Effect of high-voltage pulsed current and alternating current on macromolecular leakage in hamster cheek pouch microcirculation.

BACKGROUND AND PURPOSE: Electrical stimulation (ES) is supposed to affect edema formation by inhibiting macromolecular leakage from microvessels. The purpose of the study was to determine the effects of various forms of ES on macromolecular leakage from microvessels. SUBJECTS: Fifty-three hamsters were randomly assigned to one of seven groups: a control group (histamine only); groups that received histamine with cathodal high-voltage pulsed current (HVPC) at intensities of 90%, 50%, and 10% of visible motor threshold (VMT); groups that received anodal HVPC at intensities of 90% and 50% of VMT; and a group that received alternating current (AC) at 90% of VMT. METHODS: Anesthetized animals were injected with fluorescein-labeled dextran. Macromolecular leakage was determined by computer analysis of fluorescence microscopy images for 5 minutes after treatment. RESULTS: When compared with controls, leakage was less in groups treated with cathodal HVPC at 90% and 50% of VMT and anodal HVPC at 90% of VMT. CONCLUSION AND DISCUSSION: Cathodal and anodal HVPC, but not AC, curb macromolecular leakage from the microvessels of histamine-treated hamsters. [Taylor K, Mendel FC, Fish DR, et al. Effect of high-voltage pulsed current and alternating current on macromolecular leakage in hamster cheek pouch microcirculation.

High-voltage pulsed current: its influence on diameters of histamine-dilated arterioles in hamster cheek pouches.

Results from five independent studies from our laboratory indicate that cathodal high-voltage pulsed current (HVPC) significantly curbs posttraumatic edema formation in several animal models. Conversely, anodal HVPC did not curb edema formation. The mechanism by which HVPC reduces edema formation is unknown. We hypothesize that HVPC causes a decrease in local blood flow by active vasoconstriction of arterioles. Because we had previously observed positive effects with cathodal HVPC but not anodal HVPC, we further hypothesized that cathodal but not anodal HVPC would reduce diameters of histamine-dilated arterioles. Changes in diameters of resistance arterioles (5 to 30 microns internal diameter) were measured directly in cheek pouches of anesthetized hamsters, using in vivo video microscopy. Three minutes after superfusion with the inflammatory mediator (histamine) was begun, sensory-level HVPC at 120pps was applied concurrently for 30 minutes. Five animals received cathodal HVPC and five received anodal HVPC. Four other animals received 30-minute treatments of both cathodal and anodal HVPC in random order. Three control animals received histamine without HVPC for 30 minutes. Diameter changes of one arteriole from each cheek pouch was measured every 20 seconds throughout the treatment period. One-way analysis of variance (ANOVA) with repeated measures showed that diameters of histamine-dilated controls varied little over 30 minutes, and that adding cathodal HVPC did not significantly alter diameters of arterioles superfused with histamine. However, applying anodal HVPC to histamine-dilated arterioles significantly reduced arteriolar diameters. These results do not support the hypothesis that cathodal HVPC curbs edema formation by increasing arteriolar tone in the injured area.

Alterations in intramuscular connective tissue after limb casting affect contraction-induced muscle injury.

This study examined the effect of alterations in rat intramuscular connective tissue (CT), secondary to limb immobilization, on the muscle's susceptibility to contraction-induced injury. Hindlimbs were casted for 3 wk with the extensor digitorum longus muscle fixed in a shortened (IM-SP) or lengthened position (IM-LP). An age-matched control group remained uncasted. Extensor digitorum longus muscles were injured in vivo by using a motorized foot pedal that repeatedly flexed and extended the foot while the muscle was electrically stimulated during plantar flexion. Four hours postinjury, maximum isometric tetanic force (Po) was measured in vitro and was used as a functional index of muscle injury. Muscles were fixed, sectioned, and stained for later analysis. Intramuscular CT concentration, expressed as the ratio of CT area to muscle fiber area, was significantly higher in both IM-SP (0.153 +/- 0.003) and IM-LP (0.174 +/- 0.003) groups compared with controls (0.104 +/- 0.003). Po values of injured muscles both IM-LP and IM-SP were higher than the injured controls' Po of 9.41 +/- 0.63 N/cm2 (P < 0.05). Injured IM-LP muscle forces were significantly higher than those of IM-SP. This study demonstrated that limb immobilization increases intramuscular CT concentration, which is accompanied by attenuation of muscle injury. We conclude that remodeling of intramuscular CT affects the muscle's resistance to contraction-induced injury.

Delayed recovery of endothelium-dependent vasodilation in regenerating arterioles of skeletal muscle autografts.

This study investigated the responses of regenerating arterioles in grafted skeletal muscle to the endothelium-dependent dilator acetylcholine (ACh); the substrate for endothelium-derived relaxing factor (EDRF), L-arginine (L-Arg); and sodium nitroprusside (SNP). Additionally, responses of graft arterioles to the endothelium-independent substances adenosine (Ado) and norepinephrine (NE) were measured. The retractor muscle of hamsters was removed, placed in a myotoxic solution, and grafted into its original site. The graft revascularized spontaneously by sprouting of vessels in surrounding tissue. Quantification of changes in arteriolar luminal diameter was accomplished using in vivo video microscopy at 30, 45, and 60 days postgrafting. In 30-day grafts, there was little or no response to topically applied ACh or L-Arg. By 45 days, arteriole response to 10(-6), 10(-5), and 10(-4) M ACh and 10(-4) L-Arg was 0, 4, 20, and 17% of the control response, respectively. SNP (10(-6)-10(-4) M) produced approximately 50% of the control response at both 30 and 45 days. By 60 days the response of graft arterioles to ACh, L-Arg, and SNP was not different from controls. Arteriolar response in 30-day grafts to Ado (10(-6)-10(-4) M) was significantly attenuated, but was not different from control by 45 days. Responsiveness of arterioles to NE had recovered to control levels by 30 days postgrafting. These data indicate that the restoration of endothelium-dependent regulation of arteriolar dilation lags behind endothelium-independent, receptor-mediated mechanisms. The diminished response of regenerating arterioles may be related to dysfunctions in both the synthetic pathway for EDRF and the guanylate cyclase/cGMP mechanism for vascular smooth muscle cell relaxation and/or flow-dependent mechanisms of blood flow regulation.

Effect of electrically induced muscle contractions on posttraumatic edema formation in frog hind limbs.

We tested the hypothesis that repeated muscle contractions induced by high voltage pulsed current (HVPC) would limit volume increases in traumatized frog hind limbs. Twelve frogs were anesthetized, and both hind limbs of each frog were traumatized by impact. Limb volumes were measured via water displacement over a 24-hour period. Four 30-minute treatments of continuous 1-pulse per second HVPC were applied to one limb selected randomly. Stimulation produced muscle contractions that resulted in minimal joint movements. Volume changes from pretrauma limb volumes (in milliliters per kilogram) were analyzed by an analysis of variance for repeated measures. Our hypothesis was rejected (ie, repeated muscle contractions, as induced in this study, did not limit posttraumatic edema formation in frogs). Further investigation of the relative influences of limb position and varying pulse rates, pulse durations, and intensities of HVPC on edema formation may provide valuable insights on effective treatment of edema in humans.

Attenuation of contraction-induced skeletal muscle injury by bromelain.

The proteolytic enzyme, bromelain, reportedly has therapeutic effects in the treatment of inflammation and soft tissue injuries. We tested the hypothesis that bromelain attenuates skeletal muscle injury induced by lengthening contractions. The left extensor digitorum longus (EDL) muscle of anesthetized hamsters was injured using a motorized foot pedal which repeatedly flexed/extended the foot through a range of 125 degrees. The EDL muscle was electrically stimulated for 400 ms during plantarflexion. Animals were assigned randomly to either a 0-d group (evaluated 3-h post-injury) or to untreated (UT) or bromelain-treated (T) groups, evaluated 3, 7, or 14 d post-injury. Following injury, T received 5 mg.kg-1 b.w. of bromelain, twice daily. Maximum isometric tetanic force (Po) was measured in vitro, then muscles were fixed, sectioned, and examined for evidence of fiber damage. The Po of injured muscles from T were higher than Po of injured muscles from UT at 3 (18.7 +/- 0.4 vs 16.5 +/- N.cm-2 and 14 d (20.5 +/- 0.6 vs 18.2 +/- 0.6 N.cm-2) (P less than 0.05), but not 7 d (19.5 +/-0.7 vs 17.7 +/- 0.8 N.cm-2). The Po of UT injured muscles were significantly lower than Po of contralateral control muscles at all time periods. Po of injured muscles from T were lower than Po from control muscles at 3 and 7 d (P less than 0.05), but not 14 d. The number of intact fibers of 3-d UT injured muscles was lower than the number of intact fibers in control muscles (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of a single 30-minute treatment of high voltage pulsed current on edema formation in frog hind limbs.

The purpose of this study was to investigate the effect of a single treatment of high voltage pulsed current (HVPC) on edema formation. Twenty-four frogs were anesthetized, and both hind limbs of each frog were traumatized by impact. Limb volumes were measured by water displacement immediately before and after trauma and at predetermined intervals for 24.0 hours posttrauma. One limb of each frog was randomly selected to receive 30 minutes of continuous, 120-pulse per second, cathodal HVPC at voltages 10% less than motor threshold levels. Data were analyzed by an analysis of variance for repeated measures. Sources of significant differences were determined by paired t tests (probability values determined by Bonferroni adjustment). A single 30-minute application of HVPC significantly curbed edema formation for between 4.0 and 7.5 hours following treatment (ie, volumes of treated limbs were significantly less than those of untreated limbs). These results suggest that regimens currently applied to humans (ie, one treatment per day or three times per week) may be insufficiently aggressive to provide sustained treatment effects.

Acoustic myography as an indicator of force during sustained contractions of a small hand muscle.

To test the hypothesis that muscle sound amplitudes would remain constant during sustained submaximal isometric contractions, we recorded acoustic myograms from the abductor digiti minimi muscle in 12 subjects at 15, 25, 50, and 75% of a maximum voluntary contraction (MVC). Muscle sounds were detected with an omni-directional electret microphone encased in closed-cell foam and attached to the skin over the muscle. Acoustic amplitudes from the middle and end of the sustained contractions were compared with the amplitudes from the beginning of contractions to determine whether acoustic amplitudes varied in magnitude as force remained constant. Physiological tremor was eliminated from the acoustic signal by use of a Fourier truncation at 14 Hz. The amplitudes of the acoustic signal at a contraction intensity of 75% MVC remained constant, reflecting force production over time. At 50% MVC, the root-mean-square amplitude decreased from the beginning to the end of the contraction (P less than 0.05). Acoustic amplitudes increased over time at 15 and 25% MVC and were significantly higher at the end of the contractions than at the beginning (P less than 0.05). Alterations in the acoustic amplitude, which reflect changes in the lateral vibrations of the muscle, may be indicative of the different recruitment strategies used to maintain force during sustained isometric contractions.

Force deficit of vascularized skeletal muscle grafts in rabbits.

Through autografting experiments on 9-g rectus femoris (RFM) muscles in rabbits, we substantiated a previous observation that the maximum isometric tetanic force (Po) and specific Po (N/cm2) of neurovascular-intact grafts are not different from grafts made with neurovascular repair. We then tested the hypotheses that the specific Po of vascularized grafts is significantly less than that of control RFM muscles and the deficit in the specific Po is associated with increases in connective tissue and interstitial space. The specific Po of the grafts was 65% of the value for control RFM muscles. Connective tissue protein concentration of grafts was 3.8 times greater than the control value of 16.6 +/- 3 micrograms/mg wet mass, but this only accounted for a 5% correction in specific Po. The volume of interstitial space did not differ between grafts and control muscles. We conclude that the deficit of 35% in specific Po of vascularized grafts compared with control values is partially explained by an increase in connective tissue, but a 30% unresolved deficit remains.

Total and regional blood flows in vascularized skeletal muscle grafts in rabbits.

The transplantation of whole skeletal muscles is a common clinical procedure. Although atypical blood flows have been reported in small free muscle grafts, the blood flow of large neurovascular-intact (NVI) and neurovascular-anastomosed (NVA) grafts have not been measured. Because the maximum specific force (N/cm2) of NVI and NVA grafts is 65% that of control muscles, we hypothesized that total and regional blood flows (ml.min-1.100g-1) of NVI and NVA grafts at rest and during twitch contractions are significantly lower than lower flows of control muscles. In rabbits, blood flows of control rectus femoris (RFM) muscles and NVI and NVA grafts of RFM muscles were measured by the radioactive-microsphere technique. In control muscles, blood flow increased linearly from 6.8 +/- 1 ml.min-1.100 g-1 at rest to 64.4 +/- 7 ml.min-1. 100 g-1 at a stimulation frequency of 3 Hz with no further increase at 4 Hz. Total blood flows in grafts were not different from the control RFM muscle values, except for a higher resting flow in NVA grafts and a lower flow at 3 Hz in NVI grafts. Minor variations in regional flows were observed. We conclude that the operative procedures of grafting and repair of blood vessels affect the vascular bed of muscles minimally, and the deficits observed in grafts do not arise from inadequate perfusion.

The response of arterioles in skeletal muscle grafts to vasoactive agents.

Arteriolar responses to the vasodilator adenosine and to the vasoconstrictor norepinephrine (NE) were examined in small bundles of extensor digitorum longus muscle grafted onto the cheek pouches of hamsters. Responses of arterioles to topically applied adenosine or NE were measured from 30 to 180 days after grafting and compared with the response of arterioles in control cheek pouches. Verapamil and potassium chloride (KCl) were applied to 120- and 180-day grafts to determine the response of arterioles to vasoactive agents not mediated by receptors. Arterioles in grafted muscle did not respond to adenosine until 60 days. The response increased with time but was significantly less than the control value even after 180 days. Arterioles in grafts did not respond to NE until 90 days. At both 90 and 120 days the degree of constriction in response to NE was not uniform along the length of a given arteriole (punctate response). By 180 days, the response of a given arteriole to NE was uniform but significantly less than the control value. In 120- and 180-day grafts the responses of arterioles to verapamil and KCl were similar to the responses of arterioles to adenosine and NE, respectively. We conclude that the diminished response of arterioles in small EDL muscle grafts to vasoactive agents may be caused by either a structural or a functional impairment in the smooth muscle layer of arterioles.

Microcirculatory adaptation to skeletal muscle transplantation.

A standard whole muscle graft, in which all neural and vascular connections with the host tissue are severed, reinnervates and revascularizes spontaneously. The extent to which a standard graft regains structure and function depends largely on the degree and timing of revascularization. Whether the endothelial cells that form the first capillary tubes in a whole muscle graft originate inside or outside of the graft is unknown. Blood vessel growth into a graft proceeds in a centripetal fashion and precedes muscle fiber regeneration. Abnormalities have been observed in the structure of capillaries and of the larger vessels in stable muscle grafts. The ability of the vascular bed in large standard grafts to regulate blood flow in response to twitch contractions is impaired, as is the ability of arterioles in small grafts to respond following topical application of norepinephrine and adenosine. The dysfunction of arterioles in standard grafts may be related to structural abnormalities. Blood flow at rest and in response to twitch contractions in stable grafts in which nerves and blood vessels are left intact is not different from control muscles. The following should be investigated to provide further insights into the revascularization process in regenerating skeletal muscle and the structure and function of the microcirculation in stable muscle grafts: the origin of mitotic endothelial and smooth muscle cells; the stimulus for blood vessel growth; the overall architecture and organization of the vascular bed; the presence or absence of adrenergic innervation of blood vessels; and the development of receptors involved in blood flow regulation in muscle grafts.

Metabolic factors from exercising muscle and the proliferation of endothelial cells.

In order to identify factors that may be involved in angiogenesis in mammalian skeletal muscle after endurance exercise, the growth of cultured vascular endothelial cells derived from the canine hindlimb was measured in response to hypoxia and changes in temperature and concentrations of hydrogen ion and adenosine. Endothelial cells were freed from vessels of exsanguinated canine hindlimbs using collagenase. Cultures were grown on plastic culture dishes at 37 degrees C and 40 torr CO2 in air. Primary cultures reached confluency by 21 days. Cell number in secondary cultures was significantly increased in 7 days by exposure to 35 torr oxygen for 1 h daily, but cell growth rate was unaffected by 65 torr oxygen. Cell growth was inhibited when hydrogen ion concentration was increased by adding lactic acid daily (5 mmol X l-1). Exposing cell cultures for 1 h daily to an elevated PCO2 (70 torr) or to an environmental temperature of 41 degrees C, adding sodium lactate (5 mmol X l-1), or adenosine (10 or 100 mumol X l-1) daily did not affect cell growth rate. Thus, hypoxia increased the growth rate of cultured vascular endothelial cells and may play a role in blood vessel growth in vivo.

Selection for high specific gravity of eggs in a flock of Australorp hens.

1. A realised heritability of 0.23 was obtained in an Australorp flock (S) selected for five generations for high egg specific gravity. 2. A comparison with an unselected control flock (C) over 50 weeks of lay in the final generation indicated a number of statistically significant correlated responses in commercially important traits in the S line in addition to the direct response of +0.004 in specific gravity. 3. With an increase in specific gravity, there was a decrease of 3.4 in the percentage of soft-shelled eggs laid. 4. The weight and albumen height of eggs measured within 1 hour of lay declined by 1.8 g and 2.1 Haugh units respectively. 5. There were reductions in the weight and albumen height losses of eggs stored over a 10-d period (C 0.74, S 0.62 g and C 17.9, S 15.0 Haugh units respectively), so that at the end of this period the albumen heights in both lines were the same. 6. Average body weight and daily food intake were less by 0.28 kg and 7.5 g respectively. 7. Although there was no change in egg production, the average age at first egg was reduced by 11.3 d.