Summary

The most common body changes that occur with aging are gradual and generalized skeletal muscle disorders that involve an accelerated loss of muscle mass and function. Recently, the muscle atrophy was defined as a pathological degenerative disease caused by a reduction in muscle fiber amount, as well as reduced fiber size. Diagnosis of musculoskeletal disorders is possible through physical, laboratory, and imaging examinations. Early diagnosis of skeletal muscle disorders in the elderly is important and development of analytical techniques for these diagnoses using molecular imaging is urgently required.

Muscle mass is regulated by protein synthesis and degradation. Muscle atrophy in the elderly is associated with sarcopenia and can occur in cases where starvation/lack of proper diet is an issue. In particular, the physiological response to starvation promotes the breakdown of muscle proteins, thereby resulting in reduced muscle mass. Muscle atrophy is related to the metabolism of carbon nutrients, such as choline. A lack of choline also causes muscle atrophy. Therefore, the detection of fat-related factors or choline may be useful for diagnosing muscle atrophy.

Choline is an essential component of cell membrane phospholipids in skeletal muscle cells. Choline is involved in muscle contraction, a precursor to acetylcholine (ACh), a major neurotransmitter in alpha motor neurons. Consequently, it has clinical implications in the finding that healthy people with low serum choline levels show poor physical performance. The use of ¹⁸F-fluorocholine (¹⁸F-FCH) for the diagnosis of muscle atrophy has not been studied and needs to be verified in various aspects.

Imaging techniques for diagnosing muscle atrophy and sarcopenia remain insufficient, although various advanced diagnostic methods have been established. In the present study, the amount of ¹⁸F-FCH uptake was analyzed, using both in-vivo and in-vitro models of starvation-induced muscle atrophy, and explored the possibility of its use as a diagnostic biomarker. The feasibility of ¹⁸F-FCH positron emission tomography/computed tomography (PET/CT) was explored for evaluating skeletal muscle atrophy, as an imaging technique that tracks choline level changes in muscles. Small-animal-dedicated PET/CT imaging with ¹⁸F-FCH was examined in in-vivo models with rats that were starved to cause muscle atrophy. ¹⁸F-FCH uptake in the starvation-induced cells was vary from the untreated group, and in-vivo PET uptake also revealed a similar tendency. Based on the results of this analysis, main goal was to provide basic data for diagnosing muscle atrophy via ¹⁸F-FCH PET in the skeletal muscle.

Results from nanoScan® PET/CT

Sprague–Dawley rats were maintained in a controlled environment. Prior to the experiments, the rats were randomly divided into two groups: the untreated group (UN,) and the group starved for 48 h (ST). The ST group was provided with free access to water during the period of starvation, whereas the UN group was maintained under standard conditions. The UN group was fasted for 4 h prior to the ¹⁸F-FCH injection.

All rats in each group were administered a single dose of ¹⁸F-FCH (14.8 ± 2.96 MBq) intravenously, following which PET images were acquired for the diagnosis of muscle atrophy in the untreated and starved rats. After 40 min of conscious radiotracer uptake, rats were anesthetized and sequential PET-CT scans were acquired for 20 min. using a dedicated small animal PET/CT scanner (NanoPET/CT, Mediso Medical Imaging Systems, Budapest, Hungary). CT scans were used for attenuation correction and anatomical localization of the PET signals. The acquired PET images were reconstructed using the three-dimensional Adjoint Monte Carlo method with scatter and random corrections. The volume of interest (VOI) in both legs (hindlimb) was delineated by the intensely visualized region in the summed image. VOIs were drawn on CT images of individual animals in a slice-by-slice manner. Regional uptake of radioactivity was decay-corrected to the injection time and expressed as the average standardized uptake value (SUV), which was normalized to the amount of radioactivity injected and the animal’s body weight. InterView Fusion software (Mediso Medical Imaging Systems, Budapest, Hungary) was used to analyse the data of the SUV in the VOIs after reconstruction and quantification.

To test the hypothesis that tracking changes in choline uptake can confirm the diagnosis of muscle atrophy in-vivo experiments were performed.

To assess choline uptake in the skeletal muscle, small-animal PET/CT imaging with ¹⁸F-FCH was performed in a rat model of muscle atrophy. As shown in Figure 3A, a clear accumulation of ¹⁸F-FCH was observed in the muscle tissue of rats.

Figure 3. ¹⁸F-FCH uptake and choline acetylase expression in skeletal muscle atrophic tissue in rats. (A) Choline uptake in the atrophic skeletal muscle was determined using CT and PET with ¹⁸F-FCH tracer. The volume of interest (VOI; white arrows) indicates both legs (hindlimb). The graph indicates SUV_mean in VOI in the UN (n = 6) and ST groups (n = 7).

• As evaluated by ¹⁸F-FCH PET/CT, the quantified mean SUVs of the UN and ST groups were significantly different at 0.37 ±07 and 0.26 ± 0.06, respectively (p < 0.001).
• The results showed that the uptake of ¹⁸F-FCH radiotracer is involved in lipid metabolism and is significant in atrophy modelling, in vitro and in vivo. ¹⁸F-FCH PET/CT is a possible molecular imaging diagnostic tool for muscle atrophy.
• In this study, fasting-induced muscle atrophy showed a significant decrease in ¹⁸F-FCH uptake. In particular, in image acquisition within 60 min, ¹⁸F-FCH PET/CT showed the tracking of muscle loss.

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