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In this study, we propose a novel three-criteria performance score to semiquantitatively classify the running style, the degree of involvement and compliance and the validity of electric shock count for rats exercising on a treadmill. Each score criterion has several style-marks that are based on the observational registry of male Sprague-Dawley rats running for 4-7 weeks. Each mark was given a score value that was averaged throughout a session-registry and resulting in a session score for each criterion, ranging from "0" score for a hypothetical "worst runner", to score "1" for a hypothetical "perfect runner" rat. We found significant differences throughout a training program, thus providing evidence of sufficient sensitivity of this score to reflect the individual evolution of performance improvement in exercise capacity due to training. We hypothesize that this score could be correlated with other physiological or metabolic parameters, thus refining research results and further helping researchers to reduce the number of experimental subjects.

Altitude training has become increasingly popular in recent decades. Its central and peripheral effects are well-described; however, few studies have analyzed the effects of intermittent hypobaric hypoxia (IHH) alone on skeletal muscle morphofunctionality. Here, we studied the effects of IHH on different myofiber morphofunctional parameters, investigating whether contractile activity is required to elicit hypoxia-induced adaptations in trained rats. Eighteen male Sprague-Dawley rats were trained 1 month and then divided into three groups: (1) rats in normobaria (trained normobaric inactive, TNI); (2) rats subjected daily to a 4-h exposure to hypobaric hypoxia equivalent to 4,000 m (trained hypobaric inactive, THI); and (3) rats subjected daily to a 4-h exposure to hypobaric hypoxia just before performing light exercise (trained hypobaric active, THA). After 2 weeks, the tibialis anterior muscle (TA) was excised. Muscle cross-sections were stained for: (1) succinate dehydrogenase to identify oxidative metabolism; (2) myosin-ATPase to identify slow- and fast-twitch fibers; and (3) endothelial-ATPase to stain capillaries. Fibers were classified as slow oxidative (SO), fast oxidative glycolytic (FOG), fast intermediate glycolytic (FIG) or fast glycolytic (FG) and the following parameters were measured: fiber cross-sectional area (FCSA), number of capillaries per fiber (NCF), NCF per 1,000 μm2 of FCSA (CCA), fiber and capillary density (FD and CD), and the ratio between CD and FD (C/F). THI rats did not exhibit significant changes in most of the parameters, while THA animals showed reduced fiber size. Compared to TNI rats, FOG fibers from the lateral/medial fields, as well as FIG and FG fibers from the lateral region, had smaller FCSA in THA rats. Moreover, THA rats had increased NCF in FG fibers from all fields, in medial and posterior FIG fibers and in posterior FOG fibers. All fiber types from the three analyzed regions (except the posterior FG fibers) displayed a significantly increased CCA ratio compared to TNI rats. Global capillarisation was also increased in lateral and medial fields. Our results show that IHH alone does not induce alterations in the TA muscle. The inclusion of exercise immediately after the tested hypoxic conditions is enough to trigger a morphofunctional response that improves muscle capillarisation.

Unaccustomed eccentric contractions induce muscle damage, calcium homeostasis disruption, and mitochondrial alterations. Since exercise and hypoxia are known to modulate mitochondrial function, we aimed to analyze the effects on eccentric exercise-induced muscle damage (EEIMD) in trained rats using 2 recovery protocols based on: (i) intermittent hypobaric hypoxia (IHH) and (ii) IHH followed by exercise. The expression of biomarkers related to mitochondrial biogenesis, dynamics, oxidative stress, and bioenergetics was evaluated. Soleus muscles were excised before (CTRL) and 1, 3, 7, and 14 days after an EEIMD protocol. The following treatments were applied 1 day after the EEIMD: passive normobaric recovery (PNR), 4 h daily exposure to passive IHH at 4000 m (PHR) or IHH exposure followed by aerobic exercise (AHR). Citrate synthase activity was reduced at 7 and 14 days after application of the EEIMD protocol. However, this reduction was attenuated in AHR rats at day 14. PGC-1α and Sirt3 and TOM20 levels had decreased after 1 and 3 days, but the AHR group exhibited increased expression of these proteins, as well as of Tfam, by the end of the protocol. Mfn2 greatly reduced during the first 72 h, but returned to basal levels passively. At day 14, AHR rats had higher levels of Mfn2, OPA1, and Drp1 than PNR animals. Both groups exposed to IHH showed a lower p66shc(ser36)/p66shc ratio than PNR animals, as well as higher complex IV subunit I and ANT levels. These results suggest that IHH positively modulates key mitochondrial aspects after EEIMD, especially when combined with aerobic exercise.

Unaccustomed eccentric exercise leads to muscle morphological and functional alterations, including microvasculature damage, the repair of which is modulated by hypoxia. We present the effects of intermittent hypobaric hypoxia and exercise on recovery from eccentric exercise-induced muscle damage (EEIMD). Soleus muscles from trained rats were excised before (CTRL) and 1, 3, 7, and 14 days after a double session of EEIMD protocol. A recovery treatment consisting of one of the following protocols was applied 1 day after the EEIMD: passive normobaric recovery (PNR), a 4-h daily exposure to passive hypobaric hypoxia at 4,000 m (PHR), or hypobaric hypoxia exposure followed by aerobic exercise (AHR). EEIMD produced an increase in the percentage of abnormal fibers compared with CTRL, and it affected the microvasculature by decreasing capillary density (CD, capillaries per mm2) and the capillary-to-fiber ratio (CF). After 14 days, AHR exhibited CD and CF values similar to those of CTRL animals (789 and 3.30 vs. 746 and 3.06) and significantly higher than PNR (575 and 2.62) and PHR (630 and 2.92). Furthermore, VEGF expression showed a significant 43% increase in AHR when compared with PNR. Moreover, after 14 days, the muscle fibers in AHR had a more oxidative phenotype than the other groups, with significantly smaller cross-sectional areas (AHR, 3,745; PNR, 4,502; and PHR, 4,790 µm2), higher citrate synthase activity (AHR, 14.8; PNR, 13.1; and PHR, 12 µmol·min−1·mg−1) and a significant 27% increment in PGC-1α levels compared with PNR. Our data show that hypoxia combined with exercise attenuates or reverses the morphofunctional alterations induced by EEIMD. NEW & NOTEWORTHY Our study provides new insights into the use of intermittent hypobaric hypoxia combined with exercise as a strategy to recover muscle damage induced by eccentric exercise. We analyzed the effects of hypobaric exposure combined with aerobic exercise on histopathological features of muscle damage, fiber morphofunctionality, capillarization, angiogenesis, and the oxidative capacity of damaged soleus muscle. Most of these parameters were improved after a 2-wk protocol of intermittent hypobaric hypoxia combined with aerobic exercise.

An analytical method for the analysis of six fluoroquinolones (FQs) in animal feeds was developed. The sample treatment consists of a simple and rapid extraction of the analytes by manual shaking with an acetonitrile-water mixture containing hydrochloric acid without further sample cleanup. Matrix effects were minimized by diluting the extract with water. Determination was carried out by liquid chromatography using fluorimetric detection. The method was validated in-house in four different feed matrices (poultry, cow, pig, and lamb feed). Mean recoveries ranging from 80 to 105%, with relative standard deviations below 12%, were achieved from spiked animal feed samples on the 0.2-2.0 μg/g level. No relevant differences were observed between the studied feeds, this ensuring that the method was reliable for a wide variety of feed matrices. Decision limit and detection capability values are below 0.08 and 0.13 mg/kg, respectively, for most FQs. The results obtained demonstrate the feasibility of the analytical method developed for a routine use to control the illegal use of these substances in feeding stuffs.

Unaccustomed eccentric exercise is a well-documented cause of exercise-induced muscle damage. However, in trained subjects muscle injury involves only light or moderate tissue damage. Since trained rats are widely used as a model for skeletal muscle injury, here we propose a semiquantitative scoring tool to evaluate muscle damage in trained rats. Twenty male Sprague-Dawley rats were trained fortwo weeks following a two-week preconditioning period, and randomly divided into two groups: control rats (CTL; n=5) and rats with eccentric exercise-induced muscle damage (INJ; n=15). Injured rats were sacrificed at three time points: 1, 3 and 7 days post injury (n=5 each). Transverse sections from the right soleus were cut (10 µm) and stained with haematoxylin-eosin. Samples were evaluated by two groups of observers (four researchers experienced in skeletal muscle histopathology and four inexperienced) using the proposed tool, which consisted of six items organised in three domains: abnormal fibre morphology, necrotic/(re)degenerating fibres (muscle fibre domain), endomysial and perimysial infiltration (inflammatory state domain) and endomysium and perimysium distension (interstitial compartment domain). We observed the expected time course in the six evaluated items. Furthermore, agreement among observers was evaluated by measuring the Intraclass Correlation Coefficient (ICC). Within the experienced group, items from the muscle fibre and interstitial compartment domains showed good agreement and the two items from the infiltration compartment domain showed excellent agreement. in conclusion, the proposed tool allowed quick and correct evaluation of light to moderate muscle damage in trained rats with good agreement between observers.

Intermittent hypobaric hypoxia (IHH) exposure, accompanied or not with active recovery, can help to skeletal muscle repair. However, the erythropoietic response elicited can disturb blood rheology and thus alter the oxygen delivery to tissues. Male Sprague-Dawley rats were studied in two basal states: untrained and trained and compared with early (1-3 days) and late (7-14 days) stages of damage recovery in three groups of trained rats that had suffered skeletal muscle injury: Control, passive recovery rats; HYP, rats exposed to IHH after muscle damage; and EHYP, trained rats that performed light aerobic exercise sessions in addition to IHH. Hematocrit, RBC count and hemoglobin were only elevated in the late stage of recovery in HYP (13%; 14% and 8%) and EHYP (18%; 13% and 15%) groups. Blood viscosity increased about double for EHYP rats. It is concluded that intermittent exposure to hypobaric hypoxia in combination with light aerobic exercise in normoxia has an erythropoietic effect, but also provides advantageous hemorheological conditions for the perfusion of damaged muscle.

An analytical method for the analysis of six fluoroquinolones (FQs) in animal feeds was developed. The sample treatment consists of a simple and rapid extraction of the analytes by manual shaking with an acetonitrile-water mixture containing hydrochloric acid without further sample cleanup. Matrix effects were minimized by diluting the extract with water. Determination was carried out by liquid chromatography using fluorimetric detection. The method was validated in-house in four different feed matrices (poultry, cow, pig, and lamb feed). Mean recoveries ranging from 80 to 105%, with relative standard deviations below 12%, were achieved from spiked animal feed samples on the 0.2-2.0 μg/g level. No relevant differences were observed between the studied feeds, this ensuring that the method was reliable for a wide variety of feed matrices. Decision limit and detection capability values are below 0.08 and 0.13 mg/kg, respectively, for most FQs. The results obtained demonstrate the feasibility of the analytical method developed for a routine use to control the illegal use of these substances in feeding stuffs.

Our goal was to address if intermittent hypobaric hypoxia (IHH) exposure can help to increase the number of peripheral blood circulating progenitor cells and side population (SP) stem cells, in order to establish the usefulness of this intervention for skeletal muscle repair, because these cells play a role in tissue regeneration. Male Sprague-Dawley rats were studied in two basal states: untrained and trained and compared with 1, 3, 7 and 14 days stages of damage recovery of trained rats that had suffered skeletal muscle injury. Three experimental groups were studied: rats with passive recovery (CTRL); rats exposed to IHH after muscle damage (HYP); and, trained rats that, in addition to IHH, performed light aerobic exercise sessions (EHYP). We observed an increase in hematopoietic stem cells (HSCs) (mean = 0.153% of cells) and endothelial progenitor cells (EPCs) (mean = 0.0020% of cells) in EHYP on day 7. Also these cells showed characteristics of more primitive progenitors in comparison to the other experimental groups (mean = 0.107% of cells), as deduced by retention of the promising fluorescent probe Vybrant Dye Cycle Violet. We concluded that intermittent exposure to hypobaric hypoxia in combination with light aerobic exercise increased the number of HSCs and EPCs on the 7th day in EHYP group, although the exercise-induced stimulus showed a reverse effect on SP kinetics.

Introduction: Studies are needed to understand the role of CD34 expressing cells with regard to efficient engraftment, especially in the adjuvant treatment of cancer. Materials and Methods: In this study we have used a modified method in our laboratory for routinely counting CD34+ cells. Unlysed whole blood samples were stained with the DNA-selective and cell membrane-permeant Vibrant DyeCycle Violet stain. Results: CD34+ cells exhibit a consistent and differential Vybrant Dye Cycle Violet staining pattern. Based on their different DCV intensity, we classified these subpopulations as CD34+/DCV high and CD34+/DCV low cells. In general, DCV high cells are about 12-times brighter than DCV low cells. Conclusion: DCV staining may be used to discriminate subsets of CD34+ cells similarly to other methods which have previously defined different functional properties that can be related to the characterization, resolution, and purification of primitive hematopoietic stem cells in combination with specific useful markers for multicolor flow cytometric measurements.