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Distribution and characteristics of poststimulus effects in proximal and distal forelimb muscles from red nucleus in the monkey.

We used stimulus-triggered averaging (StTA) of electromyographic (EMG) activity to investigate two major questions concerning the functional organization of the magnocellular red nucleus (RNm) for reaching movements in the macaque monkey. The first is whether the clear preference toward facilitation of extensor muscles we have reported in previous studies for distal (wrist and digit) forelimb muscles also exists for proximal muscles (shoulder and elbow). The second question is whether distal and proximal muscles may be cofacilitated from RNm suggesting the representation of functional muscle synergies for coordinated reaching movements. Two monkeys were trained to perform a prehension task requiring multijoint coordination of the forelimb. EMG activity was recorded from 24 forelimb muscles including 5 shoulder, 7 elbow, 5 wrist, 5 digit, and 2 intrinsic hand muscles. Microstimulation (20 microA at 20 Hz) was delivered throughout the movement task. From 137 microstimulation sites in the RNm, a total of 977 poststimulus effects was obtained including 733 poststimulus facilitation effects (PStF) and 244 poststimulus suppression effects (PStS). Of the PStF effects, 58% were obtained from distal muscles; 42% from proximal muscles. Digit muscles were more frequently facilitated (35%) than the wrist, elbow, or shoulder muscles (20, 24, and 18%, respectively). The intrinsic hand muscles were infrequently facilitated (3%). At all joints tested, PStF was more common in extensor muscles than flexor muscles. This extensor preference was very strong for shoulder (85%), wrist (85%), and digit muscles (94%) and weaker for elbow muscles (60%). Of the PStS effects, 65% were in distal muscles and 35% in proximal muscles. Interestingly, the flexor muscles were more frequently inhibited from RNm than extensor muscles. At 72% of stimulation sites, at least two muscles were facilitated. The majority of these sites (61%) cofacilitated both proximal and distal muscles. At the remaining sites (39%), PStF was observed in either the proximal (17%) or distal muscles (22%). Facilitation most often involved combinations of shoulder, elbow, and distal muscles (30%) or shoulder and distal muscles (26%). Only rarely were intrinsic hand muscles part of the total muscle synergy. Our results show that the RNm 1) controls both proximal and distal muscles but the strength of influence is biased toward distal muscles, 2) preferentially controls extensor muscles not only at distal forelimb joints but also at proximal joints, and 3) output zones cofacilitate synergies of proximal and distal muscles involved in the control of forelimb reaching movements.

Animals↗

Disynaptic pyramidal excitation in forelimb motoneurons mediated via C(3)-C(4) propriospinal neurons in the Macaca fuscata.

In contrast to findings in the cat, it recently has been shown that disynaptic pyramidal EPSPs only rarely are observed in forelimb motoneurons of the macaque monkey in the intact spinal cord or after a corticospinal transection in C(5). This finding has been taken to indicate that the disynaptic pyramidal excitatory pathway via C(3)-C(4) propriospinal neurons (PNs) is weakened through phylogeny when the monosynaptic cortico-motoneuronal connection has been strengthened. We reinvestigate this issue with special focus on the possibility that the inhibitory control of the C(3)-C(4) PNs may be stronger in the macaque monkey than in the cat. The effect in forelimb motoneurons of electrical stimulation in the contralateral pyramid was investigated in anesthetized macaque monkeys (Macaca fuscata). We confirmed the low frequency of disynaptic pyramidal EPSPs in forelimb motoneurons. However, after intravenous injection of strychnine, disynaptic EPSPs could be evoked in 39 of 41 forelimb motoneurons recorded after lesion of the corticospinal fibers in C5. After a corresponding lesion in C(2), disynaptic pyramidal EPSPs were observed in 2 of 25 motoneurons. In contrast to previous reports, we conclude that C(3)-C(4) PNs can mediate disynaptic pyramidal excitation in high frequency of occurrence to forelimb motoneurons in the C(6)-C(8) segments and that this transmission is under a stronger inhibitory control than in the cat. Thus, the hypothesis that the disynaptic excitatory cortico-motoneuronal pathway via the C(3)-C(4) PNs is weakened in parallel with the strengthened monosynaptic connection through phylogeny is not supported by the present findings.

Anesthesia↗

Changes in kinematic variables observed during pressure-induced forelimb lameness in adult horses trotting on a treadmill.

OBJECTIVE: To determine whether kinematic changes induced by heel pressure in horses differ from those induced by toe pressure. ANIMALS: 10 adult Quarter Horses. PROCEDURE: A shoe that applied pressure on the cuneus ungulae (frog) or on the toe was used. Kinematic analyses were performed before and after 2 levels of frog pressure and after 1 level of toe pressure. Values for stride displacement and time and joint angles were determined from horses trotting on a treadmill. RESULTS: The first level of frog pressure caused decreases in metacarpophalangeal (fetlock) joint extension during stance and increases in head vertical movement and asymmetry. The second level of frog pressure caused these changes but also caused decreases in stride duration and carpal joint extension during stance as well as increases in relative stance duration. Toe pressure caused changes in these same variables but also caused maximum extension of the fetlock joint to occur before midstance, maximum hoof height to be closer to midswing, and forelimb protraction to increase. CONCLUSION AND CLINICAL RELEVANCE: Decreased fetlock joint extension during stance and increased head vertical movement and asymmetry are sensitive indicators of forelimb lameness. Decreased stride duration, increased relative stance duration, and decreased carpal joint extension during stance are general but insensitive indicators of forelimb lameness. Increased forelimb protraction, hoof flight pattern with maximum hoof height near midswing, and maximum fetlock joint extension in cranial stance may be specific indicators of lameness in the toe region. Observation of forelimb movement may enable clinicians to differentiate lameness of the heel from lameness of the toe.

Animals↗

Moment arms about the carpal and metacarpophalangeal joints for flexor and extensor muscles in equine forelimbs.

OBJECTIVE: To determine whether muscle moment arms at the carpal and metacarpophalangeal joints can be modeled as fixed-radius pulleys for the range of motion associated with the stance phase of the gait in equine forelimbs. SAMPLE POPULATION: 4 cadaveric forelimbs from 2 healthy Thoroughbreds. PROCEDURE: Thin wire cables were sutured at the musculotendinous junction of 9 forelimb muscles. The cables passed through eyelets at each muscle's origin, wrapped around single-turn potentiometers, and were loaded. Tendon excursions, measured as the changes in lengths of the cables, were recorded during manual rotation of the carpal (180 degrees to 70 degrees) and metacarpophalangeal (220 degrees to 110 degrees) joints. Extension of the metacarpophalangeal joint (180 degrees and 220 degrees) was forced with an independent loading frame. Joint angle was monitored with a calibrated potentiometer. Moment arms were calculated from the slopes of the muscle length versus joint angle curves. RESULTS: At the metacarpophalangeal joint, digital flexor muscle moment arms changed in magnitude by < or = 38% during metacarpophalangeal joint extension. Extensor muscle moment arms at the carpal and metacarpophalangeal joints also varied (< or = 41% at the carpus) over the range of joint motion associated with the stance phase of the gait. CONCLUSIONS AND CLINICAL RELEVANCE: Our findings suggest that, apart from the carpal flexor muscles, muscle moment arms in equine forelimbs cannot be modeled as fixed-radius pulleys. Assuming that muscle moment arms at the carpal and metacarpophalangeal joints have constant magnitudes may lead to erroneous estimates of muscle forces in equine forelimbs.

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Two-dimensional link-segment model of the forelimb of dogs at a walk.

OBJECTIVE: To calculate normative joint angle, intersegmental forces, moment of force, and mechanical power at elbow, antebrachiocarpal, and metacarpophalangeal joints of dogs at a walk. ANIMALS: 6 clinically normal mixed-breed dogs. PROCEDURE: Kinetic data were collected via a force platform, and kinematic data were collected from forelimbs by use of 3-dimensional videography. Length, location of the center of mass, total mass, and mass moment of inertia about the center of mass were determined for each of 4 segments of the forelimb. Kinematic data and inertial properties were combined with vertical and craniocaudal ground reaction forces to calculate sagittal plane forces and moments across joints of interest throughout stance phase. Mechanical power was calculated as the product of net joint moment and the angular velocity. Joint angles were calculated directly from kinematic data. RESULTS: All joint intersegmental forces were similar to ground reaction forces, with a decrease in magnitude the more proximal the location of each joint. Flexor moments were observed at metacarpophalangeal and antebrachiocarpal joints, and extensor moments were observed at elbow and shoulder joints, which provided a net extensor support moment for the forelimb. Typical profiles of work existed for each joint. CONCLUSIONS AND CLINICAL RELEVANCE: For clinically normal dogs of a similar size at a walk, inverse dynamic calculation of intersegmental forces, moments of force, and mechanical power for forelimb joints yielded values of consistent patterns and magnitudes. These values may be used for comparison in evaluations of gait in other studies and in treatment of dogs with forelimb musculoskeletal disease.

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Pressure-dependent factors in edema formation in canine forelimbs.

Acetycholine (10 mug/min) infused intra-arterially for 30 minutes into naturally perfused forelimbs increased forelimb weight 23 g. largely due to edema formation. The weight gain was associated with markedly elevated skin and skeletal muscle blood flows and small vein pressures, suggesting that the edema was attributable, in part, to a rise in microvascular pressures. Mechanically increasing venous pressure and blood flow to similar levels for 30 minutes in pump-perfused forelimbs produced a weight gain of 27 g. The rate of weight gain for the acetylcholine and mechanical alterations was nearly identical. Acetylcholine and mechanical alterations both increased forelimb lymph flow rate but failed to increase lymph total protein concentration significantly. These studies indicate that in the dog forelimb elevated microvascular pressures result in edema formation by increasing the transcapillary hydrostatic pressure gradient without producing an important decrease in the transcapillary colloid osmotic pressure gradient. Increased pressure is not associated with a large increase in microvascular permeability to plasma proteins as is seen with the administration of high doses of histamine and bradykinin.

Acetylcholine↗

[Recovery of motor skills after the caudate lesion in rats with the different prefered forelimb: the role of intense retraining].

Rats were trained for instrumental reaching (17 mm) of a sunflower seed from a horizontal tube 12 mm in diameter. After training, rats were divided into groups, "right-" and "left-handers", by the forelimb preference. Unilateral electrolytic lesions of the head of the caudate nucleus contralateral to the prefered forelimb were performed. After surgery, animals were retrained to perform the food-retrieval reaction by the same forelimb (reaching reaction by the "intact" forelimb was prohibited by a special bracelet). Both right- and left-handers were divided into groups of rare and intense retraining. Over the course of five months, animals of the groups of rare training were tested once a week, whereas the intense retraining was performed three or four times a week. Rats with right and left forelimb preference were shown to recover the reaching skill with different rates. In general, animals with left-side caudate lesion (right-handers) recovered the skill better both under condition of spontaneous recovery (rare testing) and intense retraining. The results suggest different mechanism of skill recovery after the right- and left-side brain lesion.

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Neurokinin A and B: potent vasodilators in the canine forelimb.

Neurokinin A and neurokinin B may play a role in control of the peripheral circulation in either physiological or pathophysiological conditions. We have infused these peptides intra-arterially at three infusion rates each to assess their actions on vascular pressures, blood flows and total and segmental resistances in skin and skeletal muscle in the canine forelimb. Neurokinin A infusions (.01, .1, and 1 micrograms/min) decreased total forelimb resistance; transiently, 26% and 57%, respectively. The decrease in resistance was equally distributed between the skin and skeletal muscle circulations and was manifest in both large artery and small vessel resistances. Systemic and forelimb arterial pressures were decreased in a dose-dependent manner. Neurokinin B infusions (.5, 1 and 5 micrograms/min) decreased total forelimb resistance 29%, 31%, and 52%, respectively. The decrease in resistance was equally distributed between the skin and skeletal muscle circulations and was the result of decreases in both large artery and small vessel resistances. Systemic and forelimb arterial pressures were decreased in a dose-dependent manner. The potent effect of neurokinins on vascular resistance and their concentration in perivascular nerves innervating the resistance vessels of the circulation suggests a potential role for these neuropeptides in circulatory control.

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The recovery of forelimb-placing behavior in rats with neonatal unilateral cortical damage involves the remaining hemisphere.

Following unilateral lesions of the somatic sensorimotor cortex (SMC) in neonatal, but not adult, rats, an aberrant ipsilateral corticospinal projection originates from the undamaged hemisphere (Hicks and D'Amato, 1970; Leong and Lund, 1973; Castro, 1975). We have evaluated the contribution of the hemisphere contralateral to a unilateral lesion of the SMC in the recovery of tactile forelimb-placing behavior. Neither adult-lesioned or neonatally lesioned animals show evidence for placing deficits with either forelimb when tested 30 or 42 d after the lesion. However, in adult-lesioned animals, a subsequent lesion of the undamaged SMC on postlesion day 42 produces placing deficits only with the forelimb contralateral to the second lesion, while such a second lesion in the neonatally lesioned rats results in placing deficits with both forelimbs. Anatomical observations in the animals used for behavioral analyses confirm previous reports of a substantial ipsilateral corticospinal projection in rats with unilateral SMC damage as neonates and demonstrate that many of these aberrant fibers recross the midline within the spinal cord to arborize extensively within the ipsilateral spinal gray. These findings indicate that, following unilateral SMC lesions in neonates, the contralateral hemisphere mediates some aspects of the recovery of forelimb placing. The aberrant ipsilateral corticospinal projection may provide the anatomical substrate through which the cortex effects this recovery.

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The comparative anatomy of the forelimb veins of primates.

One hundred and thirteen forelimbs taken from 62 individuals belonging to 17 primate genera were dissected to reveal the entire course of the superficial venous system. The course of the deep venous system was also documented in at least one forelimb of each primate genus, and the number and location of perforating veins was recorded in 18 human and 45 non-human primate limbs. In Pan, Gorilla and in about 25% of human specimens the lateral superficial vein was confined to the forearm, while in all other primates, and in the majority of humans, this vein extended from the carpus to the clavicular region. Only Pongo and humans exhibited a second main superficial vein on the medial side of the forearm. In all primates the deep veins of the forelimb usually accompanied the arteries. Thus variation in the deep venous system reflected the different arterial patterns exhibited by these primates. The number of perforating veins in the forelimb was related to the length of the limb. Primate genera with longer forelimbs had more perforators, though not as many as would be expected if the number of perforators scaled linearly with limb length.

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Aminophylline attenuates the edemogenic actions of histamine in the canine forelimb.

Xanthines have been employed clinically to treat asthma and related pulmonary conditions because of their bronchodilator properties. In addition, xanthines have been reported to block and/or attenuate the increase in microvascular permeability to macromolecules produced by some putative inflammatory mediators. In order to more completely assess the anti-inflammatory capabilities of xanthines, we have infused aminophylline intra-arterially in the canine forelimb prior to and during a local intra-arterial infusion of histamine. Forelimb prenodal lymph flow, protein concentration and protein transport were used as indices of transvascular fluid and protein flux. Infusion of histamine (4 micrograms/min) significantly decreased forelimb arterial pressures and increased lymph flow, protein concentration and protein transport. Aminophylline infusion (10 mg/min) decreased forelimb arterial pressures but did not affect lymph parameters. Histamine infusion during infusion of aminophylline increased lymph parameters but the increases were markedly less than with histamine infusion alone. Infusion of aminophylline (20 mg/min) decreased forelimb arterial pressures and systemic pressure. Subsequent histamine infusion resulted in small but significant increases in lymph parameters. These data indicate that aminophylline infusion can blunt the ability of subsequently administered histamine to increase microvascular permeability as evidenced by the attenuation of the increases in lymph flow, protein concentration and protein transport.

Aminophylline↗

Differential sensitivity of ethanol withdrawal signs in the rat to gamma-aminobutyric acid (GABA)mimetics: blockade of audiogenic seizures but not forelimb tremors.

Intraperitoneal injection of ethanol (1-2 g/kg) and chlordiazepoxide (2-16 mg/kg) suppressed susceptibility to audiogenically induced, clonic-tonic seizures and antagonized forelimb tremor in rats undergoing ethanol withdrawal, 30 min after treatment. However, a smaller dose of ethanol (0.5 g/kg) actually increased clonic seizure frequency, suggesting that ethanol exerts a biphasic proconvulsant/anticonvulsant action. Direct activation of gamma-aminobutyric acid (GABA) receptors by intracisternal administration of GABA (100-1000 micrograms), muscimol (0.3-1.0 micrograms) or 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) (0.3-3.0 micrograms) 5 to 10 min before testing also reduced susceptibility to audiogenic clonic-tonic seizures. In sharp contrast to these anticonvulsant actions, GABA, muscimol and THIP had no effect on withdrawal-induced forelimb tremors. Blockade of GABA uptake with 1-2,4-diaminobutyric acid (300 and 600 mg/kg i.p.) and inhibition of GABA transaminase with aminooxyacetic acid (12.5 and 25.0 mg/kg i.p.) both reduced susceptibility to seizures. However, anticonvulsant doses of these two drugs, unlike GABA, muscimol and THIP, also reduced forelimb tremor. Three other GABA transaminase inhibitors, gamma-vinyl GABA (450 and 900 mg/kg i.p.), gamma-acetylenic GABA (50-150 mg/kg i.p.) and ethanolamine-O-sulfate (250-750 mg/kg i.p.), were inactive against ethanol withdrawal audiogenic seizures and forelimb tremors. These results indicate that direct GABA receptor activation can selectively suppress one type of ethanol withdrawal response (i.e., audiogenic seizure susceptibility) while failing to influence another (forelimb tremors).

4-Aminobutyrate Transaminase↗

Histamine-induced fluid efflux in the canine forelimb as affected by carotid occlusion or hemorrhage.

Infusion of catecholamines concurrently with histamine into the forelimb of the dog prevents histamine-induced increases in lymph flow, protein concentration and forelimb weight. This study tested whether the sympathoadrenal discharge of catecholamines induced by carotid occlusion or hemorrhage would similarly prevent histamine's actions in the canine forelimb. Carotid occlusion or hemorrhage, begun after 30 minutes of histamine infusion (4 micrograms base/min), resulted in a reduction in lymph flow but lymph protein concentration was not changed. Infusion of histamine into the forelimb for 30 minutes in control studies caused the forelimb to gain weight. In experimental studies hemorrhage, begun at one minute after the start of histamine significantly reduced this weight gain but occlusion of the carotid arteries at one minute after the start of histamine did not significantly affect weight gain. These results show that sympathetic nerve activity can lower elevated lymph flow and that sympathetic activity due to hemorrhage reduces the accumulation of interstitial fluid caused by histamine. It seems probable that endogenous catecholamines act directly on filtering vessels to lessen histamine-induced increases in permeability. However, a constriction of lymphatic vessels could have contributed to the reduction in lymph flow and we have no evidence against this possibility.

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Forelimb vascular pressures, skin lymph flow and lymph protein concentration as affected by vasoactive intestinal polypeptide and bombesin.

Vasoactive intestinal polypeptide (VIP) and bombesin are peptides that have been identified in several mammalian tissues including skin. In this study, we have examined the actions of these peptides on forelimb vascular pressures, skin lymph flow, lymph total protein concentration and lymph total protein transport in the canine forelimb perfused at constant arterial inflow. Local intraarterial infusion of sequentially increasing infusion rates of VIP of 300, 600, and 1500 ng/min for twenty minutes at each infusion rate resulted in dose-dependent decreases in forelimb arterial pressures but no change in skin small vein pressure. At the two higher infusion rates, systemic pressure was significantly decreased whereas heart rate significantly increased. Skin lymph parameters were not a significantly altered with the exception of a small but significant decrease in lymph flow at the highest infusion rate of VIP. Infusion of bombesin at 500 ng/min for sixty minutes resulted in significant increases in forelimb arterial and systemic pressures, no change in skin small vein pressure and a significant bradycardia. Skin lymph flow, protein concentration and protein transport were not significantly changed during the infusion of bombesin. These data indicate that while VIP and bombesin possess potent vasodilator and vasoconstrictor effects respectively, they do not significantly affect transmicrovascular fluid and macromolecular efflux in the canine forelimb perfused at constant flow, as assessed by changes in lymph flow and protein concentration.

Animals↗

Forelimb muscle contraction induced by cerebral cortical stimulation after callosotomy: a myographic study in the mouse.

The effect of callosotomy upon motor control by the cerebral cortex on the forelimb was examined in the mouse. On the 10th day after callosotomy in the rostral or caudal part of the corpus callosum, the forelimb area of the cerebral motor cortex was stimulated intracortically with a microelectrode and muscle contraction of the forelimb was recorded by electromyography. Muscle contraction was observed in the contralateral forelimb in the mice of which callosal fibers were cut in the caudal part of the corpus callosum including the splenium corporis callosi as well as in the normal mice. On the other hand, muscle contraction was observed in the ipsilateral forelimb in the mice of which callosal fibers were incised in the rostral part of the corpus callosum including the genu, rostrum and trunk. The latency of the muscle contraction was about 0.5 msec in the both groups of the callosotomized mice, as well as in the normal mice.

Animals↗

Dimensions and moment arms of the hind- and forelimb muscles of common chimpanzees (Pan troglodytes).

This paper supplies quantitative data on the hind- and forelimb musculature of common chimpanzees (Pan troglodytes) and calculates maximum joint moments of force as a contribution to a better understanding of the differences between chimpanzee and human locomotion. We dissected three chimpanzees, and recorded muscle mass, fascicle length, and physiological cross-sectional area (PCSA). We also obtained flexion/extension moment arms of the major muscles about the limb joints. We find that in the hindlimb, chimpanzees possess longer fascicles in most muscles but smaller PCSAs than are predicted for humans of equal body mass, suggesting that the adaptive emphasis in chimpanzees is on joint mobility at the expense of tension production. In common chimpanzee bipedalism, both hips and knees are significantly more flexed than in humans, necessitating muscles capable of exerting larger moments at the joints for the same ground force. However, we find that when subject to the same stresses, chimpanzee hindlimb muscles provide far smaller moments at the joints than humans, particularly the quadriceps and plantar flexors. In contrast, all forelimb muscle masses, fascicle lengths, and PCSAs are smaller in humans than in chimpanzees, reflecting the use of the forelimbs in chimpanzee, but not human, locomotion. When subject to the same stresses, chimpanzee forelimb muscles provide larger moments at the joints than humans, presumably because of the demands on the forelimbs during locomotion. These differences in muscle architecture and function help to explain why chimpanzees are restricted in their ability to walk, and particularly to run bipedally.

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Directional asymmetry in the forelimb of Macaca mulatta.

Asymmetry was investigated in the forelimbs of 150 rhesus monkey (Macaca mulatta) skeletons using measurements of right and left humerii, radii, ulnae, second metacarpals, and femora. Seven of the ten forelimb dimensions were larger on the right than on the left side. Paired t-tests revealed that the mean of the right side was significantly larger than that for the left for two measurements of the ulna and two of the humerus. No measurement was significantly larger on the left than on the right side. These results indicate a small but significant asymmetry in the forelimb bones of rhesus monkeys and, as is the case for humans, the direction of asymmetry favors the right side. Our findings are consistent with an interpretation of hypertrophy of certain muscles and opens the question of whether rhesus monkeys preferentially use their right forelimbs for manipulative tasks that require manual dexterity, as is the case for humans. These forelimb skeletal asymmetries are discussed in light of the recent literature on cortical asymmetry and handedness in nonhuman primates.

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Use of the rat forelimb compression model to create discrete levels of bone damage in vivo.

Skeletal responses to damage are significant for understanding the etiology of stress fractures and possibly osteoporotic fractures. We refined the rat forelimb-loading model to produce a range of sub-fracture damage levels during in vivo cyclic loading. A total of 98 right forelimbs of anesthetized, male, 5-month old Fischer rats were loaded cyclically (2 Hz) in axial compression. Rats were killed immediately after loading. In the first experiment, forelimbs were loaded to fracture, which occurred after an increase in peak displacement of 2.0+/-0.2 mm, independent of peak force or cycle number. In the next experiment, we loaded forelimbs at a constant peak force until the peak displacement increased by 0.6-1.8 mm (30-90% of fracture displacement). Mechanical properties of the loaded (right) and contralateral control (left) ulnae were determined ex vivo using three-point bending, and cracks were analyzed using micro-computed tomography. Results demonstrated a dose-response between increased forelimb displacement and increased ulnar damage, with four discrete damage levels. "Low" damage was produced by cyclic loading to 30% of fracture displacement, with no visible cracks and a 10% strength loss. "Mild" damage was produced by loading to 45% of fracture displacement, with variable linear cracks and 20% strength loss. "Moderate" damage was produced by loading to 60-75% of fracture displacement, with consistent linear cracks and 40% strength loss. "High" damage was produced by loading to 85-90% of fracture displacement, with branching cracks and 60% strength loss. This loading model will be useful for examining biological responses to a range of sub-fracture damage levels in future experiments.

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