From a non-targeted metabolomics approach to a targeted biomarkers strategy to highlight testosterone abuse in equine. Illustration of a methodological transfer between platforms and laboratories

In order to overcome the challenge associated with the screening of Anabolic-Androgenic Steroids abuses in animal competitions, a non-targeted liquid chromatography coupled to high resolution mass spectrometry based metabolomics approach was implemented on equine urine samples to highlight potential biomarkers associated with the administration of such compounds, using testosterone esters as model steroids. A statistical model relying on four potential biomarkers intensity could be defined to predict the status of the samples. With a routine application perspective, the monitoring of the highlighted potential biomarkers was first transferred into high-throughput liquid chromatography-selected reaction monitoring (LC-SRM). The model's performances and robustness of the approach were preserved and providing a first demonstration of metabolomics-based biomarkers integration within a targeted workflow using common benchtop MS instrumentation. In addition, with a view to the widespread implementation of such biomarker-based tools, we have transferred the method to a second laboratory with similar instrumentation. This proof of concept allows the development and application of biomarker-based strategies to meet current doping control needs.     

Inflammation contributes to the atherogenic role of intermittent hypoxia in apolipoprotein-E knock out mice

Rationale

Obstructive sleep apnea results in nocturnal intermittent hypoxia (IH) as a main trigger for cardiovascular morbidity, including atherosclerosis. IH induces hemodynamic, hormono-metabolic and also immuno-inflammatory alterations that could differentially contribute to atherosclerosis. Our study aimed at examining their respective contribution to the proatherogenic role of IH in atherosclerosis-prone mice.

Methods

Fifteen-week-old male apolipoprotein E-deficient (ApoE^−/−) mice fed on a high-cholesterol diet (HCD) for 6 weeks and exposed for the last 14 days to IH (21–5% FiO₂, 60 s cycle, 8 h/day) or air, were investigated for aortic atherosclerosis and lipid alterations. Then IH proatherogenicity was assessed in 15- and 20-week-old ApoE^−/− mice fed on a standart-chow diet (SCD) exposed to IH or air for 14 days and assessed for atherosclerosis, lipid, hemodynamic and inflammation alterations.

Results

IH aggravated atherosclerosis in HCD-fed mice, whereas the extremely high cholesterol levels due to HCD were not different between normoxic and hypoxic animals. In SCD-fed mice, IH also aggravated atherosclerosis, more severely in 20 compared to 15-week-old animals. However, cholesterol levels that increased with IH were not different in the two SCD-fed groups. IH slightly elevated arterial blood pressure in 20-week-old animals only, and induced systemic and vascular inflammation, including increased splenocyte proliferation with decreased IL-10 secretion, and increased T-lymphocytes within atherosclerotic plaques.

Conclusions

A short IH exposure without HCD has proatherogenic effects. In contrast to blood pressure or plasma lipids which were slightly or inconstantly affected by IH, inflammation at systemic and vascular levels appears as a potential contributing factor to IH atherogenicity.     

Dorsal and ventral respiratory groups of neurons in the medulla of the rat

The aim of the present work was to identify and localize in rat the medullary neurons involved in respiration. Neural activity was recorded in ketamine-anesthetized, paralyzed and artificially ventilated rats. Active sites were marked by electrocoagulation. Neurons firing in relation to phrenic nerve activity were located between 0.5 and 2 mm lateral to the midline, extending from 0.5 mm caudal to 2 mm rostral to the posterior end of the area postrema. Two groups of respiratory neurons were found: a dorsal group located ventrolateral to the tractus solitarius and a ventral group located in the ventrolateral reticular formation close to the nucleus ambiguus. Neurons were classified as bulbospinal or laryngeal if stimulation of the spinal cord or the vagus nerve, respectively, elicited antidromic action potentials, or as propriobulbar if they were not activated. Neurons firing synchronously with lung inflation were termed pump (P) cells. The dorsal respiratory group includes inspiratory (I) bulbospinal and propriobulbar neurons, P cells, but few expiratory (E) propriobulbar neurons. The ventral respiratory group includes bulbospinal, laryngeal and propriobulbar I and E neurons. Laryngeal motoneurons project ipsilaterally whereas bulbospinal neurons project contralaterally. Cross-correlations between inspiratory bulbospinal neuronal activity and phrenic discharge suggest that bulbospinal I neurons of dorsal and ventral groups project monosynaptically to contralateral phrenic motoneurons. These results indicate a similarity of the medullary respiratory centers of rats and cats, suggesting that rats may profitably be used in studies of respiratory rhythmogenesis.     

Excitatory interactions between phrenic motoneurons in the cat

Summary 1. Interactions between phrenic motoneurons have been analysed in anaesthetized, paralyzed cats after C3 to C7 deafferentation. Effects of electrical stimulation of the C5 phrenic axons have been studied on thin filaments dissected from the stimulated nerve. Repetitive stimulation could elicit, after the primary direct response of the stimulated axons, a secondary response named Recurrent Response, RR. 2. RRs have been obtained in 117/186 phrenic axons. They appear sporadically (mean occurrence: 3.75 RRs elicited by 100 shocks of stimulation) at a constant latency. They originate from a spinal mechanism since they persist after C2 transection and disappear after section of the ventral roots. 3. The mechanism responsible for RR shows spatial and temporal facilitation. The RR probability increases with the number of antidromically invaded motoneurons as revealed by changes either of stimulation intensity or of central respiratory drive. However, RR could be evoked in a motoneuron without an antidromic volley in its axon. 4. Systemic injections of nicotinic blocking drugs such as dihydro--erytroidin or mecamylamine decrease or suppress the occurrence of RR; therefore, cholinergic synapses are involved in the RR generating process. 5. RR are assumed to be due to direct excitatory interactions between homonymous motoneurons. Recurrent axon collaterals impinging directly on neighbouring motoneurons would link together the different motoneurons of the phrenic pool. The functional significance of this phenomenon is discussed.     

Central determination of recruitment order: intracellular study of phrenic motoneurons

Simultaneous recordings were made intracellularly from phrenic motoneurons (PMs) and extracellularly from their central drivers, the inspiratory bulbospinal neurons (IBSNs) of the dorsal respiratory nucleus. On the basis of their order of recruitment from the beginning of inspiration (as estimated by the phrenic discharge). PMs and IBSNs were classified as early (E) or late (L) units. Unitary excitatory postsynaptic potentials (EPSPs) evoked in the PM by the IBSN were frequently observed between IBSNs and PMs with a similar recruitment, both E, 11/28 or both L, 10/13, and were scarce between IBSNs and PMs with different recruitment. E and L, 8/28, or L and E, 2/11. Since measured membrane resistances of E and L PMs were not statistically different, the recruitment order of PMs may be considered as mainly determined by the timing (E or L) of the central drive that they received.     

Onset and control of expiratory laryngeal discharge: cross-correlation analysis

In anaesthetized, paralysed and artificially ventilated cats, spontaneous activity of pairs of expiratory laryngeal motoneurones was recorded and submitted on line to cross-correlation analysis in order to reveal the underlying mechanisms which govern their discharge. In 45/52 pairs, flat cross-correlograms were obtained suggesting independency between the expiratory laryngeal motoneurones. Synchronization by common central respiratory drive was never observed. This negative result is in agreement with the post-inhibitory rebound hypothesis put forward to explain the discharge of expiratory laryngeal motoneurones. In 7/52 pairs, cross-correlation revealed a bell-shaped increase of probability of firing which is relevant with broad synchronization of the discharges by common peripheral afferents.     

Spontaneous respiratory activity of phrenic and intercostal Renshaw cells

Activity of Renshaw cells evoked by electrical stimulation of either phrenic or internal intercostal axons was extracellularly recorded in anaesthetized spontaneously breathing cats. The response of all the studied units to antidromic invasion of the corresponding motoneurones was related to the respiratory cycle and some units displayed spontaneous respiratory activity. Recurrent IPSPs were recorded on phrenic and intercostal motoneurones.