Biomedical subjects

H J Helminen

Publications and source records attributed to H J Helminen.

At least 19 recordsLinked to original sources

PubMed1996

Distribution of hyaluronan in articular cartilage as probed by a biotinylated binding region of aggrecan.

The proportion of total tissue hyaluronan involved in interactions with aggrecan and link protein was estimated from extracts of canine knee articular cartilages using a biotinylated hyaluronan binding region-link protein complex (bHABC) of proteoglycan aggregate as a probe in an ELISA-like assay. Microscopic sections were stained with bHABC to reveal free hyaluronan in various sites and zones of the cartilages. Articular cartilage, cut into 20 microns-thick sections, was extracted with 4 M guanidinium chloride (GuCl). Aliquots of the extract (after removing GuCl) were assayed for hyaluronan, before and after papain digestion. The GuCl extraction residues were analyzed after solubilization by papain. It was found that 47-51% of total hyaluronan remained in the GuCl extraction residue, in contrast to the 8-15% of total proteoglycans. Analysis of the extract revealed that 24-50% of its hyaluronan was directly detectable with the probe, while 50-76% became available only after protease digestion. The extracellular matrix in cartilage sections was stained with the bHABC probe only in the superficial zone and the periphery of the articular surfaces, both sites known to have a relatively low proteoglycan concentration. Trypsin pretreatment of the sections enhanced the staining of the intermediate and deep zones, presumably by removing the steric obstruction caused by the chondroitin sulfate binding region of aggrecans. Enhanced matrix staining in these zones was also obtained by a limited digestion with chondroitinase ABC. The results indicate that a part of cartilage hyaluronan is free from endogenous binding proteins, such as aggrecan and link protein, but that the chondroitin sulfate-rich region of aggrecan inhibits its probing in intact tissue sections. Therefore, hyaluronan staining was more intense in cartilage areas with lower aggrecan content. A large proportion of hyaluronan resists GuCl extraction, even from 20-micrograms-thick tissue sections.

Aggrecans↗

PubMed1995

Lifelong moderate running training increases the incidence and severity of osteoarthritis in the knee joint of C57BL mice.

BACKGROUND: Inbred C57BL male mice express a high incidence of spontaneous osteoarthritis of the knee joint at the age of 18 months. We used this strain of mice to find out the effects of life-long, moderate running exercise on the health of articular cartilage and the incidence of osteoarthritis. METHODS: Male mice (294) were divided into controls and runners. The runners were trained daily between 2 and 18 months of age. The speed was 13.3 m/min and the distance on a flatbelt treadmill was 1,000 m/day. The mice were sacrificed at the ages of 2, 6, 10, 14, and 18 months. The knee joints were sectioned in frontal direction and the osteoarthritic changes were graded using a conventional light microscope. The reproducibility of the grading method was tested by calculating the extended kappa-coefficient for the results of six researchers. RESULTS: The incidence of osteoarthritis at the age of 18 months increased from 72% in controls to 88% in runners in the medial tibial condyles (P < 0.05), and from 80 to 96% in the lateral tibial condyles (P < 0.001). The incidence of the most severe osteoarthritic changes rose from 16% in controls to 36% in runners in the medial tibial condyles, and from 4 to 36% in the lateral tibial condyles. CONCLUSION: According to our results, the moderate, long-lasting running exercise accelerates the development of osteoarthritis in the knee joints of C57BL mice.

Animals↗

PubMed1995

Influence of short-term hydrostatic pressure on organization of stress fibers in cultured chondrocytes.

The present study describes changes in the organization of stress fibers that occur in articular cartilage chondrocytes subjected to hydrostatic pressure. Primary cultures of chondrocytes from bovine articular cartilage, grown on coverslips, were subjected to 5, 15, or 30 MPa hydrostatic pressure at 37 degrees C. The pressure was applied continuously or cyclically at two frequencies: 0.125 Hz (4 seconds of pressure and 4 seconds of no pressure) or 0.05 Hz (1 second of pressure and 19 seconds of no pressure) for a period of 2 hours. Control chondrocytes showed a polygonal form with prominent stress fibers extending across the cells. The exposure of cells to 30 MPa pressure caused a nearly total disappearance of stress fibers and retraction of the cells from each other. With pressure at 15 MPa or cyclic pressure, the number of cells with stress fibers was decreased. In cells subjected to 5 MPa pressure, the stress fibers resembled those in control chondrocytes. The pressure effects were reversible after 2 hours. Pressure had no effect on the staining pattern of vinculin, which suggests that microfilaments are more vulnerable to pressure than vinculin. The results indicate that cytoskeletal changes may be an integral part of the response of chondrocytes to hydrostatic pressure.

Animals↗

PubMed1995

Long-distance running alters bone anthropometry, elemental composition and mineral density of young dogs.

Ten young beagle dogs were run on a treadmill for 1 year, the last 15 weeks 40 km/day. Thereafter bone anthropometric variables, elemental composition, mineral density (BMD) and serum markers of bone metabolism were analyzed and compared with matched-paired control dogs. More osteophytes developed, weight-bearing bones grew larger and BMD of axial skeleton was reduced in the trained group. Zinc and sodium accumulated in bones of the runners. Zinc correlated negatively with BMD. Serum osteocalcin and C-terminal propeptide of type I procollagen did not correlate with the observed differences in BMD. This experiment implies that intensive endurance training in adolescence may reduce the peak bone mass and alter the structural properties of bone.

Animals↗

PubMed1994

Articular cartilage thickness and glycosaminoglycan distribution in the young canine knee joint after remobilization of the immobilized limb.

The recovery of articular cartilage from atrophy induced by joint immobilization was investigated in immature dogs. In a previous study, we showed that 11 weeks of immobilization of the knee (stifle) joint of young dogs reduced the concentration of articular cartilage glycosaminoglycans (GAGs) by 13-47%. In the present study, right hindlimbs from six female beagles were immobilized for 11 weeks, as in the previous study, and then were remobilized for 15 weeks. Cartilage from the knee joint was compared with cartilage from nonimmobilized knees of eight age-matched control beagles. Histological samples taken from 11 different locations of the knee joint were stained with safranin O, and microspectrophotometry was used to demonstrate distribution of GAGs in the tissue. After remobilization, GAG concentration was restored in the patellofemoral region and tibial condyles. On the summits of the femoral condyles, and especially at the periphery of the femoral condyles, GAG concentration remained 8-26% less than the control values. On the summits, the thickness of the uncalcified cartilage was as much as 15% less than in the age-matched controls. Consequently, the changes induced by unloading were reversible to a great extent, but a full restoration of articular cartilage was not obtained at all sites of the knee joint within the 15 weeks of remobilization. Immobilization of the skeletally immature joint therefore may affect the development of articular cartilage in such a way that very slow recovery or permanent alterations are induced.

Animals↗

PubMed1994

Running training alters fiber type composition in spinal muscles.

The issue of whether exercise can induce changes in muscle fiber types has been long debated. Knowledge about the alterations in spinal muscle fiber types is scarce. In this study, the alterations initiated by long-distance running on spinal muscle fiber type distribution was studied. Ten young dogs were run on a treadmill for 55 weeks, 5 days a week, and ten dogs from the same litters served as controls. The daily running distance was gradually increased to 40 km and maintained at that level for the final 15 weeks. Histological sections were prepared from the cervical, thoracic, and lumbar multifidus muscles and the medial and lateral heads of triceps brachii and analyzed for the fiber type composition and cross-sectional area of fibers. In the lumbar multifidus, the numerical percentage of the muscle fibers with low oxidative capacity (type II) increased significantly in the running group. However, in the thoracic and cervical spine multifidus, the response to running resembled more of the significant shift from type II to type I fibers (with high oxidative capacity), which was also observed in the triceps brachii muscle. In these muscles, the quantitative image analysis of nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR) reaction also demonstrated a shift towards a higher oxidative capacity within the type II fibers. The results show that training can induce changes in fiber type composition not only in limb muscles but also in the stabilizing spinal muscles.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Softening of the lateral condyle articular cartilage in the canine knee joint after long distance (up to 40 km/day) running training lasting one year.

The effects of long distance running training (up to 40km/day) on the biomechanical properties of young canine articular cartilage were investigated using in situ indentation creep technique. The stiffness of articular cartilage was determined in the form of instantaneous and equilibrium shear moduli. The rate of cartilage deformation was also computed. Microspectrophotometric and polarized light microscopic analyses were made for determination of local glycosaminoglycan content and collagen organization, respectively. During a period of one year, the runner dogs (n = 10) underwent a gradually increased treadmill exercise up to 40 km/day, while the littermate control dogs (n = 10) lived normally in their cages. The equilibrium shear modulus of articular cartilage decreased significantly by 12 to 14% (p < 0.05) in the lateral, but not in the medial, condyles of femur and tibia. In the lateral condyles of the runner dogs, the deformation rate of cartilage increased by 16% (p < 0.05). Consequently, the articular cartilage response to running training was site-dependent. The simultaneous changes of the equilibrium shear modulus or the retardation time spectrum and the glycosaminoglycan content confirm the key role of proteoglycans in modulating the cartilage equilibrium stiffness and creep rate. The changes in the instant shear modulus appeared to be predominantly related to the alterations in the organization of the superficial collagen network. Although the running training did not create cartilage damage, we assume that the softening of the cartilage may with time jeopardize the ability of articular cartilage to maintain its normal structural and functional properties.

Animals↗

PubMed1994

Effect of endurance training on atrial natriuretic peptide gene expression in normal and hypertrophied hearts.

We studied the effects of physical endurance training on atrial natriuretic peptide (ANP) gene expression in beagle dogs, Wistar rats, and spontaneously hypertensive rats (SHR). The dogs underwent a gradually increased running training up to 40 km/day on a treadmill for 55 wk while the nontrained sibling control dogs were kept in their cages throughout the study. Endurance training caused a significant 13% (P < 0.05) increase in ventricular hypertrophy but did not change plasma immunoreactive (ir)-ANP levels at rest or ventricular ANP mRNA or irANP levels. When normotensive Wistar rats ran up to 2,200 m/day for 8 wk, no significant change was seen in ventricular hypertrophy or in plasma or ventricular irANP levels at rest compared with nontrained controls. However, endurance training caused a 2.2-fold increase in epicardial ANP mRNA levels (P < 0.05). In the SHR strain, running training up to 900 m/day for 31 wk increased ventricular hypertrophy of trained SHR by 7% (P < 0.01) and caused a concomitant 1.6- to 1.7-fold elevation in ventricular irANP and ANP mRNA levels (P < 0.01-0.001) compared with nontrained SHR. In contrast, changes in atrial ANP mRNA or irANP levels in response to training were small in all three protocols. This study shows that in the normal heart induction of ANP synthesis by endurance training is not associated with ventricular hypertrophy. Moreover, the common stimulus for ventricular ANP synthesis induced by both chronic pressure overload and physical training may be mechanical stretching of cardiac myocytes, because endurance training further stimulated ANP synthesis in hypertrophied ventricles in SHR.

Animals↗

PubMed1994

Proteoglycan and collagen alterations in canine knee articular cartilage following 20 km daily running exercise for 15 weeks.

The composition of extracellular matrix was studied at 11 different sites in the knee (stifle) articular cartilage of young beagle dogs after running exercise of 15 weeks, 20 km/day. Water content was significantly elevated by 5-17% in the patellofemoral groove and in anterior and intermediate sites on the lateral condyle of femur. Collagen content was decreased by 14 to 20% in the same sites of the lateral condyle. Proteoglycan (PG) content was not significantly changed except in the posterior edge of the medial condyle of femur with a 30% decrease. The proportion of PGs capable of reaggregation with hyaluronan was increased in tibial and femoral surfaces (mean of all sites +18%). Also, the aggregating PG monomers were larger at all sites, as studied by agarose gel electrophoresis. The chondroitin-6 to 4-sulphate ratio was reduced at the summits of femoral condyles and patella by 10 to 25%, but increased in the patellar surface of femur and tibial medial condyle, fitting to a previous finding that strenuous running depleted proteoglycans at the summits of femoral condyle from the superficial zone that is rich in chondroitin-6-sulphate. The increased water content, accompanied with a decreased concentration of collagen in the lateral femoral condyle, suggests loosening of the collagenous framework, an idea compatible with an earlier notion of superficial depletion of PGs in these sites, and possibly predisposing to degeneration. The size increase of the aggregating PGs probably indicate that a larger proportion of matrix PGs were newly synthesized and hence the turnover rate of the PGs was enhanced. It is concluded that the strenuous running program induced locally restricted changes resembling early degeneration of articular cartilage, while simultaneously caused alterations that suggest a general stimulation of proteoglycan metabolism.

Animals↗

PubMed1993

Long-distance running causes site-dependent decrease of cartilage glycosaminoglycan content in the knee joints of beagle dogs.

OBJECTIVE: To study the effects of a long-term (1-year) program of running exercise (up to 40 km/day) on the thickness and glycosaminoglycan (GAG) content of articular cartilage in the knee and humeral head cartilage of young dogs. METHODS: Samples for histologic analysis were obtained from 12 different locations of the joints. We conducted a detailed, area-specific analysis, measuring the thickness of articular cartilage and analyzing the distribution of Safranin O stain that binds stoichiometrically to GAG as determined by quantitative microspectrophotometry. RESULTS: Running exercise decreased the GAG content of the uncalcified articular cartilage in the weight-bearing summits of the femoral condyles by 5-13% (P < 0.05), while at margins of these areas the GAG content was equivalent to control levels. In the lateral condyle of the femur, the reduction was most prominent in the superficial zone (up to 28% decrease; P < 0.05), and extended into the intermediate zone (11% decrease; P < 0.05). GAG content was also significantly reduced in the superficial zone at the lateral condyle of the tibia and the head of the humerus, by 35% (P < 0.01) and 15% (P < 0.05), respectively. Running did not alter GAG concentration in the patellofemoral region. CONCLUSION: The GAG depletion caused by 40-km/day running exercise is restricted to prominent weight-bearing areas of the joint and begins from the superficial cartilage without signs of degeneration. The different degree and type of joint loading can explain the site-dependent cartilage response to long-distance running. The loss of GAGs was possibly due to breakdown of proteoglycans, which could not be compensated for by neosynthesis of molecules. With time, this may affect the condition of articular cartilage, especially if the joint is exposed to loading for lengthy periods.

Animals↗

PubMed1993

Proteoglycan synthesis in canine intervertebral discs after long-distance running training.

The alterations and distribution of proteoglycan (PG) synthesis in the intervertebral discs of young dogs exercised with long-distance running (40 km/day) were studied with a method based on image analysis of tissue sections. Ten dogs were run on a treadmill daily for 55 weeks, and 10 dogs from the same litters served as controls. The daily running distance gradually was increased to 40 km and was maintained at that level for the final 15 weeks. Midsagittal disc segments C7-T1, T8-9, and L1-2 were labeled with 35SO4, and histological sections of the segments were apposed against autoradiographic film to determine the synthesis of PGs. Next, the same sections were stained with safranin O to estimate possible alterations in PG concentration. The radiographs and stained sections were digitized with a flatbed scanner and measured by image analysis. The lumbar discs of runners displayed a significantly lower rate of 35SO4 incorporation, while a tendency toward enhanced incorporation was seen in the cervical and thoracic discs. Safranin O staining showed a pattern just opposite to 35SO4 incorporation: decreased staining in the cervical and thoracic discs and increased staining in the lumbar discs of the runners. The results demonstrate qualitatively different influences of long-term running training on PG metabolism at the cervical, thoracic, and lumbar levels in young dogs.

Animals↗

PubMed1993

Effects of cyclic hydrostatic pressure on proteoglycan synthesis in cultured chondrocytes and articular cartilage explants.

Primary chondrocyte cell cultures and explants of bovine articular cartilage were subjected to cyclic hydrostatic pressure in a novel computer-controlled pressure chamber designed for this purpose. The cultures were labeled with 5 microCi/ml 35SO4 and simultaneously pressurized with 5 MPa load for 1.5 or 20 h with pressure cycles of 0.0167, 0.05, 0.25, and 0.5 Hz. The chondrocyte cell cultures were also subjected to 0.0082 and 0.0034 Hz cycles. Sulfate incorporation was significantly inhibited in cell cultures subjected to the 0.5, 0.25, or 0.05 Hz cyclic loads for 1.5 h, but stimulated in explant cultures with a 0.5 Hz cyclic 1.5-h load. Chondrocyte cultures subjected to longer (20 h) loading showed a stimulation of sulfate incorporation with 0.5 and 0.25 Hz cycles, but an inhibition with 0.0167 Hz. The results indicate that cyclic hydrostatic pressures of presumably physiological magnitude have significant influences on proteoglycan synthesis in articular cartilage chondrocytes. Comparison of the cell and explant cultures under identical pressure conditions suggested that chondrocyte interactions with extracellular matrix are involved in this regulation by cyclic hydrostatic pressure. The responses of the chondrocytes to pressurization also varied according to the total length of the treatment, a finding compatible with the idea of multiple metabolic steps in chondrocytes, both pre- and post-translational, controlled by the ambient hydrostatic pressure.

Animals↗

PubMed1993

Effects of long-term running exercise on canine femoral head articular cartilage.

After long-term running program (40 km/day) anterior and posterior tissue samples from canine femoral head were labeled ex vivo in the presence of 35S-SO4. Sulfate incorporation rates did not differ between runner and control groups. The statistically significant changes in runners included a decreased uronic acid concentration (p < or = 0.02) and proportion of extractable proteoglycans (p < or = 0.05) as well as increased concentration of tissue uronic acid after 4 M GuCl extraction (p < or = 0.05) in the posterior area. These results support an idea of strengthened cartilage tissue after this kind of motion and load.

Animals↗

PubMed1993

Flat bed scanner in the quantitative assay of 35SO4-incorporation by X-ray film autoradiography of intervertebral disc sections.

A rapid quantitation of proteoglycan synthesis distribution in intervertebral disc and endplates is described. Tissue blocks of disc (C7-Th1) in the midsagittal plane from ten female beagles were incubated in the presence of 35SO4 and prepared as histological slides. For comparison, sulphate incorporation rates in the C5-C6 discs were assayed by liquid scintillation. Autoradiographic film exposed against the labelled sections was developed and digitized for image analysis using a 256 grey level flat bed table scanner connected to a microcomputer. The film density versus dpm (disintegrations per minute) calibration was performed using a set of 35SO4-labelled glycosaminoglycan standards applied on the same film. Since section thickness, dpm calibration of the film density and the specific activity of sulphate in the medium were known, the incorporations per tissue volume could be calculated. The average incorporation rates of the anterior and posterior annulus fibrosus, nucleus pulposus and vertebral endplates were 5.2 +/- 0.9, 5.2 +/- 0.8, 4.5 +/- 0.6 and 4.1 +/- 0.8 pmol/mm3 per h (+/- SE, n = 10), respectively and closely corresponded to those obtained by liquid scintillation. This method offers a convenient and reproducible way to measure the rate of proteoglycan synthesis in large tissue sections but also in thin cartilaginous tissues such as the vertebral endplate.

Animals↗

PubMed1993

Effects of aerobic long distance running training (up to 40 km.day-1) of 1-year duration on blood and endocrine parameters of female beagle dogs.

The effects of long distance running training on blood parameters, hormone responses and bone growth were studied in young growing dogs. A genetically uniform group of female beagles matched with respect to age and body mass were used. The runner dogs (n = 10) underwent gradually increased running exercise up to 40 km.day-1 on a treadmill with 15 degrees uphill gradient 5 days each week during a period of 1 year, while the littermate control dogs (n = 10) were kept in their cages throughout the study. Low plasma lactate concentrations of the runners measured immediately after the running training indicated the aerobic metabolism of the dogs while running. Significant decreases of blood haemoglobin concentrations (11%), blood erythrocyte number (10%), and erythrocyte packed cell volume (12%) were found in the runner group. Throughout the experiment, the value of thyroxine was slightly lower (13%) in the runners but no changes were found in tri-iodothyronine, free thyroxine, or cortisol serum concentrations. Serum oestradiol concentration at 56 weeks was significantly lower (42%) in the runner group than in the control group but was not as low (27%) at 70 weeks. Somatomedin-C concentration had decreased significantly by 37% at the age of 56 weeks in the runner group but was again at the level of the control dogs at the end of experiment (at 70 weeks). Ulna and radius bone mass as a ratio to the body mass had significantly increased in the runners. It would seem from our study that long distance running has a positive effect on bone growth.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Effect of running exercise on proteoglycans and collagen content in the intervertebral disc of young dogs.

Collagen and proteoglycans in the intervertebral disc (LI-II) of young beagle dogs (age 55 weeks) were analyzed following a 15 weeks' daily 20 km running training on a treadmill with 15 degree uphill inclination. In nucleus pulposus no statistically significant alterations were found in the content of proteoglycans or collagen. In annulus fibrosus the total tissue wet weight and total amount of collagen (hydroxyproline) increased by 34-36% in the runners as compared to age-matched, untrained controls. Since the total amount of proteoglycans did not increase, the annulus fibrosus became relatively depleted of proteoglycans, as indicated by the 27% reduction in uronic acid concentration, expressed either per wet weight or hydroxyproline. The average molecular size of the remaining nonaggregating proteoglycans was larger, and there was also a trend towards increased proportion of proteoglycans aggregating with hyaluronan. Most of the chondroitin sulfate side chains were 6-sulfated (65-66%). Running did not alter the sulfation or length of the chondroitin sulfate chains. The decreased proteoglycan/collagen ratio in annulus fibrosus may result in altered mechanical properties of the tissue and reflects its adaptation to enhanced motion and stress.

Animals↗

PubMed1993

Altered Golgi apparatus in hydrostatically loaded articular cartilage chondrocytes.

OBJECTIVES: Articular cartilage proteoglycan content is controlled by joint loading. This study aimed to elucidate the role of hydrostatic pressure in this regulation. METHODS: Primary cultures of chondrocytes from bovine articular cartilage, grown on coverslips, were subjected to 5, 15, or 30 MPa hydrostatic pressure, applied continuously or cyclically at 0.125 or 0.05 Hz. The Golgi apparatus was visualised either by a fluorochrome coupled wheat germ agglutinin or by transmission electron microscopy. Proteoglycan synthesis was studied by the incorporation of sulphur-35 labelled sulphate. RESULTS: After 30 MPa continuous hydrostatic pressure, the Golgi apparatus was observed in a compact form with a concomitant decrease in proteoglycan synthesis. The normal stacked appearance of the Golgi apparatus was no more visible in the electron microscopy preparation of the pressurised chondrocytes. This effect was reversible and was also noticed after 15 MPa continuous load, though to a minor extent. Cyclic pressures (5-30 MPa) caused no apparent change in the Golgi apparatus. The shape of some cells changed to a more retracted form after 30 MPa continuous pressure. Nocodazole, which causes disassembly of the microtubules, blocked the compacting influence of pressurisation on the Golgi apparatus, and reduced proteoglycan synthesis to about half of the control level. CONCLUSIONS: The packing of the Golgi apparatus is dependent on microtubules and may contribute to the inhibition of proteoglycan synthesis observed in articular cartilage subjected to high hydrostatic pressure.

Animals↗

PubMed1993

Adaptation of canine femoral head articular cartilage to long distance running exercise in young beagles.

OBJECTIVE: To study the effects of long term (one year), long distance (up to 40 km/day) running on the metabolism of articular cartilage the biosynthesis of proteoglycans was examined by in vitro labelling of anterior (weight bearing) and posterior (less weight bearing) areas of the femoral head from young beagles. METHODS: Total sulphate incorporation rates were determined and distribution of the incorporated sulphate was localised by quantitative autoradiography. Concentration and extractability of the proteoglycans were determined, and proteoglycan structures were investigated by gel filtration chromatography, agarose gel electrophoresis, and chemical determinations. RESULTS: In the less weight bearing area the amount of extractable proteoglycans was decreased (p < or = 0.02), simultaneously with an increased concentration of residual glycosaminoglycans in the tissue after 4 M GuCl extraction (p < or = 0.05). In control animals proteoglycan synthesis was most active in the deep zone of the cartilage, whereas exercise increased synthesis in the intermediate zone. There was a tendency to a lower keratan: chondroitin sulphate ratio in the running dogs. No macroscopical or microscopical signs of articular degeneration or injury were visible in any of the animals. CONCLUSION: The articular cartilage of the femoral head showed a great capacity to adapt to the increased mechanical loading. The reduced proteoglycan extractability in the less weight bearing area changed it similar to the weight bearing area, suggesting that the low extractability of proteoglycans reflects the long term loading history of articular cartilage. The congruency of the femoral head with acetabulum seems to protect the cartilage from the untoward alterations that occur in the femoral condyles subjected to a similar running programme.

Adaptation, Physiological↗