Vera Sousa

Creatine (Cr) is an amino acid derivative with an important role in the cell as energy buffer that has been largely used as dietary supplement to increase muscle strength and lean body mass in healthy individuals and athletes. However, studies in fish are scarce. The aim of this work is to determine whether dietary Cr supplementation affects muscle growth in gilthead seabream (Sparus aurata) juveniles. Fish were fed ad libitum for 69 days with diets containing three increasing levels of creatine monohydrate (2, 5, and 8%) that were compared with a non-supplemented control (CTRL) diet. At the end of the trial, the fast-twist skeletal muscle growth dynamics (muscle cellularity) and the expression of muscle-related genes were evaluated. There was a general trend for Cr-fed fish to be larger and longer than those fed the CTRL, but no significant differences in daily growth index (DGI) were registered among dietary treatments. The dorsal cross-sectional muscle area (DMA) of fish fed Cr 5 and Cr 8% was significantly larger than that of fish fed CTRL. The groups supplemented with Cr systematically had a higher relative number of both small-sized (≤20 μm) and large-sized fibers (≥120 μm). Dorsal total fibers number was highest in fish fed 5% Cr. In fish supplemented with 5% Cr, the relative expression of myogenic differentiation 1 (myod1) increased almost four times compared to those fed the CTRL diet. The relative expression of calpain 3 (capn3) was highest in fish fed diets with 2% Cr supplementation, but did not differ significantly from those fed the CTRL or Cr 5%. The myod1 gene expression had a positive and significant correlation with that of capn1, capns1a, and capn3 expression. These results suggest that the observed modulation of gene expression was not enough to produce a significant alteration in muscle phenotype under the tested conditions, as a non-significant increase in muscle fiber diameter and higher total number of fiber was observed, but still resulted in increased DMA. Additional studies may be required in order to better clarify the effect of dietary Cr supplementation in fish, possibly in conjunction with induced resistance training.

The present study aims at investigating muscle development and stress response in early stages of Siberian sturgeon when subjected to different rearing temperatures, by analysing growth and development of the muscle and by assessing the stress response of yolk-sac larvae. Siberian sturgeon larvae were reared at 16°C, 19°C and 22°C until the yolk-sac was completely absorbed. Sampling timepoints were: hatching, schooling and complete yolk-sac absorption stage. Histometrical, histochemical and immunohistochemical analyses were performed in order to characterize muscle growth (total muscle area, TMA; slow muscle area, SMA; fast muscle area, FMA), development (anti-proliferating cell nuclear antigen -PCNA or anticaspase) as well as stress conditions by specific stress biomarkers (heat shock protein 70 or 90, HSP70 or HSP90). Larvae subjected to the highest water temperature showed a faster yolk-sac absorption. Histometry revealed that both TMA and FMA were larger in the schooling stage at 19°C while no differences were observed in the SMA at any of the tested rearing temperatures. PCNA quantification revealed a significantly higher number of proliferating cells in the yolk-sac absorption phase at 22°C than at 16°C. HSP90 immunopositivity seems to be particularly evident at 19°C. HPS70 immunopositivity was never observed in the developing lateral muscle.

Lack of axon regeneration following spinal cord injury has been mainly ascribed to the inhibitory environment of the injury site, i.e., to chondroitin sulfate proteoglycans (CSPGs) and myelin-associated inhibitors (MAIs). Here, we used shiverer (shi) mice to assess axon regeneration following spinal cord injury in the presence of MAIs and CSPG but in the absence of compact myelin. Although in vitro shi neurons displayed a similar intrinsic neurite outgrowth to wild-type neurons, in vivo, shi fibers had increased regenerative capacity, suggesting that the wild-type spinal cord contains additional inhibitors besides MAIs and CSPG. Our data show that besides myelin protein, myelin lipids are highly inhibitory for neurite outgrowth and suggest that this inhibitory effect is released in the shi spinal cord given its decreased lipid content. Specifically, we identified cholesterol and sphingomyelin as novel myelin-associated inhibitors that operate through a Rho-dependent mechanism and have inhibitory activity in multiple neuron types. We further demonstrated the inhibitory action of myelin lipids in vivo, by showing that delivery of 2-hydroxypropyl-β-cyclodextrin, a drug that reduces the levels of lipids specifically in the injury site, leads to increased axon regeneration of wild-type (WT) dorsal column axons following spinal cord injury. In summary, our work shows that myelin lipids are important modulators of axon regeneration that should be considered together with protein MAIs as critical targets in strategies aiming at improving axonal growth following injury.

In the adult central nervous system, axonal regeneration is abortive. Regulators of microtubule dynamics have emerged as attractive targets to promote axonal growth following injury as microtubule organization is pivotal for growth cone formation. In this study, we used conditioned neurons with high regenerative capacity to further dissect cytoskeletal mechanisms that might be involved in the gain of intrinsic axon growth capacity.