New evidence of neuroprotection by lactate after transient focal cerebral ischaemia: extended benefit after intracerebroventricular injection and efficacy of intravenous administration.

BACKGROUND: Lactate protects mice against the ischaemic damage resulting from transient middle cerebral artery occlusion (MCAO) when administered intracerebroventricularly at reperfusion, yielding smaller lesion sizes and a better neurological outcome 48 h after ischaemia. We have now tested whether the beneficial effect of lactate is long-lasting and if lactate can be administered intravenously. METHODS: Male ICR-CD1 mice were subjected to 15-min suture MCAO under xylazine + ketamine anaesthesia. Na L-lactate (2 µl of 100 mmol/l) or vehicle was administered intracerebroventricularly at reperfusion. The neurological deficit was evaluated using a composite deficit score based on the neurological score, the rotarod test and the beam walking test. Mice were sacrificed at 14 days. In a second set of experiments, Na L-lactate (1 µmol/g body weight) was administered intravenously into the tail vein at reperfusion. The neurological deficit and the lesion volume were measured at 48 h. RESULTS: Intracerebroventricularly injected lactate induced sustained neuroprotection shown by smaller neurological deficits at 7 days (median = 0, min = 0, max = 3, n = 7 vs. median = 2, min = 1, max = 4.5, n = 5, p < 0.05) and 14 days after ischaemia (median = 0, min = 0, max = 3, n = 7 vs. median = 3, min = 0.5, max = 3, n = 7, p = 0.05). Reduced tissue damage was demonstrated by attenuated hemispheric atrophy at 14 days (1.3 ± 4.0 mm(3), n = 7 vs. 12.1 ± 3.8 mm(3), n = 5, p < 0.05) in lactate-treated animals. Systemic intravenous lactate administration was also neuroprotective and attenuated the deficit (median = 1, min = 0, max = 2.5, n = 12) compared to vehicle treatment (median = 1.5, min = 1, max = 8, n = 12, p < 0.05) as well as the lesion volume at 48 h (13.7 ± 12.2 mm(3), n = 12 vs. 29.6 ± 25.4 mm(3), n = 12, p < 0.05). CONCLUSIONS: The beneficial effect of lactate is long-lasting: lactate protects the mouse brain against ischaemic damage when supplied intracerebroventricularly during reperfusion with behavioural and histological benefits persisting 2 weeks after ischaemia. Importantly, lactate also protects after systemic intravenous administration, a more suitable route of administration in a clinical emergency setting. These findings provide further steps to bring this physiological, commonly available and inexpensive neuroprotectant closer to clinical translation for stroke.

Fast gas chromatography and negative-ion chemical ionization tandem mass spectrometry for forensic analysis of cannabinoids in whole blood.

The present work describes a fast gas chromatography/negative-ion chemical ionization tandem mass spectrometric assay (Fast GC/NICI-MS/MS) for analysis of tetrahydrocannabinol (THC), 11-hydroxy-tetrahydrocannabinol (THC-OH) and 11-nor-9-carboxy-tetrahydrocannabinol (THC-COOH) in whole blood. The cannabinoids were extracted from 500 microL of whole blood by a simple liquid-liquid extraction (LLE) and then derivatized by using trifluoroacetic anhydride (TFAA) and hexafluoro-2-propanol (HFIP) as fluorinated agents. Mass spectrometric detection of the analytes was performed in the selected reaction-monitoring mode on a triple quadrupole instrument after negative-ion chemical ionization. The assay was found to be linear in the concentration range of 0.5-20 ng/mL for THC and THC-OH, and of 2.5-100 ng/mL for THC-COOH. Repeatability and intermediate precision were found less than 12% for all concentrations tested. Under standard chromatographic conditions, the run cycle time would have been 15 min. By using fast conditions of separation, the assay analysis time has been reduced to 5 min, without compromising the chromatographic resolution. Finally, a simple approach for estimating the uncertainty measurement is presented.

Calreticulin levels determine onset of early muscle denervation by fast motoneurons of ALS model mice.

Although the precise signaling mechanisms underlying the vulnerability of some sub-populations of motoneurons in ALS remain unclear, critical factors such as metallo-proteinase 9 expression, neuronal activity and endoplasmic reticulum stress have been shown to be involved. In the context of SOD1(G93A) ALS mouse model, we previously showed that a two-fold decrease in calreticulin (CRT) is occurring in the vulnerable fast motoneurons. Here, we asked to which extent the decrease in CRT levels was causative to muscle denervation and/or motoneuron degeneration. Toward this goal, a hemizygous deletion of the crt gene in SOD1(G93A) mice was generated since the complete ablation of crt is embryonic lethal. We observed that SOD1(G93A);crt(+/-) mice display increased and earlier muscle weakness and muscle denervation compared to SOD1(G93A) mice. While CRT reduction in motoneurons leads to a strong upregulation of two factors important in motoneuron dysfunction, ER stress and mTOR activation, it does not aggravate motoneuron death. Our results underline a prevalent role for CRT levels in the early phase of muscle denervation and support CRT regulation as a potential therapeutic approach.

The Munc13 proteins differentially regulate readily releasable pool dynamics and calcium-dependent recovery at a central synapse.

The Munc13 gene family encodes molecules located at the synaptic active zone that regulate the reliability of synapses to encode information over a wide range of frequencies in response to action potentials. In the CNS, proteins of the Munc13 family are critical in regulating neurotransmitter release and synaptic plasticity. Although Munc13-1 is essential for synaptic transmission, it is paradoxical that Munc13-2 and Munc13-3 are functionally dispensable at some synapses, although their loss in other synapses leads to increases in frequency-dependent facilitation. We addressed this issue at the calyx of Held synapse, a giant glutamatergic synapse that we found to express all these Munc13 isoforms. We studied their roles in the regulation of synaptic transmission and their impact on the reliability of information transfer. Through detailed electrophysiological analyses of Munc13-2, Munc13-3, and Munc13-2-3 knock-out and wild-type mice, we report that the combined loss of Munc13-2 and Munc13-3 led to an increase in the rate of calcium-dependent recovery and a change in kinetics of release of the readily releasable pool. Furthermore, viral-mediated overexpression of a dominant-negative form of Munc13-1 at the calyx demonstrated that these effects are Munc13-1 dependent. Quantitative immunohistochemistry using Munc13-fluorescent protein knock-in mice revealed that Munc13-1 is the most highly expressed Munc13 isoform at the calyx and the only one highly colocalized with Bassoon at the active zone. Based on these data, we conclude that Munc13-2 and Munc13-3 isoforms limit the ability of Munc13-1 to regulate calcium-dependent replenishment of readily releasable pool and slow pool to fast pool conversion in central synapses.

Thermogenic effect of the new beta-adrenoreceptor agonist Ro 16-8714 in healthy male volunteers.

Previous investigations in experimental animals have shown that a new type of beta-adrenoceptor agonist (Ro 16-8714) possesses both thermogenic and antihyperglycemic properties. The aim of the study was to assess the thermogenic capacity of the compound in man after acute administration. Following an overnight fast three different doses (5, 10 and 20 mg) and a placebo were given per os to six normal-weight young men. The rate of energy expenditure (EE) and substrate utilization were determined by indirect calorimetry (hood system) before and for 6 h following the drug administration. Heart rate and blood pressure as well as plasma glucose, insulin and free fatty acid (FFA) concentrations were also measured at regular intervals throughout the study. The increment relative to base-line (mean +/- s.e.m.) in EE with placebo, 5, 10 and 20 mg was 4 +/- 3, 10 +/- 2, 11 +/- 2 and 21 +/- 2 percent respectively whereas heart rate was enhanced by 2 +/- 2, 8 +/- 3, 22 +/- 2, and 49 +/- 8 percent. Systolic blood pressure increased less (1 +/- 2, 8 +/- 1, 11 +/- 1 and 13 +/- 2 percent), and diastolic blood pressure did not change significantly. Simultaneously we observed a slight and transient increase in blood glucose, insulin and FFA concentrations. It is concluded that in lean individuals Ro 16-8714 is a potent thermogenic agent; however, new beta-adrenoceptor agonists should be developed in order to avoid the tachycardia associated with the thermogenic effect.

Use of the dried blood spot sampling process coupled with fast gas chromatography and negative-ion chemical ionization tandem mass spectrometry: application to fluoxetine, norfluoxetine, reboxetine, and paroxetine analysis.

The objective of this work was to combine the advantages of the dried blood spot (DBS) sampling process with the highly sensitive and selective negative-ion chemical ionization tandem mass spectrometry (NICI-MS-MS) to analyze for recent antidepressants including fluoxetine, norfluoxetine, reboxetine, and paroxetine from micro whole blood samples (i.e., 10 microL). Before analysis, DBS samples were punched out, and antidepressants were simultaneously extracted and derivatized in a single step by use of pentafluoropropionic acid anhydride and 0.02% triethylamine in butyl chloride for 30 min at 60 degrees C under ultrasonication. Derivatives were then separated on a gas chromatograph coupled with a triple-quadrupole mass spectrometer operating in negative selected reaction monitoring mode for a total run time of 5 min. To establish the validity of the method, trueness, precision, and selectivity were determined on the basis of the guidelines of the "Société Française des Sciences et des Techniques Pharmaceutiques" (SFSTP). The assay was found to be linear in the concentration ranges 1 to 500 ng mL(-1) for fluoxetine and norfluoxetine and 20 to 500 ng mL(-1) for reboxetine and paroxetine. Despite the small sampling volume, the limit of detection was estimated at 20 pg mL(-1) for all the analytes. The stability of DBS was also evaluated at -20 degrees C, 4 degrees C, 25 degrees C, and 40 degrees C for up to 30 days. Furthermore, the method was successfully applied to a pharmacokinetic investigation performed on a healthy volunteer after oral administration of a single 40-mg dose of fluoxetine. Thus, this validated DBS method combines an extractive-derivative single step with a fast and sensitive GC-NICI-MS-MS technique. Using microliter blood samples, this procedure offers a patient-friendly tool in many biomedical fields such as checking treatment adherence, therapeutic drug monitoring, toxicological analyses, or pharmacokinetic studies.

Cerebral Hypoperfusion in Posterior Reversible Encephalopathy Syndrome is Different from Transient Ischemic Attack on CT Perfusion.

BACKGROUND: PRES is a reversible neurotoxic state presenting with headache, altered mental status, visual loss, and seizures. Delayed diagnosis can be avoided if radiological patterns could distinguish PRES from cerebral ischemia. METHODS: Clinical and radiological data were collected on all hospitalized patients who had (1) discharge diagnosis of PRES and (2) acute CTP/CTA. Data were compared with 10 TIA patients with proven cytotoxic edema on MRI. RESULTS: Of the four PRES patients found, three were correlated with acute blood pressure and one with chemotherapy. At the radiological level, quantitative analyses of the CTP parameters showed that 2 out of 4 patients had bilaterally reduced CBF-values (23.2-47.1 ml/100g/min) in occipital regions, as seen in the pathological regions of TIA patients (27.3 ± 13.5 ml/100g/min). When compared with TIA patients, the pathological ROI's demonstrated decreased CBV-values (3.4-5.6 ml/100g). Vasogenic edema on MRI FLAIR imaging was seen in only one PRES patient, and cytotoxic edema on DWI-imaging was never found. CT angiography showed in one PRES patient a vasospasm-like unilateral posterior cerebral artery. CONCLUSIONS: If confirmed by other groups, CTP and CTA imaging in patients with acute visual loss and confusion may help to distinguish PRES from bi-occipital ischemia. These radiological parameters may identify PRES patients at risk for additional tissue infarction.

Transient ocular motor nerve palsies associated with presumed cranial nerve schwannomas.

BACKGROUND: Cranial nerve schwannomas are radiologically characterized by nodular cranial nerve enhancement on magnetic resonance imaging (MRI). Schwannomas typically present with gradually progressive symptoms, but isolated reports have suggested that schwannomas may cause fluctuating symptoms as well. METHODS: This is a report of ten cases of presumed cranial nerve schwannoma that presented with transient or recurring ocular motor nerve deficits. RESULTS: Schwannomas of the third, fourth, and fifth nerves resulted in fluctuating deficits of all 3 ocular motor nerves. Persistent nodular cranial nerve enhancement was present on sequential MRI studies. Several episodes of transient oculomotor (III) deficts were associated with headaches, mimicking ophthalmoplegic migraine. CONCLUSIONS: Cranial nerve schwannomas may result in relapsing and remitting cranial nerve symptoms.

Long-term effects of Roux-en-Y gastric bypass on postprandial plasma lipid and bile acids kinetics in female non diabetic subjects: A cross-sectional pilot study.

BACKGROUND AND AIMS: Formerly obese patients having undergone Roux-en-Y gastric bypass (RYGB) display both an accelerated digestion and absorption of carbohydrate and an increased plasma glucose clearance rate after meal ingestion. How RYGB effects postprandial kinetics of dietary lipids has yet not been investigated. METHODS: Plasma triglyceride (TG), apoB48, total apoB, bile acids (BA), fibroblast growth factor 19 (FGF19), and cholecystokinin (CCK) were measured in post-absorptive conditions and over 4-h following the ingestion of a mixed test meal in a cross-sectional, pilot study involving 11 formerly obese female patients 33.8 ± 16.4 months after RYGB surgery and in 11 weight- and age-matched female control participants. RESULTS: Compared to controls, RYGB patients had faster (254 ± 14 vs. 327 ± 7 min, p < 0.05) and lower (0.14 ± 0.04 vs. 0.35 ± 0.07 mM, p < 0.05) peak TG responses, but their peak apoB48 responses tended to be higher (2692 ± 336 vs. 1841 ± 228 ng/ml, p = 0.09). Their postprandial total BA concentrations were significantly increased and peaked earlier after meal ingestion than in controls. Their FGF19 and CCK concentrations also peaked earlier and to a higher value. CONCLUSIONS: The early postprandial apoB48 and BA responses indicate that RYGB accelerated the rate of dietary lipid absorption. The lower postprandial peak TG strongly suggests that the RYGB simultaneously increased the clearance of TG-rich lipoproteins. CLINICAL TRIAL REGISTRATION: NCT01891591.