Simulated altitude is medicine: intermittent exposure to hypobaric hypoxia and cold accelerates injured skeletal muscle recovery.

Muscle injuries are the leading cause of sports casualties. Because of its high plasticity, skeletal muscle can respond to different stimuli to maintain and improve functionality. Intermittent hypobaric hypoxia (IHH) improves muscle oxygen delivery and utilization. Hypobaria coexists with cold in the biosphere, opening the possibility to consider the combined use of both environmental factors to achieve beneficial physiological adjustments. We studied the effects of IHH and cold exposure, separately and simultaneously, on muscle regeneration. Adult male rats were surgically injured in one gastrocnemius and randomly assigned to the following groups: (1) CTRL: passive recovery; (2) COLD: intermittently exposed to cold (4°C); (3) HYPO: submitted to IHH (4500 m); (4) COHY: exposed to intermittent simultaneous cold and hypoxia. Animals were subjected to these interventions for 4 h/day for 9 or 21 days. COLD and COHY rats showed faster muscle regeneration than CTRL, evidenced after 9 days at histological (dMHC-positive and centrally nucleated fibre reduction) and functional levels after 21 days. HYPO rats showed a full recovery from injury (at histological and functional levels) after 9 days, while COLD and COHY needed more time to induce a total functional recovery. IHH can be postulated as an anti-fibrotic treatment since it reduces collagen I deposition. The increase in the pSer473Akt/total Akt ratio observed after 9 days in COLD, HYPO and COHY, together with the increase in the pThr172AMPKα/total AMPKα ratio observed in the gastrocnemius of HYPO, provides clues to the molecular mechanisms involved in the improved muscle regeneration. KEY POINTS: Only intermittent hypobaric exposure accelerated muscle recovery as early as 9 days following injury at histological and functional levels. Injured muscles from animals treated with intermittent (4 h/day) cold, hypobaric hypoxia or a simultaneous combination of both stimuli regenerated histological structure and recovered muscle function 21 days after injury. The combination of cold and hypoxia showed a blunting effect as compared to hypoxia alone in the time course of the muscle recovery. The increased expression of the phosphorylated forms of Akt observed in all experimental groups could participate in the molecular cascade of events leading to a faster regeneration. The elevated levels of phosphorylated AMPKα in the HYPO group could play a key role in the modulation of the inflammatory response during the first steps of the muscle regeneration process.

Bioactivity of Macronutrients from Chlorella in Physical Exercise.

Chlorella is a marine microalga rich in proteins and containing all the essential amino acids. Chlorella also contains fiber and other polysaccharides, as well as polyunsaturated fatty acids such as linoleic acid and alpha-linolenic acid. The proportion of the different macronutrients in Chlorella can be modulated by altering the conditions in which it is cultured. The bioactivities of these macronutrients make Chlorella a good candidate food to include in regular diets or as the basis of dietary supplements in exercise-related nutrition both for recreational exercisers and professional athletes. This paper reviews current knowledge of the effects of the macronutrients in Chlorella on physical exercise, specifically their impact on performance and recovery. In general, consuming Chlorella improves both anaerobic and aerobic exercise performance as well as physical stamina and reduces fatigue. These effects seem to be related to the antioxidant, anti-inflammatory, and metabolic activity of all its macronutrients, while each component of Chlorella contributes its bioactivity via a specific action. Chlorella is an excellent dietary source of high-quality protein in the context of physical exercise, as dietary proteins increase satiety, activation of the anabolic mTOR (mammalian Target of Rapamycin) pathway in skeletal muscle, and the thermic effects of meals. Chlorella proteins also increase intramuscular free amino acid levels and enhance the ability of the muscles to utilize them during exercise. Fiber from Chlorella increases the diversity of the gut microbiota, which helps control body weight and maintain intestinal barrier integrity, and the production of short-chain fatty acids (SCFAs), which improve physical performance. Polyunsaturated fatty acids (PUFAs) from Chlorella contribute to endothelial protection and modulate the fluidity and rigidity of cell membranes, which may improve performance. Ultimately, in contrast to several other nutritional sources, the use of Chlorella to provide high-quality protein, dietary fiber, and bioactive fatty acids may also significantly contribute to a sustainable world through the fixation of carbon dioxide and a reduction of the amount of land used to produce animal feed.

Histomorphological and functional contralateral symmetry in the gastrocnemius muscles of the laboratory rat.

It is usual in anatomical and physiological research to assess the effects of some intervention on extremities (e.g., training programmes or injury recovery protocols) using one muscle for the intervention and its contralateral as control. However, the existence of laterality (left-handedness or right-handedness) in athletes of different specialities is widely recognized. In rats, gastrocnemius is one of the muscles most widely used because of its importance in locomotion and high relative limb mass. Since we have not found studies reporting laterality assessment on the morphology and function in rat gastrocnemius, our study aimed to evaluate the fibre histochemical, morphometrical and muscle force contractile properties between right and left gastrocnemius of the laboratory rat. Fibre-type proportion, fibre morphometrical measurements, muscle capillarization and muscle force properties were analysed in the right and left gastrocnemius of six male rats. No statistically significant differences (p = 0.265) were found in gastrocnemius to body weight ratio (‰) between right (6.55 ± 0.40) and left (6.49 ± 0.40) muscles. The muscles analysed showed a great degree of heterogeneity in fibre type distribution, having three clearly distinguished regions named red, mixed and white. In the three regions, there were no statistical differences in fibre type proportions between right and left gastrocnemius, as is indicated by the p-values (from 0.203 to 0.941) obtained after running t-Student paired tests for each fibre type. When analysing fibre cross-sectional area, individual fibre capillarization and fibre circularity, no significant differences between right and left gastrocnemius in any of these morphometrical parameters were found in any muscle region or fibre type. Most of the p-values (70%) resulting from running t-Student paired tests were higher than 0.400, and the lowest p-value was 0.115. Seemingly, global capillary and fibre densities were not statistically different between right and left sides in all muscle regions with p-values ranging from 0.337 to 0.812. Force parameters normalized to gastrocnemius mass (mN g-1 ) did not show any significant difference between right (PF = 74.0 ± 13.4, TF = 219.4 ± 13.0) and left (PF = 70.9 ± 10.7, TF = 213.0 ± 18.0) muscles with p = 0.623 (PF) and p = 0.514 (TF). Twitch time parameters (ms) also lacked significant differences between the two sides (CT: 43.4 ± 8.6 vs. 45.0 ± 14.3, p = 0.639; HRT: 77.6 ± 15.0 vs. 82.3 ± 25.3, p = 0.475). Finally, both muscles also showed similar (p = 0.718) fatigue properties. We did find an absence of laterality at the morphological and functional levels, which raises the possibility of using right and left gastrocnemius muscles interchangeably for experimental designs where one muscle is used to analyse data after a physiological intervention and its contralateral muscle plays the control role, thus allowing unbiased paired comparisons to derive accurate conclusions.

Physiological Effects of Intermittent Passive Exposure to Hypobaric Hypoxia and Cold in Rats.

The benefits of intermittent hypobaric hypoxia (IHH) exposure for health and its potential use as a training tool are well-documented. However, since hypobaric hypoxia and cold are environmental factors always strongly associated in the biosphere, additive or synergistic adaptations could have evolved in animals' genomes. For that reason, the aim of the present study was to investigate body composition and hematological and muscle morphofunctional responses to simultaneous intermittent exposure to hypoxia and cold. Adult male rats were randomly divided into four groups: (1) control, maintained in normoxia at 25°C (CTRL); (2) IHH exposed 4 h/day at 4,500 m (HYPO); (3) intermittent cold exposed 4 h/day at 4°C (COLD); and (4) simultaneously cold and hypoxia exposed (COHY). At the end of 9 and 21 days of exposure, blood was withdrawn and gastrocnemius (GAS) and tibialis anterior muscles, perigonadal and brown adipose tissue, diaphragm, and heart were excised. GAS transversal sections were stained for myofibrillar ATPase and succinate dehydrogenase for fiber typing and for endothelial ATPase to assess capillarization. Hypoxia-inducible factor 1α (HIF-1α), vascular endothelial growth factor (VEGF), and glucose transporter 1 (GLUT1) from GAS samples were semi-quantified by Western blotting. COLD and HYPO underwent physiological adjustments such as higher brown adipose tissue weight and increase in blood-related oxygen transport parameters, while avoiding some negative effects of chronic exposure to cold and hypoxia, such as body weight and muscle mass loss. COHY presented an additive erythropoietic response and was prevented from right ventricle hypertrophy. Intermittent cold exposure induced muscle angiogenesis, and IHH seems to indicate better muscle oxygenation through fiber area reduction.

Implication of gut microbiota in the physiology of rats intermittently exposed to cold and hypobaric hypoxia.

This study examines the influence of intermittent exposure to cold, hypobaric hypoxia, and their combination, in gut microbiota and their metabolites in vivo, and explores their effects on the physiology of the host. Sprague-Dawley rats were exposed to cold (4°C), hypobaric hypoxia (462 torr), or both simultaneously, 4 h/day for 21 days. Biometrical and hematological parameters were monitored. Gut bacterial subgroups were evaluated by qPCR and short-chain fatty acids were determined by gas chromatography in caecum and feces. Cold increased brown adipose tissue, Clostridiales subpopulation and the concentration of butyric and isovaleric acids in caecum. Hypobaric hypoxia increased hemoglobin, red and white cell counts and Enterobacteriales, and reduced body and adipose tissues weights and Lactobacilliales. Cold plus hypobaric hypoxia counteracted the hypoxia-induced weight loss as well as the increase in white blood cells, while reducing the Bacteroidetes:Firmicutes ratio and normalizing the populations of Enterobacteriales and Lactobacilliales. In conclusion, intermittent cold and hypobaric hypoxia exposures by themselves modified some of the main physiological variables in vivo, while their combination kept the rats nearer to their basal status. The reduction of the Bacteroidetes:Firmicutes ratio and balanced populations of Enterobacteriales and Lactobacilliales in the gut may contribute to this effect.

Self-Paced Free-Running Wheel Mimics High-Intensity Interval Training Impact on Rats' Functional, Physiological, Biochemical, and Morphological Features.

Free-running wheel (FRW) is an animal exercise model that relies on high-intensity interval moments interspersed with low-intensity or pauses apparently similar to those performed in high-intensity interval training (HIIT). Therefore, this study, conducted over a 12-weeks period, aimed to compare functional, thermographic, biochemical and morphological skeletal and cardiac muscle adaptations induced by FRW and HIIT. Twenty-four male Wistar rats were assigned into three groups: sedentary rats (SED), rats that voluntarily exercise in free wheels (FRW) and rats submitted to a daily HIIT. Functional tests revealed that compared to SED both FRW and HIIT increased the ability to perform maximal workload tests (MWT-cm/s) (45 ± 1 vs. 55 ± 2 and vs. 65 ± 2). Regarding thermographic assays, FRW and HIIT increased the ability to lose heat through the tail during MWT. Histochemical analyzes performed in tibialis anterior (TA) and soleus (SOL) muscles showed a general adaptation toward a more oxidative phenotype in both FRW and HIIT. Exercise increased the percentage of fast oxidative glycolytic (FOG) in medial fields of TA (29.7 ± 2.3 vs. 44.9 ± 4.4 and vs. 45.2 ± 5.3) and slow oxidative (SO) in SOL (73.4 ± 5.7 vs. 99.5 ± 0.5 and vs. 96.4 ± 1.2). HITT decreased fiber cross-sectional area (FCSA-µm2) of SO (4350 ± 286.9 vs. 4893 ± 325 and vs. 3621 ± 237.3) in SOL. Fast glycolytic fibers were bigger across all the TA muscle in FRW and HIIT groups. The FCSA decrease in FOG fibers was accompanied by a circularity decrease of SO from SOL fibers (0.840 ± 0.005 vs. 0.783 ± 0.016 and vs. 0.788 ± 0.010), and a fiber and global field capillarization increase in both FRW and HIIT protocols. Moreover, FRW and HIIT animals exhibited increased cardiac mitochondrial respiratory control ratio with complex I-driven substrates (3.89 ± 0.14 vs. 5.20 ± 0.25 and vs. 5.42 ± 0.37). Data suggest that FRW induces significant functional, physiological, and biochemical adaptations similar to those obtained under an intermittent forced exercise regimen, such as HIIT.

Additive Effects of Intermittent Hypobaric Hypoxia and Endurance Training on Bodyweight, Food Intake, and Oxygen Consumption in Rats.

Cabrera-Aguilera, Ignacio, David Rizo-Roca, Elisa A. Marques, Garoa Santocildes, Teresa Pagès, Gines Viscor, António A. Ascensão, José Magalhães, and Joan Ramon Torrella. Additive effects of intermittent hypobaric hypoxia and endurance training on bodyweight, food intake, and oxygen consumption in rats. High Alt Med Biol. 19:278-285, 2018.-We used an animal model to elucidate the effects of an intermittent hypobaric hypoxia (IHH) and endurance exercise training (EET) protocol on bodyweight (BW), food and water intake, and oxygen consumption. Twenty-eight young adult male rats were divided into four groups: normoxic sedentary (NS), normoxic exercised (NE), hypoxic sedentary (HS), and hypoxic exercised (HE). Normoxic groups were maintained at an atmospheric pressure equivalent to sea level, whereas the IHH protocol consisted of 5 hours per day for 33 days at a simulated altitude of 6000 m. Exercised groups ran in normobaric conditions on a treadmill for 1 hour/day for 5 weeks at a speed of 25 m/min. At the end of the protocol, both hypoxic groups showed significant decreases in BW from the ninth day of exposure, reaching final 10% (HS) to 14.5% (HE) differences when compared with NS. NE rats also showed a significant weight reduction after the 19th day, with a decrease of 7.4%. The BW of hypoxic animals was related to significant hypophagia elicited by IHH exposure (from 8% to 12%). In contrast, EET had no effect on food ingestion. Total water intake was not affected by hypoxia but was significantly increased by exercise. An analysis of oxygen consumption at rest (mL O2/[kg·min]) revealed two findings: a significant decrease in both hypoxic groups after the protocol (HS, 21.7 ± 0.70 vs. 19.1 ± 0.78 and HE, 22.8 ± 0.80 vs. 17.1 ± 0.90) and a significant difference at the end of the protocol between NE (21.3 ± 0.77) and HE (17.1 ± 0.90). These results demonstrate that IHH and EET had an additive effect on BW loss, providing evidence that rats underwent a metabolic adaptation through a reduction in oxygen consumption measured under normoxic conditions. These data suggest that the combination of IHH and EET could serve as an alternative treatment for the management of overweight and obesity.