INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

MANY TERRESTRIAL ANIMALS live in environments that are topographically diverse and must often move up, down, and across slopes of varying degree. Grade-related changes in gravitational potential energy require that an animal expend more energy to move uphill at the same speed as on the level and dissipate energy to maintain this speed downhill. In accord with this, many physiological studies have shown that energetic costs, as measured by levels of oxygen consumption, increase during incline locomotion in diverse animal species (1, 9-11, 25, 30, 38-41) and decrease during decline locomotion (1, 6, 25, 30, 38, 41). Such shifts in locomotor energetics are largely due to changes in the recruitment and actions of various limb muscles. Whereas limb muscle recruitment typically increases on an incline (7, 8, 13, 20, 29, 31, 33), the reverse is true for decline locomotion relative to on the level (e.g., Ref. 18). In fact, Smith and Carlson-Kuhta (36) noted that several major hip extensor muscles in cats remain completely inactive during decline walking on grades as low as 10%.

In addition to these changes in muscle recruitment, alterations in limb posture and kinematics often accompany a change in surface grade (7, 17, 20, 37). For example, recent work on incline walking in cats demonstrated that, as grade increased from 25 to 100%, joint extension at the hip, knee, and ankle increased concomitant with electromyographic (EMG) intensity in extensors acting at those joints (7). In comparison, Smith et al. (37) showed that knee joint extension was reduced, whereas yield-related flexion at the ankle was increased on declined slopes of varying degree (also in cats). Such kinematic results suggest that limb muscles likely undergo relatively more active shortening on an incline but more stretching while active on a decline, which implies that shifts in muscle strain as well as recruitment are important for mediating changes in net muscle work and gravitational potential energy.

Although quantification of joint kinematics provides insight into the length-change patterns of muscle-tendon units spanning those joints, kinematic data cannot directly resolve the specific muscle and/or tendon strains that underlie a given joint excursion in vivo. Sonomicrometry permits direct measurements of limb muscle fascicle strain during dynamic behaviors, but few studies have employed this technique to quantify alterations in muscle strain regimes associated with shifts in locomotor surface grade. Recent work by Roberts et al. (32) and Gabaldon et al. (12) has shown that lateral gastrocnemius fascicles in running turkeys actively shorten on an incline, remain nearly isometric on the level, and lengthen while active on a decline. These data support the notion that shifts in distal limb muscle strain are important for mediating mechanical work output or absorption in response to changes in surface grade, but the role of more proximal limb muscles in this context remains unexplored.

To what extent might more proximal limb muscles also augment mechanical work output or dissipate energy in response to changes in surface grade? Hip and knee extensors generally have longer fibers than more pinnate ankle extensors, which suggests the capacity to actively shorten or stretch over relatively large distances, thus facilitating substantial contributions to energy production or absorption. However, aside from the conceptual understanding that incline and decline locomotion tend to bias muscles toward concentric vs. eccentric contractions, respectively, we have little appreciation for how length-change patterns in proximal limb muscles actually change with grade.

In this study we use sonomicrometry and electromyography to measure patterns of muscle strain and activation in the biceps femoris (a biarticular muscle that acts in hip extension and knee flexion) and vastus lateralis (a major uniarticular knee extensor) of rats during uphill and downhill locomotion by having the rats move on a treadmill over a range of speeds and gaits. Our specific goals are threefold. First, we wish to test whether proximal muscles acting at the hip and knee exhibit different strain patterns depending on the surface grade. More specifically, we sought to determine whether these muscles shorten more on an incline than on the level and shorten less, or perhaps stretch, on a decline. Second, we address whether deactivation of the biceps femoris during stance, as observed in cats (36, 37), is characteristic of decline locomotion in rats as well. If both rats and cats exhibit this pattern of deactivation, this may represent a relatively widespread neuromotor response to downhill grades among mammalian quadrupeds. Finally, by examining a range of speeds, we can explore the interaction between gait and grade on limb muscle actions during locomotion. For example, are differences observed among grades during walking also observed during running, or are the effects of grade more prominent at a particular gait or range of speeds?

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Animals. Female Sprague-Dawley rats weighing 225-305 g (mean = 257 g) were obtained from Charles River Laboratories. Rats were housed in pairs in cages and maintained on a diet of IsoPro 3000. The room in which rats were held was kept at 21°C, and a 12:12-h light-dark cycle was established. Individuals were initially trained to walk, trot, and gallop on a small level treadmill with a 60 × 20-cm working section. Once individuals could maintain speed for 1 min at each gait, rats were then trained to move over a range of speeds with the treadmill either inclined or declined 15° (27% grade). Many animals were resistant to declined treadmill locomotion at high speeds, and thus the sample size of rats that performed trotting and galloping gaits downhill is relatively low. Animals ranged between 8 and 15 mo of age at the time of experiments, and all experimental procedures were approved by the University Committee for the Use and Care of Animals at Harvard University.

Implantation procedures. To record patterns of muscle electrical activity and length change, fine-wire bipolar electrodes and piezoelectric sonomicrometry crystals were implanted unilaterally into the cranial or anterior aspect of the biceps femoris and central region of the vastus lateralis. The biceps is the largest muscle in the rat's hindlimb (3). Although it is biarticular, fascicles within the most anterior region of the biceps act primarily in hip extension and have little, if any, effect in flexing the knee. The vastus is the largest muscle of the quadriceps complex (3) and acts as a major extensor of the knee. Thus the two muscles of interest act as major extensors of the proximal hindlimb.

Locomotor trials and data collection. The miniature female connectors fastened to the epoxy block on the rat's skull were connected via male counterparts (Microconnectors, GM-6) to lightweight shielded cables (Cooner Wire) that were attached to the EMG and sonomicrometry amplifiers. EMG signals were amplified (×1,000) and filtered (100- to 3,000-Hz band pass; 60-Hz notch) by using Grass P-511 preamplifiers. Sonomicrometry signals were amplified by using a Triton sonomicrometer (model 120-1001). Raw voltage data were digitized at 5,000 Hz with a 12-bit analog-to-digital converter (Digidata 1200B; Axon Instruments) and recorded onto computer with Digidata software (Axoscope). Table 1 shows the individuals for which successful EMG and strain data were collected for the biceps and vastus.

Statistical analyses. Basic statistics (i.e., mean and standard error) were calculated for each of the variables mentioned above to quantitatively assess aspects of the step cycle and patterns of strain and EMG activity in the biceps and vastus on incline and decline grades. Our recent work (14) on these proximal muscles during level locomotion allows for a direct quantitative assessment of the influence of surface grade on muscle recruitment and strain. To determine the effects of surface grade on these variables, repeated-measures ANOVAs were computed by using the mean values from each individual. Because slow walking was the only gait for which trials were obtained from all individuals on level, incline, and decline grades, this was the only gait for which repeated-measures ANOVAs were performed incorporating all three grades. Most other gaits had enough data from two of the grades to facilitate quantitative comparison. In these cases, t-tests were used to compare between grades, and they allowed for determination of whether trends observed in slow walking were also present at faster speeds and gaits. t-Tests were only used if data from at least the same four individuals were available at each grade. Thus comparisons were made between level and decline fast walking, level and incline trotting, and level and incline galloping (second limb). We used regression to analyze the relationships between speed and EMG intensity and relative duration. Analysis of covariance (ANCOVA) was then used, when appropriate, to compare these relationships among grades. A P value of <0.05 was used for all statistical tests to determine significance.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Step cycle. Step-cycle duration decreased in a curvilinear fashion with locomotor speed on level, incline, and decline surfaces (Fig. 1A). At all grades, these decreases are mirrored by similar reductions in stance-phase duration (Fig. 1B). Swing-phase duration decreases minimally but significantly (P < 0.001; Fig. 1C). As a result, the proportion of the step cycle occupied by the stance phase (i.e., the duty cycle) exhibits a shallow curvilinear decrease with increasing speed regardless of grade (Fig. 1D).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Graphs of step-cycle duration (A), stance-phase duration (B), swing-phase duration (C), and duty cycle (D) as a function of speed for incline, level, and decline locomotion (black, gray, and white symbols, respectively). Different symbols represent different individuals (n = 7 rats). Note the comparable decrease in step-cycle and stance-phase duration, whereas swing-phase durations remain relatively constant or decrease only slightly.

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Effects of surface grade on step-cycle duration (A), stance-phase duration (B), swing-phase duration (C), and duty cycle (D) during slow walking. Black bars, incline locomotion; gray bars, level locomotion; white bars, decline locomotion. Data are from the same 7 rats as Fig. 1 at each grade. Speeds averaged 25 cm/s on the incline, 26 cm/s on the level, and 26 cm/s on the decline. Repeated-measures ANOVAs demonstrate a significant effect of grade on step-cycle, stance-phase, and swing-phase duration (P < 0.05). In all cases, durations are longer on an incline than on a decline. No significant effect of grade on duty cycle is detected (P > 0.05). Error bars denote standard errors.

Muscle activity patterns. EMG activity in both the biceps and vastus typically begins near the time the foot makes ground contact during each stride and ends in the second half of the stance phase, regardless of gait or grade (Fig. 3). Biceps activity, on average, begins slightly before the stance phase starts (Fig. 3). The major burst of vastus activity can begin slightly before or after the foot makes ground contact, but on average it starts after the onset of biceps activity (Fig. 3). Although a small burst of electrical activity is often present in the vastus late in the swing phase, this burst does not appear to be affected much by speed or grade. Absolute EMG burst durations in both muscles decrease with speed in a manner similar to the speed-dependent decrease in stance-phase duration (Fig. 4, A and C). As a result, on a given surface grade, EMG duration remains a nearly constant fraction of stance-phase duration, regardless of speed or gait (Fig. 4, B and D),

View larger version (38K): [in this window] [in a new window]   Fig. 3.   Schematic diagrams indicating the average timing of electromyographic (EMG) activity in the biceps and vastus during the step cycle for various gaits and grades. Within each gait, the stance and swing phases are distinguished by dark and light gray shading, respectively. Biceps data are shown in the top half of each diagram (above solid line), and vastus data are shown in the bottom half (below solid line). Average periods of EMG activity from level, incline, and decline locomotion are shown using gray, black and white bars, respectively. Numbers within each bar reflect the average speed of trials (in cm/s), followed by the number of rats (in parentheses) used to derive the EMG timing data. Data are only shown if at least 3 rats exhibited measurable EMG signals for that gait and grade. Note that EMG data from downhill trials for the biceps are often lacking due to little or no EMG activity in this muscle during downhill locomotion and not because of faulty recordings. Similarly, few animals exhibited downhill trotting or used the experimental limb as the trailing limb during uphill galloping. Error bars denote standard errors.

View larger version (35K): [in this window] [in a new window]   Fig. 4.   Effects of locomotor speed and surface grade on absolute EMG burst durations and durations as a fraction of stance-phase duration for the biceps (A and B; n = 4 rats) and vastus (C and D; n = 5 rats). Different symbols represent different rats (black = incline, gray = level). Best-fit linear-regression lines are shown in B and D for all incline and level locomotion trials. Data for decline locomotion are not included because EMG activity was rarely present across the entire range of speeds for an individual. For all regression lines shown, the slope is not significantly different from zero (see graphs for statistics). Thus, although absolute EMG burst duration decreases with speed, bursts occupy a consistent fraction of the stance-phase duration at all speeds. Analysis of covariance (ANCOVA) results show that this fraction is greater in the biceps on an incline than on the level (P = 0.006); however, the fraction of the stance phase occupied by vastus EMG activity is similar across these 2 grades (P = 0.34).

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Effects of speed and grade on the relative intensity of EMG signals from the biceps (A; n = 4 rats) and vastus (B; n = 5 rats). Relative intensity values were obtained for each individual by dividing the absolute intensity of every EMG signal by the maximum intensity observed during level locomotion for that individual. Different symbols represent different individuals (black = incline, gray = level, white = decline). Best-fit linear-regression lines are shown for each grade (dashed line corresponds to decline data). Except for decline biceps data, all regression lines have a positive slope significantly different from zero that demonstrate increasing intensities with increasing speed (see graphs for statistics). ANCOVA results demonstrate that EMG intensities are significantly greater, on average, on an incline than on the level in the biceps (P = 0.0001) and vastus (P < 0.0001) and that vastus intensities are significantly greater on the level than on a decline (P < 0.0001). A lack of homogeneity among slopes precluded the use of ANCOVA for comparing biceps data from decline locomotion with other grades.

View larger version (42K): [in this window] [in a new window]   Fig. 6.   In vivo recordings of biceps strain and activation from the same rat during walking (top) and galloping (bottom) (all recordings from second limb down) for level, incline, and decline surfaces (left, middle, and right, respectively). Dark gray shading represents the stance phase, and light gray shading represents the swing phase. Note that the biceps largely shortens during stance, regardless of gait or grade, and that for both gaits stance-related shortening strains are greatest on an incline. In addition, traces from decline trials demonstrate minimal EMG activity, even at relatively fast speeds. All panels have the same scales for strain and EMG amplitude. Points of maximum strain, rather than the timing of stance onset, were used to define the intervals shown in each panel. Because maximum length was typically reached late in swing, the leftmost portion of each panel typically encompasses the very end of the swing phase (the small light gray area). L, muscle length; Lo, optimal muscle length.

Muscle-strain patterns. In both muscles, fascicles undergo consistent and cyclic patterns of length change during each stride. In the biceps, fascicles generally shorten during stance and lengthen during swing, but both gait and grade influence the magnitude and/or trajectory of this length-change pattern (Fig. 6). Biceps fascicles generally begin to shorten just before the foot makes ground contact. After a brief bout of rapid shortening, fascicles often undergo a short period in which they remain relatively isometric, creating a small "shoulder" in the biceps strain trace early in stance (Fig. 6). This shoulder is typically prominent on level and decline grades but is diminished on an incline. After this shoulder, biceps fascicles shorten more considerably throughout the rest of stance. The biceps then begins to lengthen at the stance-swing transition and is stretched passively through all of the swing phase (Fig. 6).

View larger version (23K): [in this window] [in a new window]   Fig. 7.   Histograms of average biceps shortening during stance at different gaits and grades for one individual. Gray, black, and white bars represent level, incline, and decline trials, respectively. Although variation exists among individuals with respect to shortening strain values, the grade-related changes exhibited here are generally representative of other animals. The only exception is the difference between incline and level galloping (lead limb), which is substantially greater in the animal depicted here than in the other rats (mean = 20% difference). Regardless of gait, however, shortening strains are typically highest on an incline, intermediate on the level, and lowest on a decline. Error bars represent standard errors.

View larger version (46K): [in this window] [in a new window]   Fig. 8.   In vivo recordings of vastus strain and activation from the same rat during walking (top) and galloping (bottom) (all recordings from second limb down) for level, incline, and decline surfaces (left, middle, and right, respectively). Dark gray shading represents the stance phase, and light gray shading represents the swing phase. Note that during stance the vastus always undergoes an initial period of stretch, regardless of gait or grade. After this relatively rapid stretch, fascicles exhibit a period of shortening, continued stretching, or relatively isometric behavior, depending on gait and grade. Late in stance, the muscle is again stretched rapidly as the limb transitions into swing. During the initial portion of the swing phase, fascicles continue to stretch before shortening rapidly and over a long distance until the foot again makes ground contact. The vastus typically exhibits EMG activity during the first two-thirds of stance, when the predominant strain is stretching. However, in the second limb down during galloping (bottom), the vastus typically undergoes more substantial shortening strains in the latter half of stance, and EMG activity is often present during much of this shortening. All panels have the same scales for strain and EMG amplitude.

View larger version (33K): [in this window] [in a new window]   Fig. 9.   Schematic vastus strain profiles for different gaits and grades. Vastus strain trajectories typically comprise 4 phases during each stride: 1) an intial stretch in the first half of stance; 2) a subsequent period in which fascicles exhibit variable degrees of shortening, lengthening, or remain nearly isometric; 3) another stretch during the transition from stance to swing; 4) a final period of substantial shortening over the second half of swing. Panels show the average timing and fascicle length excursions during each of these phases for different gaits and grades (n = 5 rats). Error bars are not shown in the figure because they obscure the presentation. Solid black lines reflect incline trials, gray lines represent level trials, and dashed lines represent decline trials. Within each gait, the stance and swing phases are distinguished by dark and light gray shading, respectively. Average speeds (in cm/s) and sample sizes (in parentheses) are shown at right. Animals rarely trotted downhill or used the experimental limb as the first limb down when galloping uphill; hence, strain trajectories for these gaits and grades are not shown. Note that the phase after the initial stretch in stance is what varies most across gaits and grades. Fascicles generally exhibit more shortening during this period as speed increases and as the grade inclines. In contrast, decline trials show little to no shortening over the same period.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We undertook this study to determine the response of two proximal hindlimb muscles to changes in surface grade. The vastus lateralis is an uniarticular extensor of the knee; the biceps femoris is a biarticular muscle with an extensor moment at the hip and a flexor moment at the knee. Our results demonstrate that, at all speeds and gaits, both recruitment intensity and fascicle strain change markedly with grade in these muscles. Muscle activation levels are greater, for a given speed, on an incline than on the level and lower on a decline. In fact, the biceps femoris remains inactive during decline locomotion, except at relatively high speeds. Biceps fascicles shorten during stance on all grades, but for any gait they shorten more on an incline than on the level and more on the level than on a decline. The vastus lateralis is generally stretched 8-15% over the first half of stance, regardless of grade. After this initial stretch, vastus fascicles undergo a variable amount of shortening during the second half of stance on level and incline surfaces. This shortening is higher, on average, on an incline than on the level and increases with speed, reaching a maximum in the second limb down during galloping. In contrast, on a decline, vastus fascicles are generally stretched throughout stance, except during galloping, where a brief period of nearly isometric behavior follows the initial stretch. Taken together, these results suggest that proximal hindlimb muscles are likely as important for mediating levels of mechanical work output and absorption during grade locomotion as the distal ankle extensors that have been studied previously (e.g., Refs. 12, 15, 31).

Surface grade and the step cycle. Moderate slopes can influence temporal aspects of the step cycle during locomotion. For example, rats in this study used longer stride periods (i.e., lower stride frequencies) when walking up a 27% grade than when walking on the level at comparable speeds. Grade-related increases in stride duration have also been observed in rats moving up a 30% grade over a range of speeds (33), in cats walking up a 36% grade [see Fig. 1 in Pierotti et al. (29)], and in horses trotting up a 6-10% grade (16, 35). Although fewer studies have examined the effects of a decline grade on stride parameters, both rats (this study) and cats (37) appear to adopt relatively higher stride frequencies (i.e., shorter stride periods) and shorter stride lengths during decline walking than on the level. In fact, the study by Smith et al. (37) suggests that the steeper the slope, the more stride period and stride length decrease during decline walking.

Surface grade, gait, and speed. Although we did not set out to examine the interactions among grade, gait, and speed explicitly, examination of Table 2 provides some insight into these issues. First, animals were typically resistant to moving at high speeds on a decline (e.g., only 4 of 7 animals exhibited high-speed locomotion on a decline, whereas all 7 moved at high speeds on incline and level surfaces). We doubt such results are indicative of any natural disinclination toward downhill galloping in the wild but instead likely reflect the unnatural circumstance of running downhill fast on an enclosed treadmill. Second, animals rarely used trotting on a decline (e.g., only 1 of 7 animals exhibited downhill trotting). Most rats that were willing to gallop downhill were unwilling to exhibit a trotting gait on a decline at the speeds typically used for trotting on level and incline surfaces. Instead, these animals extended use of their fast walking gait at these speeds. This could reflect either a natural disinclination toward decline trotting or a response to using an enclosed treadmill. The fact that rats were willing to gallop on a decline but were nevertheless unwilling to trot at the speeds examined suggests that the reasons underlying the lack of one gait vs. the other may be different. Additional experiments are required to tease apart these issues.

Surface grade and muscle recruitment. It is the activation and contraction of limb muscles that provide the forces and energy required to support and propel terrestrial animals during locomotion. Although there have been relatively few studies of the effects of surface grade on muscle activity patterns during animal locomotion, a number of results from this study are consistent with those observed among various vertebrate species moving up and down inclines. In general, extensor muscles become activated shortly before or at the time the foot makes ground contact, and activity persists over 60-80% of the stance phase. Although the effects of surface grade on the timing of muscle activity are relatively minor, the intensity of this activity is altered substantially. Relative to level locomotion, incline locomotion at similar speeds generally elicits an increase in EMG intensity in hindlimb extensors (7, 8, 19, 31, 32), except those composed predominantly of slow-twitch fibers (29, 33). In contrast, decline locomotion generally elicits a reduction in EMG intensity in various limb extensors (37). Such alterations are typically interpreted as reflecting shifts in the level of activation or volume of muscle recruited to mediate the mechanical output required on different grades.

Surface grade and muscle strain. It is well known that certain limb muscles exhibit biochemical and histological responses to exercise on different grades. More specifically, antigravity muscles typically experience substantial physical damage in untrained animals after bouts of downhill locomotion but less damage on the level (2). Such changes are assumed to be linked to grade-dependent differences in the mechanical actions of the underlying muscles. Limb muscles are presumed to undergo mainly concentric (i.e., shortening) contractions during uphill locomotion that result in relatively minimal damage. In contrast, downhill locomotion is known to bias limb muscles toward "eccentric" contractions, in which muscle fibers are stretched while actively generating force. Active muscle stretching dissipates energy as animals move downhill but also may result in rapid- and high-force development and injury, particularly to muscles composed mainly of slow fibers. For example, the vastus intermedius in rats, which is largely composed of slow oxidative fibers, incurs more damage after downhill locomotion than the vastus lateralis and medialis, which both have smaller proportions of slow fibers (2). Short-term training can mitigate the extent of muscle damage (34), perhaps by leading to an increase in the number of sarcomeres in series within myofibrils (23, 24) or by degeneration and/or regeneration of fibers susceptible to injury (2, 34).