Effect of hindlimb suspension on cardiovascular responses to sympathomimetics and lower body negative pressure

To determine whether hindlimb suspension is associated with the development of cardiovascular deconditioning, male rats were studied before and after undergoing one of three treatment conditions for 9 days: 1) cage control (n = 15, CON), 2) horizontal suspension (n = 15, HOZ), and 3) head-down suspension (n = 18, HDS). Testing included lower body negative pressure administered during chloralose-urethan anesthesia and graded doses of sympathomimetic agents (norepinephrine, phenylephrine, and tyramine) administered to conscious unrestrained animals. Both HDS and HOZ were associated with a small decrease in the hypotensive response to lower body negative pressure. The HOZ group, but not the HDS group, exhibited augmented reflex tachycardia. Furthermore, both HDS and HOZ groups manifested reduced pressor responses to phenylephrine after treatment. These reductions were associated with significantly attenuated increases in mesenteric vascular resistance. However, baroreflex control of heart rate was not altered by the treatment conditions. Collectively, these results indicate that 9 days of HDS in rats does not elicit hemodynamic response patterns generally associated with cardiovascular deconditioning induced by hypogravic conditions.     

Skeletal muscle response to spaceflight, whole body suspension, and recovery in rats

Comparisons of soleus and extensor digitorum longus (EDL) muscles from male Sprague-Dawley rats (350-400 g) after 7 days of weightlessness, 7 and 14 days of whole body suspension (WBS), and 7 days of recovery from WBS and from vivarium controls were made. Muscle mass loss of approximately 30% was observed in soleus after 7 and 14 days of WBS. Measurement of slow- and fast-twitch fibers showed significant alterations. Reductions in cross-sectional areas and increases in fiber densities in soleus after spaceflight and WBS were related to previous findings of muscle atrophy during unloading. Capillary density also showed a marked increase with unloading. Seven days of weightlessness were sufficient to effect a 20 and 15% loss in absolute muscle mass in soleus and EDL, respectively. However, the antigravity soleus was more responsive in terms of cross-sectional area reductions. After 7 days of recovery from WBS, with normal ambulatory loading, the parameters studied showed a reversal to control levels. Muscle plasticity, in terms of fiber and capillary responses, indicated differences in responses in the two types of muscles and further amplified that antigravity posture muscles are highly susceptible to unloading. Studies of recovery from spaceflight for both muscle metabolism and microvascular modifications are further justified.     

Effect of hindlimb suspension on VO2 max and regional blood flow responses to exercise

Male rodents were studied before and after undergoing one of three treatment conditions for 9 days: 1) cage control (n = 15, CON), 2) horizontal suspension (n = 15, HOZ), and 3) head-down suspension (n = 18, HDT). Testing included measurements of maximal O2 uptake (VO2 max) and select cardiovascular responses to graded treadmill exercise. VO2 max expressed on an absolute basis (ml/min) was significantly decreased after HOZ (-14.1 +/- 2.5%) and HDT (-14.3 +/- 2.0%), while being essentially unchanged in CON (-1.0 +/- 3.3%). Significant reductions in body weight were observed after both HOZ (-10.1 +/- 4.2 g) and HDT (-22.5 +/- 3.3 g), whereas CON animals exhibited a significant increase in weight (10.4 +/- 3.8 g). As a result, when VO2 max was normalized for body weight, all groups exhibited similar significant reductions of 6-7%. Although no differences in heart rate and blood pressure response to graded exercise were observed, the HDT group exhibited greater increases in mesenteric resistance at the same absolute exercise intensity. Furthermore, both suspended groups had higher iliac resistance values during exercise at similar relative exercise conditions, suggesting that muscle blood flow during treadmill running may have been reduced after suspension. In general, the decrements associated with the HOZ and HDT conditions were similar. It was concluded that reduction in exercise capacity and altered cardiovascular responses to exercise observed after 6-9 days of suspension were attributable to a combination of hypokinesia, lack of hindlimb weight bearing, or restraint, rather than to hydrostatic influences associated with HDT.     

Exercise intensity and glucose tolerance in trained and nontrained subjects

The improved glucose tolerance and increased insulin sensitivity associated with regular exercise appear to be the result, in large part, of the residual effects of the last bout of exercise. To determine the effects of exercise intensity on this response, glucose tolerance and the insulin response to a glucose load were determined in seven well-trained male subjects [maximal O2 uptake (VO2max) = 58 ml.kg-1.min-1] and in seven nontrained male subjects (VO2max = 49 ml.kg-1.min-1) in the morning after an overnight fast 1) 40 h after the last training session (control), 2) 14 h after 40 min of exercise on a cycle ergometer at 40% VO2max, and 3) 14 h after 40 min of exercise at 80% VO2max. Subjects replicated their diets for 3 days before each test and ate a standard meal the evening before the oral glucose tolerance test. No differences in the 3-h insulin or glucose response were observed between the control trial and before exercise at either 40 or 80% VO2max in the trained subjects. In the nontrained subjects the plasma insulin response was decreased by 40% after a single bout of exercise at either 40 or 80% VO2max (7.0 X 10(3) vs. 5.0 X 10(3), P less than 0.05; 3.8 X 10(3) microU.ml-1.180 min-1, P less than 0.01). The insulin response after a single bout of exercise in the nontrained subjects was comparable with the insulin responses found in the trained subjects for the control and exercise trials.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intensity-controlled treadmill running in rats: VO(2 max) and cardiac hypertrophy

Physiological studies of long-term cardiovascular adaptation to exercise require training regimens that give robust conditioning effects and adequate testing procedures to quantify the outcome. We developed a valid and reproducible protocol for measuring maximal oxygen uptake (VO(2 max)), which was reached at a 25 degrees inclination with a respiratory exchange ratio > 1.05 and blood lactate > 6 mmol/l. The effect of intensity-controlled aerobic endurance training was studied in adult female and male rats that ran 2 h/day, 5 days/wk, in intervals of 8 min at 85-90% of VO(2 max) and 2 min at 50-60% of VO(2 max), with adjustment of exercise level according to VO(2 max) every week. After 7 wk, the increase in VO(2 max) plateaued at 60-70% above sedentary controls. Ventricular weights and myocyte length were up 25-30% and 6-12%, respectively. Work economy, oxygen pulse, and heart rate were sufficiently changed to indicate substantial cardiovascular adaptation. The model mimics important human responses to training and could be used in future studies on cellular, molecular, and integrative mechanisms of improved cardiovascular function.     

Exercise training and incidence of cerebrovascular lesions in stroke-prone spontaneously hypertensive rats

To assess the effects of moderate exercise [40-70% maximal oxygen uptake (VO2max)] on resting blood pressures, the presence of cerebrovascular lesions, and the life spans of stroke-prone hypertensive rats, nontrained and trained male and female rats were assigned to two experimental groups. The first (n = 48) were exercise trained after 38 days of age, whereas the second (n = 44) initiated exercise training when the animals were 134 days of age. To facilitate cerebrovascular lesions, the sodium concentrations in the rat chow and in the drinking solutions were increased. Symptoms utilized to denote the presence of cerebrovascular lesions were irritability, hyperresponsiveness, ataxia, lethargy, unwillingness to run, and combinations thereof. All brains were removed immediately after death, fixed, and evaluated grossly and microscopically for lesions. In the study with the younger animals, training was associated with a 7-9% increase in VO2max that was statistically significant only in animals with no histological evidence of cerebrovascular lesions. For the older animals, a significant 5-8% increase in VO2max was noted for animals with or without lesions. After 42 days of training for both groups, resting blood pressures for the trained groups with histological lesions were significantly lower. However, this trend did not continue, and the older trained rats appeared to have strokes earlier and to die sooner than their nontrained controls. Although 83% of the older animals had subjective evidence for a stroke before they died, the percentage of animals with lesions ranged from 42 to 58%, with the trained groups having higher percentages.(ABSTRACT TRUNCATED AT 250 WORDS)     

Aerobic performance of female marathon and male ultramarathon athletes.

The aerobic performance of thirteen male ultramarathon and nine female marathon runners were studied in the laboratory and their results were related to their times in events ranging in distance from 5 km to 84.64 km. The mean maximal aerobic power output (VO2 max) of the men was 72.5 ml/kg . min compared with 58.2 ml/kg . min (p less than 0.001) in the women but the O2 cost (VO2) for a given speed or distance of running was the same in both sexes. The 5 km time of the male athletes was closely related to their VO2 max (r = -0.85) during uphill running but was independent of relative power output (%VO2 max). However, with increasing distance the association of VO2 max with male athletic performance diminished (but nevertheless remained significant even at 84.64 km), and the relationship between %VO2 max and time increased. Thus, using multiple regression analysis of the form: 42.2 km (marathon) time (h) = 7.445 - 0.0338 VO2 max (ml/kg . min) - 0.0303% VO2 max (r = 0.993) and 84.64 km (London-Brighton) time (h) = 16.998 - 0.0735 VO2 max (ml/kg . min) - 0.0844% VO2 max (r = 0.996) approximately 98% of the total variance of performance times could be accounted for in the marathon and ultramarathon events. This suggests that other factors such as footwear, clothing, and running technique (Costill, 1972) play a relatively minor role in this group of male distance runners. In the female athletes the intermediate times were not available and they did not compete beyond 42.2 km (marathon) distance but for this event a similar association though less in magnitude was found with VO2 max (r = -0.43) and %VO2 max (= -0.49). The male athletes were able to sustain 82% VO2 max (range 80--87%) in 42.2 km and 67% VO2 max (range 53--76%) in 84.64 km event. The comparable figure for the firls in the marathon was 79% VO2 max (ranges 68--86%). Our data suggests that success at the marathon and ultramarathon distances is crucially and (possibly) solely dependent on the development and utilisation of a large VO2 max.     

Early adaptations in blood substrates, metabolites, and hormones to prolonged exercise training in man.

This study was designed to investigate the effect of short-term, submaximal training on changes in blood substrates, metabolites, and hormonal concentrations during prolonged exercise at the same power output. Cycle training was performed daily by eight male subjects (VO2max = 53.0 +/- 2.0 mL.kg-1.min-1, mean +/- SE) for 10-12 days with each exercise session lasting for 2 h at an average intensity of 59% of VO2max. This training protocol resulted in reductions (p less than 0.05) in blood lactate concentration (mM) at 15 min (2.96 +/- 0.46 vs. 1.73 +/- 0.23), 30 min (2.92 +/- 0.46 vs. 1.70 +/- 0.22), 60 min (2.96 +/- 0.53 vs. 1.72 +/- 0.29), and 90 min (2.58 +/- 1.3 vs. 1.62 +/- 0.23) of exercise. The reduction in blood lactate was also accompanied by lower (p less than 0.05) concentrations of both ammonia and uric acid. Similarly, following training lower concentrations (p less than 0.05) were observed for blood beta-hydroxybutyrate (60 and 90 min) and serum free fatty acids (90 min). Blood glucose (15 and 30 min) and blood glycerol (30 and 60 min) were higher (p less than 0.05) following training, whereas blood alanine and pyruvate were unaffected. For the hormones insulin, glucagon, epinephrine, and norepinephrine, only epinephrine and norepinephrine were altered with training. For both of the catecholamines, the exercise-induced increase was blunted (p less than 0.05) at both 60 and 90 min. As indicated by the changes in blood lactate, ammonia, and uric acid, a depression in glycolysis and IMP formation is suggested as an early adaptive response to prolonged submaximal exercise training.     

Plasma catecholamine and lactate response during graded exercise with varied glycogen conditions.

The relationships between the lactate threshold (TLa), plasma catecholamines, and ventilatory threshold (TVE) were examined under normal and glycogen-depleted conditions. Nine male subjects performed a graded exercise test on a bicycle ergometer in a normal glycogen (NG) state and in a glycogen-depleted (GD) state to determine if manipulation of muscle glycogen content would affect their ventilatory, lactate, and catecholamine responses. High correlations were found between plasma lactate and the two catecholamines, epinephrine (r = 0.964) and norepinephrine (r = 0.965) under both conditions. The GD protocol resulted in a shift in the TLa to a later work rate; inflections in epinephrine and norepinephrine shifted in a coordinated manner. TVE and TLa occurred at similar work loads under NG conditions [67.2 +/- 1.5 and 65.6 +/- 2.3% maximal oxygen consumption (VO2max), respectively], but TLa occurred at a later work load (75.3 +/- 1.9% VO2max) compared with TVE (68.3 +/- 1.6% VO2max) under GD conditions. These results suggest a causal relationship between plasma lactate and epinephrine during a graded exercise test under the glycogen conditions studied. Although an association existed between ventilation and lactate, this relationship was not as strong.     

A program of moderate physical training for Wistar rats based on maximal oxygen consumption

Moderate physical training is often associated with improved cardiorespiratory fitness in athletes and the general population. In animals, studies are designed to investigate basic physiology that could be invasive and uncomfortable for humans. The standardization of an exercise training protocol for rats based on maximal consumption of oxygen (VO(2)max) is needed. This study validated a program of moderate physical training for Wistar rats based on VO(2)max determined once a week. A 10-stage treadmill running test was developed to measure VO(2)max through an indirect, open circuit calorimeter. Thirty male Wistar rats (210-226 g) were randomly assigned to either a nontrained group or a trained group. The animals were evaluated weekly to follow their VO(2)max during 8 weeks of moderate training and to adjust the intensity of the protocol of training. The soleus muscle was removed for determination of citrate synthase activity. Trained animals maintained their values of VO(2)max during a moderate running training and showed a significant less body weight gain. An increase of 42% in citrate synthase activity of the soleus muscle from trained rats was found after the training program. Our study presents a protocol of moderate physical training for Wistar rats based on VO(2)max. Peripheral adaptations such as the values of citrate synthase activity also responded to the moderate training program imposed as observed for VO(2)max. Other studies can use our protocol of moderate training to study the physiologic adaptations underlying this specific intensity of training. It will provide support for study with humans.     

Effect of exercise during pregnancy, graded as a percentage of aerobic capacity: maternal and fetal responses of the rat.

1. A number of variables were studied in pregnant rats that underwent strenuous exercise during pregnancy. They were: total weight gain, daily weight gain, length of pregnancy, number of offspring. Also the weight, the heart weight and fibre/capillary ratio of the newborn male rats and their VO2 max at 90 days were measured. 2. The exercise was graded in accordance to previous aerobic capacity as determined by VO2 max with relative loads of 60% (E60), 70% (E70), 80% (E80) and 90% (E90) of VO2 max being applied to the various groups (N = 6 per group). 3. The total weight gain and daily weight gain was significantly less in the E70, E80 and E90 groups. Weight gain in the anabolic phase (0-14d) was not different, but during the first week the weight gain in the E90 group was significantly less than control group. In the catabolic phase the observations were similar the first week of the anabolic phase. 4. Length of pregnancy, heart weight offspring and VO2 max of 90-day-old male rats were not significantly different. The number of offspring of the E90 group was significantly smaller than the control, E60 and E70 groups. 5. The offspring body weight was less in the E70, E80 and E90 groups than control group and was significantly less in the E90 group compared to the E60 and E70 groups. 6. The fibre/capillary ratio of the offspring was different in the E90 group compared to the control group. 7. These results suggest that the effect of exercise depends on the relative work load applied to the mother and these effects are particularly marked at high work loads.     

Chronic leptin infusion advances, and immunoneutralization of leptin postpones puberty onset in normally fed and feed restricted female rats

Does leptin play a vital role in initiating puberty in female rats and can it overrule a nutrionally imposed (i.e. a 30% feed restriction, FR) delay in puberty onset? Prepubertal female rats were chronically infused for 14 days with leptin (icv or sc) or leptin-antiserum (icv) while puberty onset was monitored by means of scoring the moment of vaginal opening (VO). Median VO age was higher (35 days versus 27 days) in FR animals but leptin levels at VO were significantly decreased (1.44 +/- 0.17 ng/ml versus 2.79 +/- 0.31 ng/ml). Centrally (icv) and peripherally (sc) infused leptin (1 microg/day) advanced VO age compared to FR controls (30 days versus 35 days and 31 days versus 41 days, respectively). Congruently, centrally (icv) administered leptin-antiserum (0.6 microg/day) delayed puberty onset. In normally fed rats median VO age was only marginally advanced (26 days versus 27 days) but only if leptin was applied centrally. The effects of FR on puberty onset are counteracted or even normalized by the infusion of leptin, whereas immunoneutralization of central leptin postpones puberty onset. We therefore conclude that central leptin is crucial for initiating puberty in female rats.     

Altered gravity downregulates aquaporin-1 protein expression in choroid plexus.

Aquaporin-1 (AQP1) is a water channel expressed abundantly at the apical pole of choroidal epithelial cells. The protein expression was quantified by immunocytochemistry and confocal microscopy in adult rats adapted to altered gravity. AQP1 expression was decreased by 64% at the apical pole of choroidal cells in rats dissected 5.5-8 h after a 14-day spaceflight. AQP1 was significantly overexpressed in rats readapted for 2 days to Earth's gravity after an 11-day flight (48% overshoot, when compared with the value measured in control rats). In a ground-based model that simulates some effects of weightlessness and alters choroidal structures and functions, apical AQP1 expression was reduced by 44% in choroid plexus from rats suspended head down for 14 days and by 69% in rats suspended for 28 days. Apical AQP1 was rapidly enhanced in choroid plexus of rats dissected 6 h after a 14-day suspension (57% overshoot, in comparison with control rats) and restored to the control level when rats were dissected 2 days after the end of a 14-day suspension. Decreases in the apical expression of choroidal AQP1 were also noted in rats adapted to hypergravity in the NASA 24-ft centrifuge: AQP1 expression was reduced by 47% and 85% in rats adapted for 14 days to 2 G and 3 G, respectively. AQP1 is downregulated in the apical membrane of choroidal cells in response to altered gravity and is rapidly restored after readaptation to normal gravity. This suggests that water transport, which is partly involved in the choroidal production of cerebrospinal fluid, might be decreased during spaceflight and after chronic hypergravity.     

Intravenous perfusion of adrenaline and adaptation to muscular exercise in man]

Eleven normal subjects underwent epinephrine perfusions (1.9; 6.1; 11.8 ng/min) during a short (20 min) and mild (50% VO2 max) exercise. VO2 was not modified by epinephrine perfusion, while heart rate ventrilation and plasmatic lactate were increased proportionally to epinephrine doses.     

Evidence for an inverse relationship between the ventilatory response to exercise and the maximum whole body oxygen consumption value

Ventilatory responses to submaximal exercise loads indicate that in a population of 895 physically active and sedentary male and female subjects, exercise ventilation is inversely related to predicted VO2max. The correlation coefficients for males and females in this relationship are 0.61 (P less than 0.0001) and 0.26 (P less than 0.0001) respectively. The slopes of regression lines for VE/VO2 and VO2max in female and male subjects are -2.59 and -0.91 respectively. This is associated with changes in composition of the expired air in that PCO2 increases and PO2 decreases with greater VO2max. The difference between the mean oxygen and carbon dioxide partial pressures in expired air of individuals in the highest and lowest VO2max ranges are 1.2 kPa (9 mm Hg) and 0.8 kPa (6 mm Hg) respectively.     

Influence of single hindlimb support during simulated weightlessness in the rat

Mature male rats (n = 16) were assigned to either 14 days of head-down suspension with one hindlimb supported (HDS) or to control cages (C) of similar dimensions. Hindlimb support during HDS preserved the muscle mass-to-body mass ratio (mg/100g) compared with C conditions for the soleus (48.3 +/- 1.0 to 41.7 +/- 1.0), plantaris (98.4 +/- 3.4 to 103.3 +/- 4.1), and gastrocnemius (484.7 +/- 18.5 to 507.2 +/- 13.9). However, the muscle mass-to-body mass ratio was significantly lower for the soleus (28.9 +/- 1.5), plantaris (83.9 +/- 3.6), and gastrocnemius (411.9 +/- 24.2) muscles from the freely hanging hindlimbs compared with the contralateral muscles from the supported hindlimbs or muscles from C animals. Citrate synthase activity (mumol.g-1.min-1) was significantly lower in soleus muscles from HDS rats in both the supported (19.4 +/- 2.3) and freely hanging (20.0 +/- 1.6) hindlimbs compared with C (28.5 +/- 3.1), whereas soleus muscle glycogen concentration (mg/g) was significantly higher in the freely hanging limbs from HDS rats (5.90 +/- 0.31) but not in the supported limbs (3.80 +/- 0.61) compared with C (4.34 +/- 0.50). Doppler flow probes were used to determine that iliac blood flow to freely hanging hindlimbs was significantly decreased after 48 (-19 +/- 5%) and 72 (-20 +/- 6%)h of HDS compared with presuspension values. In addition, iliac vascular resistance was significantly elevated at most time points during the 72 h of HDS in the freely hanging limbs but not the supported hindlimbs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolic adaptations to training precede changes in muscle mitochondrial capacity.

To determine whether increases in muscle mitochondrial capacity are necessary for the characteristic lower exercise glycogen loss and lactate concentration observed during exercise in the trained state, we have employed a short-term training model involving 2 h of cycling per day at 67% maximal O2 uptake (VO2max) for 5-7 consecutive days. Before and after training, biopsies were extracted from the vastus lateralis of nine male subjects during a continuous exercise challenge consisting of 30 min of work at 67% VO2max followed by 30 min at 76% VO2max. Analysis of samples at 0, 15, 20, and 60 min indicated a pronounced reduction (P less than 0.05) in glycogen utilization after training. Reductions in glycogen utilization were accompanied by reductions (P less than 0.05) in muscle lactate concentration (mmol/kg dry wt) at 15 min [37.4 +/- 9.3 (SE) vs. 20.2 +/- 5.3], 30 min (30.5 +/- 6.9 vs. 17.6 +/- 3.8), and 60 min (26.5 +/- 5.8 vs. 17.8 +/- 3.5) of exercise. Maximal aerobic power, VO2max (l/min) was unaffected by the training (3.99 +/- 0.21 vs. 4.05 +/- 0.26). Measurements of maximal activities of enzymes representative of the citric acid cycle (succinic dehydrogenase and citrate synthase) were similar before and after the training. It is concluded that, in the voluntary exercising human, altered metabolic events are an early adaptive response to training and need not be accompanied by changes in muscle mitochondrial capacity.     

Regulation of leptin by steroid hormones in rat adipose tissue.

We investigated if steroid hormones regulate the secretion and the expression of leptin in female and male rat adipose tissue fragments in vitro. Dexamethasone time and dose-dependently increased the secretion and mRNA expression of leptin with a half-maximal stimulation of approximately 1 nM. A time-course revealed a maximal stimulatory effect of 17 beta-estradiol after 24 hours. In male adipose tissue 17 beta-estradiol increased leptin secretion (32% by 50 nM 17 beta-estradiol, P = 0.07 and 34% by 500 nM 17 beta-estradiol, P < 1780.05) after 24 hours. An additional effect of estrogen was seen in the dexamethasone (50 nM) stimulated cells (38% with 50 nM 17 beta-estradiol, P < 0.05 and 48% by 500 nM 17 beta-estradiol, P < 0.05). Basal secretion of leptin was equal in female and male adipose tissue, whereas the effects of 17 beta-estradiol (50 nM) and dexamethasone were significantly increased in female as compared with male adipose tissue. Progesterone, testosterone, dihydrotestosterone and dehydroepiandrostendione-sulfate neither affected leptin secretion in male nor female adipose tissue in vitro. Furthermore, to investigate the effect of estrogen female rats were ovariectomized (OVX) and the adipose tissue was incubated in vitro and compared with adipose tissue leptin secretion from sham operated rats (SHAM), and with ovariectomized rats treated with 17 beta-estradiol (EST). A decreased basal and dexamethasone-stimulated leptin secretion from OVX rats compared with SHAM rats was found (P < 0.005) whereas 17 beta-estradiol treatment of ovariectomized rats maintained a normal leptin secretion. However, the dexamethasone stimulation was equally increased above basal levels in SHAM, OVX and EST rats (3.7 +/- 1.2, 2.9 +/- 0.8, 4.2 +/- 1.4, NS, ANOVA) respectively.     

The effects of estrogen on thermoregulation,heart rate,and activity in the female rat with comparisons to males

Intact female (SHAM), ovariectomized (OVX), OVX with estradiol (E2), and male (MALE) rats, had core temperature (T_c), heart rate (HR), and activity (ACT) measured every 5 min for 10 days. E2 lowered T_c (SHAM > MALE ⩾ OVX > E2) and increased ACT (E2 > all other groups). Diurnal and mean 24 h HR was reduced in resting animals by E2 (OVX > SHAM > E2 > MALE). Significant differences in T_c and HR were quantified without the need to align the SHAM female data by estrus cycle phase. Telemetered female rats provide a sensitive model with which to measure the effects of subtle thermoeffectors on temperature regulation.