A simple chemiluminescence assay for detecting oxidative stress in ischemic limb injury.

This study was designed to evaluate a simple, sensitive, nonstimulated chemiluminescence assay to measure the oxidative stress production in the whole blood of rabbits after an ischemic insult. By using an ultra-sensitive chemiluminescence (CL) analyzer and lucigenin amplification, the assay system can be performed without leukocyte isolation and stimulant administration. The blood CL levels of healthy rabbits were 122 +/- 18 counts/10 s. After 8 h of ischemia, the CL levels of whole blood immediately after reperfusion, 1 h, 2 h, 24 h, 48 h, 7 days, and 14 days of reperfusion were 409 +/- 78 counts/10 s, 283 +/- 55 counts/10 s, 256 +/- 43 counts/10 s, 228 +/- 33 counts/10 s, 185 +/- 32 counts/10 s, 160 +/- 16 counts/10 s, and 119 +/- 15 counts/10 s. The differences were statistically significant between the control and samples obtained up to 24 h after reperfusion. The corresponding creatine phosphate kinase (CPK) levels of the rabbits in this study were 938 +/- 43 U/l for the control blood sample and 5921 +/- 498 U/l, 6948 +/- 427 U/l, 6860 +/- 1115 U/l, 5763 +/- 516 U/l, 1545 +/- 291 U/l, 478 +/- 60 U/l, and 458 +/- 48 U/l for the experimental blood samples. The CPK levels of the blood samples before 24 h and after 7 days of reperfusion were significantly different to that of the control blood sample. The changes in the CL and CPK levels were quite similar before 48 h reperfusion. This assay has proved to be valuable in the quantitative measurement of ischemic insults of skeletal muscles.

Scavenging effect of benzophenones on the oxidative stress of skeletal muscle cells.

Benzophenone is an ultraviolet (UV)-absorbing agent that has been used in industry and medicine for more than 30 years. Consumers of cosmetics and sunscreens containing UV-absorbers are exposed to benzophenones on a daily basis, owing to the widespread use of these compounds. However, the efficacy of these compounds as scavengers of oxidative stress is still not well established. In the present study, we investigate the antioxidative capacity of six sunscreen benzophenone compounds. A primary myoblast culture was mixed in vitro with 100 microM menadione. The cytotoxic effect by menadione-induced oxidative stress was monitored by the lucigenin- or luminol-amplified chemiluminescence, methylthiotetrazole (MTT) assay, and the antioxidative effects of various benzophenone compounds were evaluated. The results showed that the addition of menadione can induce oxidative stress on myoblasts by superoxide and hydrogen peroxide production, which can be eradicated by superoxide dismutase (SOD) and catalase, respectively, in a dose-dependent mode. The catalase has a protective effect on the cytotoxicity induced by menadione as measured by the MTT assay, while the SOD does not. The selected benzophenones also have a significant scavenging effect on the menadione-induced cell death on the myoblasts. The ortho-dihydroxyl structure and other hydroxy groups in the same ring have a stronger scavenging effect on the superoxide anion on myoblasts; thus, a stable penoxy radical may be formed. The mechanism of this effect remains to be clarified.

Ultrastructural studies on myofibrillogenesis and neogenesis of skeletal muscles after prolonged traction in rabbits.

Little is known about the morphological response of muscle after long term traction. The purpose of this study was to investigate the morphological changes of skeletal muscle during limb lengthening. After application of mini-extraskeletal fixator, the hindlimb of New Zealand white rabbit was osteotomized and then slowly lengthened at the rate of 1 mm/day up to a 20 mm gain in length. The muscles of hindlimbs were perfused and dissected. Morphological studies were performed at electron microscopic level. Transmission electron microscopy revealed foci of microtrauma at the myotendinous junction. The distance between the muscle fibers and tendon parenchyma increased, with numerous primitive mesenchyme-like cells interposed within this gap. The cytoplasmic space of these cells was devoid of myofibril formation at the ends of stretched fibers. Within the satellite near the myotendinous junction myofilament production was observed in various gradations of maturation. It is concluded that myofibrillogenesis with traction neogenesis of skeletal muscle during limb lengthening does exist and occurs mainly near the myotendinous junction. The myotendinous junction in mature skeletal muscle actively participated in the process of limb lengthening.

Biochemical and histopathological changes in the mortality caused by acute ischemic limb injury: a rabbits' model.

Restoration of blood flow to an acute ischemic extremity may deteriorate the ischemic injury, lead to multiple organ dysfunction or even death. This paradox of continuing injury during reperfusion is not completely understood. The role of multi-organ damage in the mortality caused by ischemic limb injury is also still not clarified. The purpose of this study is to determine the biochemical and histopathological changes in the mortality caused by ischemic limb injury. After anesthesia, the hindlimbs of 14 New Zealand white rabbits were made ischemic and set into 8 hours or 12 hours of ischemia. Blood samples were obtained then the creatine kinase (CK) levels were determined and CK isoenzymes analyzed. All rabbits with 8 hours' ischemia survived well, and 5 of the 7 rabbits with 12 hours' ischemia expired within 8 hours after reperfusion. CK elevation was correlated most strongly with the time of the ischemic insults. The percentage of CK-MB isoenzyme remained unchanged after 8 hours' ischemia-reperfusion insult, while increased significantly after 12 hours' ischemia-reperfusion insult. Histologic examinations showed that the major systemic manifestation was massive destruction of the liver and kidney. The injuries are more obvious in areas with the greatest blood flow during reperfusion. We concluded that the ratio of CK-MB isoenzyme is most useful for distinguishing the risk of mortality caused by acute ischemic limb injury, and the cause of systemic complications are attributed to the multi-organ failure.

Biocompatibility and osteoconductivity of the pyrost bone substitutes.

The purpose of this study was to re-evaluate the bone regeneration power and the in vitro biocompatibility of the Pyrost bone substitute. Twenty-four adult New Zealand White rabbits were used. Bony defect over both iliac crest and mid-diaphyseal portion of the ulna bone were created. Appropriate sized-block of Pyrost bone substitute were implanted. Four of the animals were killed at each postoperative month to evaluate its bone regeneration power by histologic study. The Pyrost bones were co-cultured with osteoblasts to evaluate its biocompatibility. The results showed that Pyrost bone substitute was quite stable and incorporated well with active bone regeneration. The Pyrost heal better at the iliac crest than at the ulnar defect. The Pyrost was compatible to the osteoblasts. Osteoblasts had successfully seeded and mitotically expanded on the porous surface of the Pyrost bone graft. The result showed that Pyrost bone obviously exerts an intense stimulus on osteo-regeneration in the presence of osteoblasts. We consider Pyrost to be an alternate to the conventional preserved allografts that is occasionally necessary.

Antioxidant status following acute ischemic limb injury: a rabbit model.

Although ischemic injury to skeletal muscle is a matter of great clinical importance, relatively little is known about the mechanisms which determine systemic responses. One purpose of this study is to elucidate the systemic antioxidant status following an episode of acute ischemic limb injury and subsequent reperfusion. Twelve New Zealand white rabbits were used in this study. After the animals were anesthetized, an ischemic insult was created in the right hind limb for twelve hours, followed by four hours of reperfusion. Several series of blood samples were obtained. At the end of the experiment, the animals were killed and necropsies undertaken in order to evaluate the antioxidant status of various visceral organs. The results link ischemia and reperfusion injury to a significant decline in antioxidative activity in various tissues. The weakening in antioxidant status after ischemic limb injury was most pronounced in the heart tissue, followed in descending order by the spleen, skeletal muscle, lung, liver, and kidney tissue. The levels of specific antioxidants and reactive oxygen species in various organs changed significantly, and the changes were tissue specific. Endogenous radical scavenging systems were not entirely overwhelmed in most of the tissues studied. But higher levels of malondialdehyde (MDA) found in cardiac tissue suggest that the production of oxygen free radicals is accelerated by an ischemic injury. Based on the study, we believe that the cardiac tissue is particularly susceptible to the effects of ischemia and reperfusion injury. Damage to cardiac tissue is probably the major cause of mortality following acute ischemic injury in a limb.

Contracture of the hip secondary to fibrosis of the gluteus maximus muscle.

Twenty-eight children were treated who had limited flexion of the hips and various degrees of contracture of the abductor and external rotator muscles because of fibrosis of the gluteus maximus muscle. Although the lesions could be classified as those associated with poliomyelitis, infection of the gluteus maximus muscle, and fibrosis of unknown etiology, all forty-five hips had a typical restriction of motion such that an affected hip could not be flexed in the usual sagittal plane, but had to be flexed in abduction. Poliomyelitis may have been adjunctive to the causative factor of the lesion in some cases but the probable primary etiology was multiple intramuscular injections. Excellent correction of the hip contracture was achieved in all patients by division of the fibrotic bands.

Contact areas and pressure distributions in the subtalar joint.

We investigated how foot position and ankle arthrodesis affect the contact characteristics of the subtalar joint. Nine fresh-frozen specimens of amputated lower legs were used. Pressure-sensitive films were inserted into the anterior and posterior articulation of the subtalar joint. The contact areas and pressure for various foot positions and under axial loads of 600, 1200, and 1800 N were determined based on the gray level of the digitized film. In neutral position and under a 600 N load, the maximum contact pressure in the subtalar joint was 5.13 +/- 1.16 MPa. The contact area (1.18 +/- 0.35 cm2) was only 12.7% of the whole subtalar articulation area (9.31 +/- 0.66 cm2), and the total force (348.5 +/- 41.7 N) transmitted via this contact area was about 58% of the applied load (600 N). Dorsiflexion of the foot increased the contact area and the force transmitted, but decreased the average contact pressure in the subtalar joint, while the reverse occurred in plantar flexion. Eversion increased the subtalar contact stress, whereas inversion up to 10 degrees decreased it. Ankle joint arthrodesis shifted the contact areas in the subtalar joint posteriorly in all inversion/eversion positions. Moreover, total force transmitted through the subtalar joint as well as the contact pressure increased.

An ultra-weak chemiluminescence study on oxidative stress in rabbits following acute thermal injury.

It is not easy to detect oxygen free radicals directly because of their very short half-life. In the present study, a sensitive ultra-weak chemiluminescence detector was used to detect the generation of oxygen free radicals following thermal injury. Twelve New Zealand white rabbits were used in this study. After anesthesia, the bilateral hind-limbs were exposed to 100 degrees C water for 30 s. Six control animals were exposed to 22 degrees C water to act as a control. The chemiluminescence of whole blood and visceral organs were measured with both luminol-amplified t-butyl hydroperoxide-initiated and lucigenin-initiated methods. The results showed that chemiluminescence of blood was affected significantly by acute thermal injury. The chemiluminescence of blood increased significantly at 1 h following acute thermal injury, reached a peak at 2 h, then decreased but still remained above the control level at 4 h following thermal injury. The results for TBHP-initiated chemiluminescence from visceral organs following acute thermal injury were much higher than that of the control rabbits. The effects of lucigenin-initiated tissue chemiluminescence following acute thermal injury were not statistically significant. It is suggested that the decreased vascular antioxidant activity following local thermal injury is partially contributed by the superoxide pathway; while, the remote pathophysiologic events are mediated by the defective scavenging defenses.

Cushioning effect of heel cups.

Three types of heel cups, two rubber and one plastic, were evaluated in this study. The vertical forces under the heel were monitored using the Computer Dyno Graphy system in 16 normal subjects. Peak force reduction in walking and running after heel cup use was found for all three types of heel cups. The shock absorbency (peak force reduction as a ratio) of heel cups was better in walking (3.5 km h(-1)) than in running (10 km h(-1)). Pressure-sensitive film under the heel revealed that the pressure concentration at the location of calcaneal tuberosity could be smoothed out by the use of heel cups. When plastic heel cups were used, pedobarography showed that the contact area of the heel while standing decreased to 61% of that when barefoot. Roentgenographic study in six patients with heel pain syndrome showed that the thickness of the heel pad increased from 14.4 mm (SD 1.4 mm) to 17.0 mm (SD 1.2 mm) when plastic heel cups were used.

Failure sites and peak tensile forces of the composite triceps surae muscle by passive extension in the rabbit.

The triceps surae muscle-tendon unit of rabbit was tested by a passive extension technique. The effects of different stretch rates were evaluated. Functioning as a composite muscle-tendon unit, the triceps surae responded viscoelastically to the tensile loads. The sites of failure were consistent at the muscular portion near the musculotendinous junction. Failure occurred within 0.1-1.0 mm of the distal musculotendinous junction for the soleus muscle and within 5-10 mm proximal to the distal musculotendinous junction in the lateral head of gastrocnemius muscle. In the medial head of gastrocnemius muscle failure occurred within 15-30 mm proximal to the distal musculotendinous junction. There was a greater peak tensile force requirement and a greater energy absorption when tested with faster stretch rates. These results furnish evidence that the biomechanical property of composite musculotendinous unit is strain-rate dependent and behaves viscoelastically.

In vivo kinematic study of normal wrist motion: an ultrafast computed tomographic study.

OBJECTIVE: To quantify in vivo carpal kinematics of a normal wrist in a dynamic continuous model. DESIGN: The instantaneous changes in the radiocarpal and midcarpal joints during normal wrist motion were analyzed using ultrafast computed tomography (CT). BACKGROUND: Wrist injuries account for a considerable and growing proportion of work-related disorders and disability. However, little is known about normal wrist kinematics. METHODS: Ten uninjured subjects were studied using ultrafast CT to measure the continuous motion of the wrist from full flexion to full extension. Sagittal plane scanning was performed mediolaterally at six different locations as the wrists were moved slowly and repeatedly from full flexion to full extension. The data were printed to X-ray film and transferred to an independent work station with a video camera. The motion of the radiocarpal, midcarpal and wrist joints was determined by an image analyzing system. RESULTS: Wrist motion was expressed as a ratio of capitate-lunate (C-L) (midcarpal) motion and radio-lunate (R-L) (radiocarpal) motion. In the volar flexion of normal wrists, the contribution of the radiocarpal joint and midcarpal joint were approximately equal; while dorsal flexion of the normal wrist occurred mainly at the midcarpal joint. CONCLUSIONS: In normal wrists, the radiocarpal joint and midcarpal joint contribute equally to volar flexion, while the midcarpal joint is more important in dorsal flexion. RELEVANCE: In this study, we demonstrated the suitability of using two-dimensional computed tomographic images in a quantitative study of flexion/extension kinematics of the normal wrist.

Effect of external load on isokinetic torque production by the knee in anterior cruciate ligament deficient patients.

The purpose of this study was to examine the effect of an external load on the isokinetic torque production of the knee directly from the anterior cruciate ligament (ACL) of deficient patients. Ten surgically proven ACL deficient patients were included in this study. Each patient was preoperatively studied using a Biodex isokinetic dynamometer. Isokinetic contraction of the quadriceps muscle and hamstring muscles were performed in five repetitions with an angular velocity of 45 degrees per second with proximal and distal pad placement, respectively. The results demonstrated that the ratio of torque production of the ACL deficient knee to the contralateral normal knee was greater in proximal pad placement than in distal placement and that the ratio of torque production of the knee in proximal pad placement to that of the knee in distal pad placement was greater in the ACL deficient knee than in the contralateral normal knee. Similar results were found for both extension and flexion of the knee. The results showed that ACL deficient patients felt more confident doing isokinetic contractions of the knee with proximal pad placement.

Quantitative test of knee laxity in Chinese.

Anterior knee laxity of 100 Chinese male subjects who had no history of previous injuries to their knees was examined with an instrumented knee arthrometer (MEDmetric Knee Arthrometer, model KT-1000). Two measurements were used to evaluate the anterior laxity: 1) anterior displacement produced by anterior force of 89 newtons (20 lbs); and 2) anterior compliance index (ACI), the displacement difference between an anterior force of 89 newtons (20 lbs) and 67 newtons (15 lbs). All knees were examined at a flexion angle of 20 +/- 5 degrees and an external tibial rotation of 10 +/- 5 degrees with the use of thigh and foot supports. The mean anterior displacement for the right and left knee were 4.5 +/- 2.0 mm and 4.3 +/- 1.9 mm, respectively, while 84% of the study subjects had right and left differences (anterior displacement difference, ADD) of less than 2 mm. The anterior compliance index was 0.85 +/- 0.4 mm for the right knee and 0.78 +/- 0.33 mm for the left knee. The mean anterior compliance index difference (CID), that is the difference between the right and left ACI, was 0.25 +/- 0.3 mm. A total of 93% of the subjects had a difference in the right-left CID of less than 0.5 mm. Variation between the right and left knee does exist, but the difference is not statistically significant (p > 0.05). Therefore, comparison of the right-left ADD and CID can be a useful reference in the assessment of a ligamentous injury.

A dynamic study of the ankle-foot complex.

A kinematic study of the ankle-foot complex in 19 healthy volunteers was performed using computerized radiocinematography. We found that the instant centers of rotation of the ankle joint fell closely within the talus in the sagittal plane. When the ankle joint moved, the talus, the calcaneus and the navicular bone performed different excursions. Except for the movement in the sagittal plane, the calcaneus assumed eversion in dorsiflexion and inversion in plantar flexion when the ankle moved in an open kinematic chain (under non-weight-bearing conditions). In a closed kinetic chain movement (under weight-bearing conditions), the lower leg exhibited internal rotation, and the talus moved plantarward when the ankle was dorsiflexed. The internal rotation of the lower leg did not cause a slippery rotation of the foot on the surface, but rather it was converted into eversion of the calcaneus in the frontal plane.

Epiphyseal scar of the femoral head: a three-dimensional anatomic observation.

The purpose of this study was to inspect the three-dimensional structure of the epiphyseal scar of the femoral head. Three adult human femoral head specimens with a dense epiphyseal scar shown by radiography were used to study the morphology of the scar. One femoral head was cut in the coronal plane to show the cross-section. A trabecular pattern in the femoral head was demonstrated by radiography. The second femoral head specimen, was cut by a wafering saw in a plane parallel to the epiphyseal scar into 1.5 mm thick slices. Microradiographs of these specimen slices were taken to trace the scar from the proximal part of the femoral head to its distal end. In the third specimen, the epiphysis was chipped away to uncover the whole epiphyseal scar for a topographic examination by dissecting microscope and scanning electron microscope. The epiphyseal scar was found to consist of multiple layers of plate-like bony structure capping the metaphysis similar to an inverted cup with a defect adjacent to the fovea. Differences between the trabecular pattern in the epiphysis and metaphysis were distinctive. These morphologic characteristics of the femoral head should be taken into account when investigating stress distribution in the femoral head.

Effect of nerve function on the failure mechanism of the triceps surae muscle by passive extension in the rabbit.

The triceps surae muscle-tendon units of rabbits were tested in vivo by a passive extension technique using an MTS Bionix 858 machine. The effect of nerve function on the failure mechanism was evaluated. The sites of failure were always at the muscular portion near the musculotendinous (MT) junction. It always occurred within 0.1 to 1.0 mm of the distal MT junction of the soleus muscle and within 5 to 10 mm of the distal MT junction in the lateral head of the gastrocnemius muscle. In the medial head of the gastrocnemius muscle, it always occurred within 5 to 10 mm of the proximal MT junction. The loss of nerve function significantly reduced the peak force and the absorption of energy before the point of peak force, but it produced no significant changes in the failure length and the total energy absorption. The functioning nerve might have a protective mechanism which works on the muscle-tendon unit by increasing the peak force and the energy requirement before the muscle is injured.

Surgical treatment of tibial shaft fracture: experience of 207 cases.

From January 1984 to March 1988, 210 tibial shaft fractures in 207 patients treated by intramedullary nails, and by plate and screws and extraskeletal fixation were studied. There were 166 men and 41 women. Of the fractures, 95 (42.2%) of the 210 tibial shaft fractures were closed fractures and 115 (54.8%) were open fractures [Grade I: 44 (21.0%), Grade II: 39 (18.6%), and Grade III: 32 (15.2%)]. The incidence of associated injury was 44.3% and it was highest in the group with extraskeletal fixation (64.7%). Intramedullary nailing was performed in the majority of these fractures (127 out of 210) and yielded the best results in this series. Kuntscher's nailing resulted in nonunion in 3 cases (2.8%), superficial infections in 3 cases (2.8%) and deep infections in 4 cases (3.8%). Interlocking nailing resulted in nonunion in 1 case (4.8%), superficial infection in 1 case (4.8%) and deep infections in 3 cases (14.3%). Plate and screws fixation was performed primarily for metaphyseal fractures (71.4%) and resulted in nonunion in 4 cases (8.1%), superficial infection in 1 case (2.0%) and deep infection in 5 (10.2%). In the group treated with extraskeletal fixation, inasmuch as there were more cases of compromised soft tissue (open fracture, 97.1%), a higher incidence of associated major injuries (64.7%) and more cases of comminution of the bone (67.6%), the final results were less satisfactory. Nonunion was noted in 26.5% of the cases, superficial infection in 2.9% of the cases, and deep infection in 32.4% of the cases. From our study, we conclude that in appropriately selected patients, surgical fixation of tibial shaft fractures can produce a good functional recovery with an acceptable low rate of complications.

Evaluation of hip disorders by radiography, radionuclide scanning and magnetic resonance imaging.

Thirty-six hips were studied because of significant hip pain. Radiography of the hip and bone scintigraphy showed subtle changes. Magnetic resonance imaging (MRI) was performed using a 1.5 Tesla superconductive unit. All MRI findings were confirmed by surgical or pathologic results. Twenty-nine hips had a single lesion, including: infection (one), fracture (eight), avascular necrosis of the femur(two), or contralateral hip (four), transient osteoporosis (six), osteoporosis (one), post-irradiation myositis (one), metastasis (four), and synovitis (two). Twenty-six lesions (89.6%) appeared normal on the radiographs of the hip, while three lesions (10.4%) showed only osteoporotic change. Another seven hips had more than one lesion, including: avascular necrosis and fracture (four), fracture foci (two), and metastasis and fracture (two). Radiography of the hip showed either a negative finding or detected only a single lesion, missing other important pathologic foci. MRI is extremely sensitive to alterations in the bone marrow that may represent pathology occult to plain radiography and bone scintigraphy of the hips. For diagnosis and treatment planning, MRI of the hips should be performed early in patients with persistent pain and negative radiography findings.

Magnetic resonance imaging of rotator cuff lesions.

The purpose of this study was to evaluate the diagnostic performance of magnetic resonance imaging (MRI) in rotator cuff diseases. Forty consecutive patients studied by MRI from September 1990 to September 1992 were included in the study. MRI was performed on a 1.5 tesla MR scanner. Each MRI study was reviewed by two radiologists experienced in rotator cuff tendons. The MRI finding was compared with the surgical result. MRI sensitivity for all tears (partial and complete) was 1.0 (22/22), specificity 0.89 (16/18), accuracy rate 0.95 (38/40), and the predictive value 0.92 (22/24). Signal changes of the rotator cuff tendon (primary signs) were the most reliable criteria in diagnosis of the tear. Changes of subacromial and subdeltoid fat planes and bursa (secondary signs) were complimentary when primary signs were indeterminant. The direction of the long axis of abnormal signal intensity in the rotator cuff tendon may be a good indicator when differentiating tears from tendinitis. We conclude that MRI shows excellent performance in the determination of the presence or absence of a rotator cuff tear.