Marguerite Izquierdo

High‐resolution myocardial perfusion mapping in small animals in vivo by spin‐labeling gradient‐echo imaging

An ECG and respiration‐gated spin‐labeling gradient‐echo imaging technique is proposed for the quantitative and completely noninvasive measurement and mapping of myocardial perfusion in small animals in vivo. In contrast to snapshot FLASH imaging, the spatial resolution of the perfusion maps is not limited by the heart rate. A significant improvement in image quality is achieved by synchronizing the inversion pulse to the respiration movements of the animals, thereby allowing for spontaneous respiration. High‐resolution myocardial perfusion maps (in‐plane resolution = 234 × 468 μm2) demonstrating the quality of the perfusion measurement were obtained at 4.7 T in a group of seven freely breathing Wistar‐Kyoto rats under isoflurane anesthesia. The mean perfusion value (group average ± SD) was 5.5 ± 0.7 ml g–1min–1. In four animals, myocardial perfusion was mapped and measured under cardiac dobutamine stress. Perfusion increased to 11.1 ± 1.9 ml g–1min–1. The proposed method is particularly useful for the study of small rodents at high fields. Magn Reson Med 51:62–67, 2004.

In vivo reduction in ATP cost of contraction is not related to fatigue level in stimulated rat gastrocnemius muscle

1 We tested whether the reduction in ATP cost of contraction during in vivo stimulation of rat gastrocnemius muscle was related to fatigue level. 2 Muscles (n= 44) were electrically stimulated to perform 6 min repeated isometric contractions at different frequencies; one non‐fatiguing protocol (stimulation at 0.8 Hz) and five fatiguing protocols (2, 3.2, 4, 5.2 and 7.6 Hz) were used. Anaerobic and oxidative ATP turnover rates were measured non‐invasively using 31P‐magnetic resonance spectroscopy. 3 At the onset of the stimulation period, no signs of fatigue were measured in the six protocols and ATP cost of contraction did not differ significantly (P= 0.45) among protocols (mean value of 1.76 ± 0.11 mm (N s)−1). 4 For the six protocols, ATP cost of contraction was significantly reduced (P < 0.05) at the end of the stimulation period when compared with the initial value. This reduction did not differ significantly (P= 0.61) among the five fatiguing protocols (averaging 35 ± 3 % of initial value), whereas isometric force decreased significantly as stimulation frequency increased. No significant correlation (P= 0.87, r2= 0.01) was observed between isometric force and ATP cost of contraction at the end of the stimulation period. In addition, this reduction was significantly lower (P < 0.05) for the non‐fatiguing protocol (67 ± 9 % of initial value) when compared with the fatiguing protocols. 5 These results demonstrate that (i) the reduction in ATP cost of contraction during in vivo stimulation of rat gastrocnemius muscle is not related to the fatigue level; (ii) surprisingly, this reduction was significantly larger during the fatiguing protocols compared with the non‐fatiguing protocol.

New experimental setup for studying strictly noninvasively skeletal muscle function in rat using 1H-magnetic resonance (MR) imaging and 31P-MR spectroscopy.

Traditional setups for in situ MR investigation of skeletal muscle function in animals use invasive systems for muscle stimulation and force measurement. These systems require surgical preparation and therefore exclude repetitive investigations on the same animal. This article describes a new experimental setup allowing strictly noninvasive MR investigations of muscle function in contracting rat gastrocnemius muscle using 1H‐MR imaging and 31P‐MR spectroscopy. The novelty of this setup is the integration of two noninvasive systems allowing muscle contraction by transcutaneous stimulation and force measurement with a dedicated ergometer. Muscle function was investigated in 20 rats (275–300 g) through a fatiguing stimulation protocol, either with this noninvasive setup (n = 10) or with a traditional MR setup (n = 10). T2‐weighted images demonstrated that transcutaneous stimulation activated mainly the gastrocnemius muscle. Moreover, the changes in force development and in energy metabolism obtained with the noninvasive setup were qualitatively and quantitatively similar to those obtained with the traditional setup. This noninvasive setup is thus suitable for investigating skeletal muscle function in situ. It offers the possibility to repeat investigations in the same animal, avoiding individual variability and enabling longitudinal follow‐up studies. This opens up new perspectives in various research areas including pharmaceutical research. Magn Reson Med, 2005.

Time-dependent and indirect effect of inorganic phosphate on force production in rat gastrocnemius exercising muscle determined by 31P-MRS.

The relationship of inorganic phosphate (Pi) and its diprotonated form (H2PO4 −) to isometric force (F) was analyzed non‐invasively using 31P‐magnetic resonance spectroscopy. Rat gastrocnemius muscles were electrically stimulated at six different frequencies in order to produce different levels of fatigue. A curvilinear relationship was demonstrated between force production and [Pi] and [H2PO4 −] accumulation. [Pi] and [H2PO4 −] were correlated with F at the end of the stimulation period but not when F was maximal at the early stage of the stimulation period. Interestingly, the respective [Pi] and [H2PO4 −] did not differ significantly between these two stages demonstrating that [Pi] and [H2PO4 −] cannot be considered as direct effectors of fatigue. This time‐dependent and indirect effect of [Pi] and [H2PO4 −] on force production might be mediated by calcium ions.

Beneficial effects of citrulline malate on skeletal muscle function in endotoxemic rat.

Although citrulline malate (CM; CAS 54940-97-5, Stimol) is used against fatigue states, its anti-asthenic effect remains poorly documented. The objective of this double-blind study was to evaluate the effect of oral ingestion of CM on a rat model of asthenia, using in situ (31)Phosphorus magnetic resonance spectroscopy ((31)P-MRS). Muscle weakness was induced by intraperitoneal injections of Klebsiella pneumoniae endotoxin (lipopolysaccharides at 3 mg/kg) at t(0) and t(0)+24 h. For each animal, muscle function was investigated strictly non-invasively before (t(0)-24 h) and during (t(0)+48 h) endotoxemia, through a standardized rest-stimulation-recovery protocol. The transcutaneous electrical stimulation protocol consisted of 5.7 min of repeated isometric contractions at a frequency of 3.3 Hz, and force production was measured with an ergometer. CM supplementation in endotoxemic animals prevented the basal phosphocreatine/ATP ratio reduction and normalized the intracellular pH (pH(i)) time-course during muscular activity as a sign of an effect at the muscle energetics level. In addition, CM treatment avoided the endotoxemia-induced decline in developed force. These results demonstrate the efficiency of CM for limiting skeletal muscle dysfunction in rats treated with bacterial endotoxin.

NOS substrate during cardioplegic arrest and cold storage decreases stunning after heart transplantation in a rat model

BACKGROUND In this study, we evaluated how adding L-arginine to Centre de Résonance Magnétique Biologique et Médicale (CRMBM) solution affected myocardial performance during post-ischemic in vivo reperfusion. METHODS Experiments were conducted using a modified Lewis-Lewis heterotopic heart transplantation model, with a total ischemic time of 3 hours followed by 1 or 24 hours of blood reperfusion. Heart grafts were arrested using intra-aortic injection of CRMBM solution, either supplemented or not supplemented with 2 mmol/liter L-arginine (n = 12 in each group). We measured systolic indexes and simultaneously performed phosphorus magnetic resonance spectroscopy ((31)P MRS). We quantified total endothelial nitric oxide synthase (eNOS) protein using the Western blot test of freeze-clamped hearts. RESULTS Contractility during early reperfusion was significantly better in grafts arrested with CRMBM solution enriched with L-arginine: mean rate pressure product, 11249 +/- 1548 vs 5637 +/- 1118 mm Hg/min (p = 0.05), and maximal first derivative of the pressure signal (dP/dt(max)), 1721 +/- 177 vs 1214 +/- 321 mm Hg/sec (p = 0.013). Conversely, during late reperfusion, contractility did not relate to the nature of the preservation solution. The presence of L-arginine in the CRMBM solution did not alter time-related variations of high-energy phosphate ratios measured using in vivo (31)P MRS. The eNOS protein level decreased significantly during early compared with late reperfusion, with no effect caused by L-arginine. CONCLUSIONS During early reperfusion, the limited myocardial stunning observed with CRMBM solution containing L-arginine does not relate to energy metabolism but to better preservation of the NO pathway.

Metabolic underpinnings of the paradoxical net phosphocreatine resynthesis in contracting rat gastrocnemius muscle

Net phosphocreatine (PCr) resynthesis during muscle contraction is a paradoxical phenomenon because it occurs under conditions of high energy demand. The metabolic underpinnings of this phenomenon were analyzed non-invasively using 31P-magnetic resonance spectroscopy in rat gastrocnemius muscle (n=11) electrically stimulated (7.6 Hz, 6 min duration) in situ under ischemic and normoxic conditions. During ischemic stimulation, [PCr] initially fell to a steady state (9+/-5% of resting concentration) which was maintained for the last 5 min of stimulation, whereas isometric force production decreased to a non-measurable level beyond 3 min. Throughout normoxic stimulation, [PCr] and force production declined to a steady state after respectively 1 min (5+/-3% of resting concentration) and 3.25 min (21+/-8% of initial value) of stimulation. Contrary to the observations under ischemia, a paradoxical net PCr resynthesis was recorded during the last 2 min of normoxic stimulation and was not accompanied by any improvement in force production. These results demonstrate that the paradoxical net PCr resynthesis recorded in contracting muscle relies exclusively on oxidative energy production and could occur in inactivated fibers, similarly to PCr resynthesis during post-exercise recovery.

Endotoxemia causes a paradoxical intracellular pH recovery in exercising rat skeletal muscle

In resting skeletal muscle, endotoxemia causes disturbances in energy metabolism that could potentially disturb intracellular pH (pHi) during muscular activity. We tested this hypothesis using in situ 31P‐magnetic resonance spectroscopy in contracting rat gastrocnemius muscle. Endotoxemia was induced by injecting rats intraperitoneally at t0 and t0 + 24 h with Klebsiella pneumoniae endotoxin (lipopolysaccharides at 3 mg/kg) or saline vehicle. Muscle function was investigated strictly noninvasively at t0 + 48 h through a transcutaneous electrical stimulation protocol consisting of 5.7 minutes of repeated isometric contraction at 3.3 HZ, and force production was measured with an ergometer. At rest, endotoxin treatment did not affect pHi and adenosine triphosphate concentration, but significantly reduced phosphocreatine and glycogen contents. Endotoxemia produced both a reduction of isometric force production and a marked linear recovery (0.08 ± 0.01 pH unit/min) of pHi during the second part of the stimulation period. This recovery was not due to any phenomenon of fiber inactivation linked to development of muscle fatigue, and was not associated with any change in intracellular proton buffering, net proton efflux from the cell, or proton turnovers through creatine kinase reaction and oxidative phosphorylation. This paradoxical pHi recovery in exercising rat skeletal muscle under endotoxemia is likely due to slowing of glycolytic flux following the reduction in intramuscular glycogen content. These findings may be useful in the follow‐up of septic patients and in the assessment of therapies. Muscle Nerve, 2007

Simultaneous study of metabolism and function following cardioplegic arrest: a novel method of evaluation of the transplanted heart in the rat ☆

BACKGROUND Limitations of the isolated perfused rat heart model for heart preservation studies include short study time due to the lack of stability of the preparation. We aimed to develop a new experimental model based on heterotopic heart transplantation in the rat to achieve simultaneous (31)P magnetic resonance spectroscopy (MRS) and functional study of the transplanted heart during early and late blood reperfusion. METHODS Twenty-five Lewis rats underwent heterotopic abdominal isograft heart transplantation and were randomized in two groups. Hearts were harvested after cardioplegic arrest induced with Centre de Résonance Magnétique Biologique et Médicale (CRMBM) solution and then stored at 4 degrees C for a total ischemic time of 3 hours. Graft contractility measurement and simultaneous (31)P MRS were performed after 1 hour and 24 hours of blood reperfusion, respectively, in groups I (n = 12) and II (n =13). RESULTS Contractility improved during reperfusion. The mean rate pressure product plus or minus standard error of mean increased from 11,373 +/- 1,377 mm Hg/min in group I to 24,363 +/- 3,860 mm Hg/min in group II (P = 0.003), while mean dP/dtmax increased from 1,642 +/- 173 mm Hg/sec to 2,571 +/- 333 mm Hg/sec, respectively (p = 0.03). Simultaneously, both the phosphocreatine/adenosine triphosphate (ATP) and inorganic phosphate/ATP ratios decreased from group I to group II (p = 0.025 and p = 0.015, respectively), suggesting regeneration of the intracellular pool of ATP in group II. CONCLUSIONS Simultaneous functional and metabolic studies of the transplanted heart are feasible in rats. Improvement in contractility during late reperfusion is contemporary with significant changes in energetic metabolism. Our model should be useful for the further improvement of heart preservation, which may result in significant clinical progress.

Endotoxemia does not limit energy supply in exercising rat skeletal muscle

Although depletion in high‐energy phosphorylated compounds and mitochondrial impairment have been reported in septic skeletal muscle at rest, their impact on energy metabolism has not been documented during exercise. In this study we aimed to investigate strictly gastrocnemius muscle function non‐invasively, using magnetic resonance techniques in endotoxemic rats. Endotoxemia was induced by injecting animals intraperitoneally at t0 and t0 + 24 h with Klebsiella pneumoniae lipopolysaccharides (at 3 mg kg−1). Investigations were performed at t0 + 48 h during a transcutaneous electrical stimulation protocol consisting of 5.7 min of repeated isometric contractions at a frequency of 3.3 HZ. Endotoxin treatment produced a depletion in basal phosphocreatine content and a pronounced reduction in oxidative adenosine triphosphate (ATP) synthesis capacity, whereas the resting ATP concentration remained unchanged. During the stimulation period, endotoxemia caused a decrease in force‐generating capacity that was fully accounted for by the loss of muscle mass. It further induced an acceleration of glycolytic ATP production and an increased accumulation of adenosine diphosphate (ADP, an important mitochondrial regulator) that allowed a near‐normal rate of oxidative ATP synthesis. Finally, endotoxemia did not affect the total rate of ATP production or the ATP cost of contraction throughout the whole stimulation period. These data demonstrate that, in an acute septic phase, metabolic alterations in resting muscle do not impact energy supply in exercising muscle, likely as a result of adaptive mechanisms. Muscle Nerve, 2008