OPA1. Recently, the clinical spectrum of dominant optic atrophy has been extended to frequent syndromic forms, exhibiting various degrees of neurological and muscle impairments frequently found in mitochondrial diseases. Although characterized by a specific loss of retinal ganglion cells, the pathophysiology of dominant optic atrophy is still poorly understood. We generated an Opa1 mouse model carrying the recurrent Opa1(delTTAG) mutation, which is found in 30% of all patients with dominant optic atrophy. We show that this mouse displays a multi-systemic poly-degenerative phenotype, with a presentation associating signs of visual failure, deafness, encephalomyopathy, peripheral neuropathy, ataxia and cardiomyopathy. Moreover, we found premature age-related axonal and myelin degenerations, increased autophagy and mitophagy and mitochondrial supercomplex instability preceding degeneration and cell death. Thus, these results support the concept that Opa1 protects against neuronal degeneration and opens new perspectives for the exploration and the treatment of mitochondrial diseases.

Metabolic changes detected in vivo by 1H MRS in the MPTP-intoxicated mouse.

We used in vivo proton ((1)H) Magnetic Resonance Spectroscopy (MRS) to measure the levels of the main excitatory amino acid, glutamate (Glu) and also glutamine (Gln) and GABA in the striatum and cerebral cortex in the MPTP-intoxicated mouse, a model of dopaminergic denervation, before and after dopamine (DA) replacement. The study was performed at 9.4T on control mice (n = 8) and MPTP-intoxicated mice (n = 8). In vivo spectra were acquired in a voxel (8 microL) centered in the striatum, and in the cortex (4.6 microL). Three days after basal MRS acquisitions new spectra were acquired in the striatum and cortex, after levodopa (200 mg.kg(-1)). Glu, Gln and GABA concentrations obtained in the basal state were significantly increased in the striatum of MPTP-lesioned mice (Glu: 20.2 +/- 0.8 vs 11.4 +/- 0.9 mM, p < 0.001; Gln: 5.4 +/- 1.6 vs 2.0 +/- 0.6 mM, p < 0.05; GABA: 3.6 +/- 0.8 vs 1.6 +/- 0.2 mM, p < 0.05). Levodopa lowered metabolites concentrations in the striatum of MPTP-lesioned mice (Glu: 20.2 +/- 0.8 vs 11.2 +/- 0.4 mM (+ Ldopa), p < 0.001; Gln: 5.4 +/- 1.6 vs 1.6 +/- 0.4 mM (+ Ldopa), p < 0.05; GABA: 3.6 +/- 0.8 vs 1.7 +/- 0.4 mM (+ Ldopa), p < 0.01). Metabolite levels in the striatum of MPTP-intoxicated mice + levodopa were not significantly different from those in the striatum of controls. No change was found in the cortex after DA denervation and after DA replacement between the two animals groups. These results strongly support a predominant change in striatal Glu synaptic activity in the cortico-striatal pathway. Acute levodopa administration reverses the increase of metabolites in the striatum.

Metabolic changes detected by proton magnetic resonance spectroscopy in vivo and in vitro in a murin model of Parkinson's disease, the MPTP-intoxicated mouse.

Parkinson's disease is a neurodegenerative disorder characterized by the progressive loss of the dopaminergic neurons in the substantia nigra pars compacta, which project to the striatum. The aim of this study was to analyze in vivo and in vitro consequences of dopamine depletion on amount of metabolites in a mouse model of Parkinson's disease using proton (1)H magnetic resonance spectroscopy (MRS). The study was performed on control mice (n = 7) and MPTP-intoxicated mice (n = 7). All the experiments were performed at 9.4 T. For in vivo MRS acquisitions, mice were anesthetized and carefully placed on an animal handling system with the head centered in birdcage coil used for both excitation and signal reception. Spectra were acquired in a voxel (8 microL) centered in the striatum, applying a point-resolved spectroscopy sequence (TR = 4000 ms, TE = 8.8 ms). After in vivo MRS acquisitions, mice were killed; successful lesion verified by tyrosine hydroxylase immunolabeling on the substantia nigra pars compacta and in vitro MRS acquisitions performed on perchloric extracts of anterior part of mice brains. In vitro spectra were acquired using a standard one-pulse experiment. The absolute concentrations of metabolites were determined using jmrui (Lyon, France) from (1)H spectra obtained in vivo on striatum and in vitro on perchloric extracts. Glutamate (Glu), glutamine (Gln), and GABA concentrations obtained in vivo were significantly increased in striatum of MPTP-lesioned mice (Glu: 15.5 +/- 2.5 vs. 12.9 +/- 1.0 mmol/L, p < 0.05; Gln: 2.3 +/- 0.9 vs. 1.8 +/- 0.6 mmol/L, p < 0.05; GABA: 2.3 +/- 0.9 vs. 1.3 +/- 0.6 mmol/L, p < 0.05). The in vitro results confirmed these results, Glu (10.9 +/- 2.5 vs. 7.9 +/- 1.7 micromol/g, p < 0.05), Gln (6.8 +/- 2.9 vs. 4.3 +/- 1.0 micromol/g, p < 0.05), and GABA (2.9 +/- 0.9 vs. 1.5 +/- 0.4 micromol/g, p < 0.01). The present study strongly supports a hyperactivity of the glutamatergic cortico-striatal pathway hypothesis after dopaminergic denervation in association with an increase of striatal GABA levels. It further shows an increased of striatal Gln concentrations, perhaps as a strategy to protect neurons from Glu excitotoxic injury after striatal dopamine depletion.

Orthologous metabonomic qualification of a rodent model combined with magnetic resonance imaging for an integrated evaluation of the toxicity of Hypochoeris radicata.

In the present study, we have used metabonomics combined with magnetic resonance imaging (MRI) to investigate an orphan neurological disease, Australian stringhalt, described in horse-ingesting inflorescences of Hypochoeris radicata (HR), without any knowledge on the toxic principle and without any practical possibility to perform experiments on the target species. To get valuable candidate biomarkers, we have chosen the mouse species as a "metabolically competent" laboratory animal model. Metabonomics has been applied as a holistic approach to obtain some pertinent metabolic information about the target organs and biomarker metabolites involved in the HR-induced disruptive events. From urine, liver, and brain metabolic fingerprints, HR ingestion induced a very significant effect on the general metabolism, which is proportional to the HR dose administered and to the HR intoxication duration. The main metabolic biomarker in the mouse model of an intoxication specifically induced by HR feeding has been unambiguously identified as scyllo-inositol. A significant increase of this metabolic marker has been measured in urine and in hydrosoluble liver or brain extracts with a very significant canonical link between these two organs. MRI results obtained in the thalamus have confirmed the involvement of scyllo-inositol, a metabolite found in many neurodegenerative diseases, in some specific metabolic disruptions involved in both neuronal and glial dysfunctions as awaited from etiology of this horse disease. This brain metabolic biomarker has been clearly associated with changes in N-acetyl-aspartate, lactate, and choline cerebral concentration found in both neuronal and glial dysfunctions. Scyllo-inositol is a valuable candidate biomarker of the Australian stringhalt disease that needs now to be clinically validated in the target species.

Relevance of isotopic and molecular biomarkers for the authentication of milk according to production zone and type of feeding of the cow.

The first objective of the present paper was to assess the potential of both isotopic ( (18)O/ (16)O in milk water) and molecular biomarkers (terpenes, fatty acids, carotenoids, and vitamins) and milk color to discriminate the production zone (lowland or upland areas) from which 49 tanker bulk milks were collected over one year from a total of 204 farms. The milk water (18)O enrichment was higher in lowland (<500 m altitude) than in upland (>700 m altitude), but the delta (18)O values failed to discriminate systematically the production zone at the scale of the year because of its high variability related to the sampling period. In contrast with vitamins A and E, carotenoids, and milk color measurements, terpenes and fatty acids were confirmed to be relevant tracers of the production zone. The milk compounds with the strongest discriminative potential were fatty acids, which were determined by high-resolution gas chromatography. The calculation of fatty acid ratios, which permits the limitation of using fatty acid relative quantity expressed in percentage of total fatty acids to be overcome, was shown to be particularly relevant in discriminating upland from lowland milk ratios. The selection of two pairs of ratios, namely, iso-C17:0/C18:3 n-3 and iso-C15:0/iso-C14:0, enabled the authentication of 100% of the highland versus lowland milks whatever the season. The second objective was to evaluate the relevance of fatty acid composition to discriminate milks according to the proportion of corn silage in the diets of dairy cows. The selection of two fatty acids ratios, namely, trans11 cis15-C18:2/trans11-C18:1 and cis9-C16:1/iso-C16:0, enabled the correct classification of 100% of the milk samples according to the proportion of corn silage in the basic fodder rations (<25% vs >30%). The relationship between the milk production zone and the type of forage fed to the cows is discussed.

Insulin-dependent glycogen synthesis is delayed in onset in the skeletal muscle of food-deprived aged rats.

Insulin resistance with aging may be responsible for impaired glycogen synthesis in the skeletal muscle of aged rats and contribute to the well-known decreased ability to respond to stress with aging. For this reason, to assess the ability of the skeletal muscle to utilize glucose for glycogen synthesis during aging, the time course of glycogen synthesis was continuously monitored by 13C nuclear magnetic resonance for 2 h in isolated [13C] glucose-perfused gastrocnemius-plantaris muscles of 5-day food-deprived adult (6-8 months; n=10) or 5-day food-deprived aged (22 months; n=8) rats. [13C] glucose (10 mmol/L) perfusion was carried out in the presence or absence of an excess of insulin (1 micromol/L). Food deprivation only decreased glycogen level in adult rats (8.9+/-2.4 micromol/g in adults vs. 35.6+/-2.4 micromol/g in aged rats; P<.05). In the presence of an excess of insulin, muscle glycogen synthesis was stimulated in both adult and aged muscles, but the onset was delayed with aging (40 min later). In conclusion, this study highlights the important role of glycogen depletion in stimulating glycogen synthesis in muscles. Consequently, the absence of glycogen depletion in response to starvation in aged rats may be the origin of the delay in insulin-stimulated glycogen synthesis in the skeletal muscle. Glycogen synthesis clearly was not impaired with aging.

Glutamate and CO2 production from glutamine in incubated enterocytes of adult and very old rats.

Glutamine is the major fuel for enterocytes and promotes the growth of intestinal mucosa. Although oral glutamine exerts a positive effect on intestinal villus height in very old rats, how glutamine is used by enterocytes is unclear. Adult (8 months) and very old (27 months) female rats were exposed to intermittent glutamine supplementation for 50% of their age lifetime. Treated rats received glutamine added to their drinking water, and control rats received water alone. Jejunal epithelial cells (~300×10(6) cells) were incubated in oxygenated Krebs-Henseleit buffer for 30 min containing [1-(13)C] glutamine (~17 M) for analysis of glutamine metabolites by (13)C nuclear magnetic resonance ((13)C NMR). An aliquot fraction was incubated in the presence of [U-(14)C] glutamine to measure produced CO2. Glutamine pretreatment increased glutamate production and decreased CO2 production in very old rats. The ratio CO2/glutamate, which was very high in control very old rats, was similar at both ages after glutamine pretreatment, as if enterocytes from very old rats recovered the metabolic abilities of enterocytes from adult rats. Our results suggest that long-term treatment with glutamine started before advanced age (a) prevented the loss of rat body weight without limiting sarcopenia and (b) had a beneficial effect on enterocytes from very old rats probably by favoring the role of glutamate as a precursor for glutathione, arginine and proline biosynthesis, which was not detected in (13)C NMR spectra in our experimental conditions.

Impaired resting muscle energetics studied by (31)P-NMR in diet-induced obese rats.

OBJECTIVE: Mitochondrial activity is altered in skeletal muscle of obese, insulin-resistant or type 2 diabetic patients. We hypothesized that this situation was associated with profound adaptations in resting muscle energetics. For that purpose, we used in vivo (31)P-nuclear magnetic resonance ((31)P-NMR) in male sedentary Wistar rats fed with obesogenic diets known to induce alterations in muscle mitochondrial activity. METHODS AND PROCEDURES: Two experimental diets (high sucrose and high fat) were provided for 6 weeks at two levels of energy (standard, N and high, H) and compared to control diet. The rates of the adenosine triphosphate (ATP) exchange between phosphocreatine (PCr) and gamma-ATP (k(a)) and beta-adenosine diphosphate (beta-ADP) to beta-ATP (k(b)) were evaluated using (31)P-NMR in resting gastrocnemius muscle. Muscle contents in phosphorylated compounds as well as creatine, were assessed using (31)P-NMR and biochemical assays, respectively. RESULTS: ATP content increased by 6.7-8.5% in standard-energy high-sucrose (NSU), high-energy high-fat (HF) and high-energy high-sucrose (HSU) groups compared to control (P < 0.05), whereas PCr content decreased by 4.2-6.4% (P < 0.01). Consequently, PCr to ATP ratio decreased in NSU, HF, and HSU groups, compared to control (P < 0.01). Furthermore in high-energy groups (HF and HSU) compared to control, creatine contents were decreased by 14-19% (P < 0.001), whereas k(a) and k(b) fluxes were increased by 89-133% (P < 0.001) and 243-277% (P < 0.01), respectively. DISCUSSION: Our in vivo data showed adaptations of resting skeletal muscle energetics in response to high-energy diets. Increased activity of enzymes catalyzing ATP production may reflect a compensatory mechanism to face impaired mitochondrial ATP synthesis in order to preserve intracellular energy homeostasis.

Proton MRS of early post-natal mouse brain modifications in vivo.

NMR provides a non-invasive tool for the phenotypic characterisation of mouse models. The aim of the present study was to apply reliable in vivo MRS techniques for non-invasive investigations of brain development in normal and transgenic mice, by monitoring metabolite concentrations in different brain regions. The conditions of anaesthesia, immobilisation and respiratory monitoring were optimized to carry out in vivo MRS studies in young mice. All the experiments were performed in normal mice, at 9.4 T, applying a point-resolved spectroscopy (PRESS) sequence (TR = 2,000 ms; TE = 130 ms). We obtained reproducible in vivo (1)H NMR spectra of wild-type mouse brains as early as post-natal day 5, which allowed us to follow brain maturation variations from post-natal days 5 to 21. The survival rate of animals was between 66 and 90% at post-natal days 5 and 21, respectively. Developmental changes of metabolite concentrations were measured in three brain regions: the thalamus, a region rich in cell bodies, the olfactory bulb, rich in fibre tracts actively myelinated during brain maturation, and the cerebellum. The voxel size varied from 2 to 8 microL according to the size of the brain structure analysed. The absolute concentrations of the total creatine, taurine, total choline, N-acetylaspartate and of the glutamate/glutamine pool were determined from (1)H NMR spectra obtained in the different brain regions at post-natal day 5, 10, 15 and 21. Variations observed during brain development were in accordance with those previously reported in mice using ex vivo MRS studies, and also in rats and humans in vivo. Possibilities of longitudinal MRS analysis in maturing mice brains provide new perspectives to characterise better the tremendous number of transgenic mutant mice generated with the aim of decrypting the complexity of brain development and neurodegenerative diseases but also to follow the impact of environmental and therapeutic factors.

Diets high in sugar, fat, and energy induce muscle type-specific adaptations in mitochondrial functions in rats.

Obesity is often associated with insulin resistance and mitochondrial dysfunction within skeletal muscles, but the causative factors are not clearly identified. The present study examined the role of nutrition, both qualitatively and quantitatively, in the induction of muscle mitochondrial defects. Two experimental diets [high sucrose (SU) and high fat (F)] were provided for 6 wk to male Wistar rats at 2 levels of energy [standard (N) and high (H)] and compared with a standard energy cornstarch-based diet (C). Insulin sensitivity (intraperitoneal glucose tolerance test, IPGTT) and intramyocellular triglyceride (IMTG) content (1H MRS) were determined at wk 5. Mitochondrial oxidative phosphorylation and superoxide anion radical (MSR) production were assessed on soleus (oxidative) and tibialis (glycolytic) muscles. Experimental diets induced hyperinsulinemia during IPGTT (P < 0.01 vs. C). Rats in the HSU and HF groups were hyperglycemic relative to the C group, P < 0.05 vs. C. The severity of insulin resistance paralleled IMTG accumulation (P < 0.05). In soleus, mitochondrial respiration and ATP production rates were lower in HSU and HF than in C (P < 0.05). By contrast, respiration was unaffected by the diets in tibialis, whereas ATP production tended to be lower in rats fed the experimental diets compared with C (P = 0.09). Mitochondrial adaptations were associated with more than a 50% reduction in MSR production in HSU and HF compared with C in both soleus (P < 0.05) and tibialis (P < 0.01). Changes in mitochondrial functions in the NSU and NF groups were intermediate and not significantly different from C. Therefore, excess fat or sucrose and more importantly, excess energy intake by rats is associated with muscle type-specific mitochondrial adaptations, which contribute to decrease mitochondrial production of ATP and reactive oxygen species.

Cerebral glutamate metabolism in Parkinson's disease: an in vivo dynamic (13)C NMS study in the rat.

The aim of this work was to explore in vivo the metabolism of the basal ganglia in a rat model of Parkinson's disease. (13)C NMR spectroscopy was used to monitor the synthesis of glutamate/glutamine from [2-(13)C] sodium acetate. (13)C label incorporation in glutamate at the carbon C4 was measured in the brain of rats in different physiopathological states and after antiparkinsonian treatment. Studies were performed in control rats (n = 6) and parkinsonian rats (n = 5) in a stable state and after acute levodopa administration (50 mg/kg iv). (13)C NMR spectra recorded using a (1)H/(13)C surface probe were acquired in the injured cerebral hemisphere. The sequence was a (13)C acquisition sequence with (1)H-decoupling during acquisition, which lasted 17 min, six spectra were obtained during the acetate infusion. Levels of glutamate C4 expressed as a percentage of the lipid resonance that appears in the same spectrum were significantly higher in parkinsonian rats than in controls after 34 min (45.1 +/- 12.8% vs. 32.0 +/- 3.7%; P < 0.05), 51 min (49.0 +/- 5.6% vs. 29.8 +/- 4.0%; P < 0.001), 68 min (61.6 +/- 12.5% vs. 43.5 +/- 13.7%; P < 0.01), and 85 min (46.8 +/- 5.8% vs. 27.4 +/- 7.4%; P < 0.05) of substrate infusion. In parkinsonian rats receiving an acute levodopa injection, the relative proportion of glutamate C4 was statistically lower than in parkinsonian rats receiving saline. Our results show that the metabolism of neuronal glutamate increases in dopamine-depleted striatum and that is restored by administration of levodopa.