Mouse Lumbar Vertebra Uniaxial Compression Testing with Embedding of the Loading Surface.

There is increasing awareness that cortical and cancellous bone differ in regulating and responding to pharmaceutical therapies, hormone therapies, and other treatments for age-related bone loss. Three-point bending is a common method used to assess the influence of a treatment on the mid-diaphysis region of long bones, which is rich in cortical bone. Uniaxial compression testing of mouse vertebrae, though capable of assessing bones rich in cancellous bone, is less commonly performed due to technical challenges. Even less commonly performed is the pairing of three-point bending and compression testing to determine how a treatment may influence a long bone's mid-diaphysis region and a vertebral centrum similarly or differently. Here, we describe two procedures to make compression testing of mouse lumbar vertebrae a less challenging method to perform in parallel with three-point bending: first, a procedure to convert a three-point bending machine into a compression testing machine, and second, an embedding method for preparing a mouse lumbar vertebra loading surface.

Dietary Hempseed Decreases Femur Maximum Load in a Young Female C57BL/6 Mouse Model but Does Not Influence Bone Mineral Density or Micro-Architecture.

Numerous seed and seed extract diets have been investigated as a means of combating age-related bone loss, with many findings suggesting that the seeds/extracts confer positive effects on bone. Recently, there has been rising interest in the use of dietary hempseed in human and animal diets due to a perceived health benefit from the seed. Despite this, there has been a lack of research investigating the physiologic effects of dietary hempseed on bone. Previous studies have suggested that hempseed may enhance bone strength. However, a complete understanding of the effects of hempseed on bone mineralization, bone micro-architecture, and bone biomechanical properties is lacking. Using a young and developing female C57BL/6 mouse model, we aimed to fill these gaps in knowledge. From five to twenty-nine weeks of age, the mice were raised on either a control (0%), 50 g/kg (5%), or 150 g/kg (15%) hempseed diet (n = 8 per group). It was found that the diet did not influence the bone mineral density or micro-architecture of either the right femur or L5 vertebrae. Furthermore, it did not influence the stiffness, yield load, post-yield displacement, or work-to-fracture of the right femur. Interestingly, it reduced the maximum load of the right femur in the 15% hempseed group compared to the control group. This finding suggests that a hempseed-enriched diet provides no benefit to bone in young, developing C57BL/6 mice and may even reduce bone strength.

Sindbis Virus Replication Reduces Dependence on Mitochondrial Metabolism During Infection.

Alphaviruses are single stranded, positive sense RNA viruses that are often transmitted through mosquito vectors. With the increasing spread of mosquito populations throughout the world, these arboviruses represent a significant global health concern. Viruses such as Sindbis Virus (SINV), Chikungunya Virus (CHIKV) and Equine Encephalitis Viruses (EEV) are all alphaviruses. As viruses, these pathogens are dependent on the host cell environment for successful viral replication. It has been observed that viruses manipulate cellular metabolism and mitochondrial shape, activity, and dynamics to favor viral infection. This report looked to understand the metabolic changes present during Sindbis virus infection of hamster and human kidney cells. Cells were infected with increasing levels of SINV and at 24 hours post infection the mitochondria morphology was assessed with staining and mitochondrial activity was measured with a real-time Seahorse Bioanalyzer. The relative amount of mitochondrial staining intensity decreased with Sindbis virus infected cells. Both oxygen consumption rate and ATP production were decreased during SINV infection while non-mitochondrial respiration and extracellular acidification rate increased during infection. Collectively, the data indicates that SINV primarily utilizes non-mitochondrial metabolism to support viral infection within the first 24 hours. This understanding of viral preference for host cell metabolism may provide critical targets for antiviral therapies and help further define the nature of alphavirus infection.

Interpreting muscle function from EMG: lessons learned from direct measurements of muscle force.

Electromyography is often used to infer the pattern of production of force by skeletal muscles. The interpretation of muscle function from the electromyogram (EMG) is challenged by the fact that factors such as type of muscle fiber, muscle length, and muscle velocity can all influence the relationship between electrical and mechanical activity of a muscle. Simultaneous measurements of EMG, muscle force, and fascicle length in hindlimb muscles of wild turkeys allow us to probe the quantitative link between force and EMG. We examined two features of the force-EMG relationship. First, we measured the relaxation electromechanical delay (r-EMD) as the time from the end of the EMG signal to time of the end of force. This delay varied with locomotor speed in the lateral gastrocnemius (LG); it was longer at slow walking speeds than for running. This variation in r-EMD was not explained by differences in muscle length trajectory, as the magnitude of r-EMD was not correlated with the velocity of shortening of the muscle during relaxation. We speculate that the longer relaxation times at slow walking speeds compared with running may reflect the longer time course of relaxation in slower muscles fibers. We also examined the relationship between magnitude of force and EMG across a range of walking and running speeds. We analyzed the force-EMG relationship during the swing phase separately from the force-EMG relationship during stance phase. During stance, force amplitude (average force) was linearly related to mean EMG amplitude (average EMG). Forces during swing phase were lower than predicted from the stance phase force-EMG relationship. The different force-EMG relationships during the stance and swing phases may reflect the contribution of passive structures to the development of force, or a nonlinear force-EMG relationship at low levels of muscle activity. Together the results suggest that any inference of force from EMG must be done cautiously when a broad range of activities is considered.

Relative shortening velocity in locomotor muscles: turkey ankle extensors operate at low V/V(max).

The force-velocity properties of skeletal muscle have an important influence on locomotor performance. All skeletal muscles produce less force the faster they shorten and typically develop maximal power at velocities of approximately 30% of maximum shortening velocity (V(max)). We used direct measurements of muscle mechanical function in two ankle extensor muscles of wild turkeys to test the hypothesis that during level running muscles operate at velocities that favor force rather than power. Sonomicrometer measurements of muscle length, tendon strain-gauge measurements of muscle force, and bipolar electromyographs were taken as animals ran over a range of speeds and inclines. These measurements were integrated with previously measured values of muscle V(max) for these muscles to calculate relative shortening velocity (V/V(max)). At all speeds for level running the V/V(max) values of the lateral gastrocnemius and the peroneus longus were low (<0.05), corresponding to the region of the force-velocity relationship where the muscles were capable of producing 90% of peak isometric force but only 35% of peak isotonic power. V/V(max) increased in response to the demand for mechanical power with increases in running incline and decreased to negative values to absorb energy during downhill running. Measurements of integrated electromyograph activity indicated that the volume of muscle required to produce a given force increased from level to uphill running. This observation is consistent with the idea that V/V(max) is an important determinant of locomotor cost because it affects the volume of muscle that must be recruited to support body weight.

Muscle strain is modulated more with running slope than speed in wild turkey knee and hip extensors.

We examined the length changes and electromyographic (EMG) activity of two hindlimb muscles in wild turkeys, to determine how these muscles modulate mechanical function with changes in running speed and slope. The muscles studied were the iliotibialis lateralis pars postacetabularis (ILPO), a biarticular knee and hip extensor, and the femorotibialis lateralis (FT), a knee extensor. Muscle length changes were recorded using sonomicrometry, and EMG activity was recorded from indwelling bipolar electrodes as the animals walked and ran at a range of speeds (1-3.5 m s(-1)). Treadmill slope was also varied, from a 12 degrees uphill slope to a downhill slope of -12 degrees. To test the hypothesis that the strain pattern in active muscles reflects the demand for mechanical work, we compared strain in the ILPO and FT across the range of slopes. Both muscles underwent active lengthen-shorten cycles during stance. We analyzed the lengthening and shortening part of the strain pattern separately to determine the response of muscle strain to surface slope. In both muscles stance phase shortening strain increased over the range of slopes studied, from 7.8+/-3.5% (ILPO) and 1.9+/-2.2% (FT) during downhill running at -12 degrees, to 30.3+/-3.9% (ILPO) and 8.2+/-5.6% (FT) during uphill running at 12 degrees. Stance-phase lengthening strain was also modulated with slope, from -15.6+/-3.2% (ILPO) and -22.1+/-9.6% (FT) during downhill running at -12 degrees, to -4.2+/-2.5% (ILPO) and -9.0+/-5.6% (FT) during uphill running at 12 degrees. The results suggest that for the ILPO and FT a change in net mechanical work output with running slope is likely mediated by a change in both the lengthening, energy absorbing portion of the contraction and the shortening, energy producing part of the contraction. We also found changes in the timing of EMG activity, and the relative portion of the stance period spent lengthening, which were consistent with a shift in muscle function from energy absorption during downhill running, to net energy production during uphill running. Generally, muscle strain was less affected by speed than by slope. Shortening strains were not significantly correlated with running speed. Only FT lengthening strain changed significantly with speed, ranging from -6.8+/-4.3% at 1 m s(-1) to -15.3+/-4.7% at 3.5 m s(-1). The consistent patterns of strain changes with running slope are evidence that strain pattern is modulated to meet the changes in demand for net mechanical work. The relatively poor relationship between strain and running speed may reflect the fact that changes in running speed during level running are not associated with a change in demand for net mechanical work. Taken together, the speed and slope results suggest that the demand for mechanical work is an important determinant of muscle length patterns in running and walking.

Mechanical function of two ankle extensors in wild turkeys: shifts from energy production to energy absorption during incline versus decline running.

We investigated the mechanical function of two ankle extensor muscles, the lateral gastrocnemius (LG) and peroneus longus (PL), in wild turkeys Meleagris gallopavo during steady speed running. We hypothesized that mechanical work output of the LG and PL during running parallels the demand for mechanical work on the body. The turkeys ran on level, inclined (+6 degrees, +12 degrees ) and declined (-6 degrees, -12 degrees ) treadmills to change the demand for mechanical work. Simultaneous measurements of muscle length (from sonomicrometry) and muscle force (from tendon strain gauges) were used to calculate mechanical work output. During level running at a speed of 2 m s(-1), the LG and PL were both active in stance but produced peak force at different times, at approximately 21% of stance duration for the LG and 70% for the PL. The LG and PL also had different length patterns in stance during level running. The LG underwent little shortening during force production, resulting in negligible net positive work (2.0+/-0.8 J kg(-1)). By contrast, the PL produced force across a stretch-shorten cycle in stance and did significant net positive work (4.7+/-1.6 J kg(-1)). Work outputs for both the LG and PL were directly proportional to running slope. When we increased the demand for net positive work by running the turkeys on an incline, the LG and PL increased stance net positive work output in direct proportion to slope (P<0.05). Stance net positive work output increased to 7.0+/-1.3 J kg(-1) for the LG and 8.1+/-2.9 J kg(-1) for the PL on the steepest incline. Increases in stance net positive work for the LG and PL were associated with increases in net shortening strain and average shortening velocity, but average force in stance remained constant. The LG and PL muscles were also effective energy absorbers during decline running, when there is demand for net negative work on the body. During decline running at 2 m s(-1) on the steepest slope, the LG absorbed 4.6+/-2.2 J kg(-1) of net work in stance and the PL absorbed 2.4+/-0.9 J kg(-1) of net work. Shifts in muscle mechanical function from energy production during incline running to energy absorption during decline running were observed over a range of running speeds from 1-3 m s(-1) for both the LG and PL. Two fundamentally different mechanisms for changing work output were apparent in the mechanical behavior of the LG and PL. The LG simply altered its length pattern; it actively shortened during incline running to produce mechanical energy and actively lengthened during decline running to absorb mechanical energy. The PL changed mechanical function by altering its length pattern and by shifting the timing of force production across its stretch-shorten cycle. During incline running, the PL produced force during late stance shortening for positive work, but during decline running, the timing of force production shifted into early stance, to align with lengthening for negative work. In addition, during decline running, the PL greatly reduced or eliminated late stance shortening, thus reducing the potential for positive work. Our results show that the changing demands for whole body work during steady speed running are met, at least in part, by an ability of single muscles to shift mechanical function from net energy production to net energy absorption.

Force-velocity properties of two avian hindlimb muscles.

Recent work has provided measurements of power output in avian skeletal muscles during running and flying, but little is known about the contractile properties of avian skeletal muscle. We used an in situ preparation to characterize the force-velocity properties of two hind limb muscles, the lateral gastrocnemius (LG) and peroneus longus (PL), in Wild Turkeys (Meleagris gallopavo). A servomotor measured shortening velocity for at least six different loads over the plateau region of the length-tension curve. The Hill equation was fit to the data to determine maximum shortening velocity and peak instantaneous power. Maximum unloaded shortening velocity was 13.0+/-1.6 L s(-1) for the LG muscle and 14.8+/-1.0 L s(-1) for the PL muscle (mean+/-S.E.M.). These velocities are within the range of values published for reptilian and mammalian muscles. Values recorded for maximum isometric force per cross-sectional area, 271+/-28 kPa for the LG and 257+/-30.5 kPa for the PL, and peak instantaneous power output, 341.7+/-36.4 W kg(-1) for the LG and 319.4+/-42.5 W kg(-1) for the PL, were also within the range of published values for vertebrate muscle. The force-velocity properties of turkey LG and PL muscle do not reveal any extreme differences in the mechanical potential between avian and other vertebrate muscle.

Norepinephrine release in brown adipose tissue remains robust in cold-exposed senescent Fischer 344 rats.

Near the end of life, old F344 rats undergo a transition, marked by spontaneous and rapidly declining function. Food intake and body weight decrease, and these rats, which we call senescent, develop severe hypothermia in the cold due in part to blunted brown fat [brown adipose tissue (BAT)] thermogenesis. We tested the hypothesis that this attenuation may involve diminished sympathetic signaling by measuring cold-induced BAT norepinephrine release in freely moving rats using linear microdialysis probes surgically implanted into interscapular BAT 24 and 48 h previously. In response to 2 h at 15 degrees C, senescent rats increased BAT norepinephrine release 6- to 10-fold but did not maintain homeothermy. This increase was comparable to that of old presenescent (weight stable) rats that did maintain homeothermy during even greater cold exposure (2 h at 15 degrees C followed by 1.5 h at 8 degrees C). Tail temperatures, an index of vasoconstrictor responsiveness to cold, exhibited similar cooling curves in presenescent and senescent rats. Thus cold-induced sympathetic signaling to BAT and tail vasoconstrictor responsiveness remain robust in senescent rats and cannot explain their cold-induced hypothermia.