Rabbit Diaphragm Research Articles

Functional basis for dose-dependent antagonism of rat and rabbit neuromuscular transmission by the bis-pyridinium oxime MMB4.

Organophosphorus (OP) compounds inhibit central and peripheral acetylcholinesterase (AChE) activity, overstimulating cholinergic receptors and causing autonomic dysfunction (e.g., bronchoconstriction, excess secretions), respiratory impairment, seizure and death at high doses. Current treatment for OP poisoning in the United States includes reactivation of OP-inhibited AChE by the pyridinium oxime 2-pyridine aldoxime (2-PAM). However, 2-PAM has a narrow therapeutic index and its efficacy is confined to a limited number of OP agents. The bis-pyridinium oxime MMB4, which is a more potent reactivator than 2-PAM with improved pharmaceutical properties and therapeutic range, is under consideration as a potential replacement for 2-PAM. Similar to other pyridinium oximes, high doses of MMB4 lead to off-target effects culminating in respiratory depression and death. To understand the toxic mechanisms contributing to respiratory depression, we evaluated the effects of MMB4 (0.25-16mM) on functional and neurophysiological parameters of diaphragm and limb muscle function in rabbits and rats. In both species, MMB4 depressed nerve-elicited muscle contraction by blocking muscle endplate nicotinic receptor currents while simultaneously prolonging endplate potentials by inhibiting AChE. MMB4 increased quantal content, endplate potential rundown and tetanic fade during high frequency stimulation in rat but not rabbit muscles, suggesting species-specific effects on feedback mechanisms involved in sustaining neurotransmission. These data reveal multifactorial effects of MMB4 on cholinergic neurotransmission, with the primary toxic modality being reduced muscle nicotinic endplate currents. Evidence of species-specific effects on neuromuscular function illustrates the importance of comparative toxicology when studying pyridinium oximes and, by inference, other quaternary ammonium compounds.

Titin stiffness modifies the force-generating region of muscle sarcomeres.

The contractile units of striated muscle, the sarcomeres, comprise the thick (myosin) and thin (actin) filaments mediating active contraction and the titin filaments determining “passive” elasticity. We hypothesized that titin may be more active in muscle contraction by directly modulating thick-filament properties. We used single-myofibril mechanical measurements and atomic force microscopy of individual sarcomeres to quantify the effects of sarcomere strain and titin spring length on both the inter-filament lattice spacing and the lateral stiffness of the actin-myosin overlap zone (A-band). We found that strain reduced the lattice spacing similarly in sarcomeres with stiff (rabbit psoas) or compliant titin (rabbit diaphragm), but increased A-band lateral stiffness much more in psoas than in diaphragm. The strain-induced alterations in A-band stiffness that occur independently of lattice spacing effects may be due to titin stiffness-sensing by A-band proteins. This mechanosensitivity could play a role in the physiologically important phenomenon of length-dependent activation of striated muscle.

Direct Evidence for the Effect of Titin Stiffness on Thick-Filament Mechanical Properties in Stretched Muscle Sarcomeres

The titin springs of muscle sarcomeres determine tensile muscle stress. However, they run not strictly in parallel with the sarcomeric axis, being anchored to actin at Z-disks and myosin at A-bands. On tensile sarcomere strain, which stretches the titin springs, a force component arises both longitudinally and laterally. The latter could contribute to increased sarcomeric transversal stiffness, decreased lateral myofilament spacing, and length-dependent activation of stretched muscle. We aimed to quantify by AFM force-mapping the titin contribution to sarcomeric transversal stiffness in comparison to the contribution from electrostatic forces modulated by osmotic compression. Single myofibrils were isolated from skeletal muscles expressing stiff titin-isoform (rabbit psoas) or compliant titin-isoform (rabbit diaphragm). Myofibrils were placed in relaxing buffer under the MFP-3D-BIO-AFM (Asylum Research) and force curves (50x50) were performed over a region-of-interest encompassing a whole sarcomere. Measurements were done in the absence or presence (5%) of dextran, and in the non-stretched (‘slack’) or stretched state (140-150% of slack). Force mapping revealed distinct transversal stiffness patterns along the sarcomere, suggesting relatively higher Z-disk stiffness and somewhat lower A-band stiffness. A-band lateral stiffness did not differ between psoas and diaphragm sarcomeres at slack. With stretch, this stiffness was increased significantly greater in psoas than in diaphragm. Osmotic compression by 5% dextran at slack increased the A-band lateral stiffness to a degree matching (psoas) or exceeding (diaphragm) that seen after stretch in 0% dextran. The stretch-induced and compression-induced contributions to A-band lateral stiffness were additive. Thus, stiff titin contributes more to A-band stiffness than compliant titin, confirming a role for titin in lateral force generation during sarcomere stretching. The electrostatic and the titin-based lateral forces compressing the A-band lattice are of the same order of magnitude in stretched muscle.

Effectiveness of diaphragmatic stimulation with single-channel electrodes in rabbits

Every year, a large number of individuals become dependent on mechanical ventilation because of a loss of diaphragm function. The most common causes are cervical spinal trauma and neuromuscular diseases. We have developed an experimental model to evaluate the performance of electrical stimulation of the diaphragm in rabbits using single-channel electrodes implanted directly into the muscle. Various current intensities (10, 16, 20, and 26 mA) produced tidal volumes above the baseline value, showing that this model is effective for the study of diaphragm performance at different levels of electrical stimulation

The Effects of Chronically Increased Intra-abdominal Pressure on the Rabbit Diaphragm

Diaphragmatic muscular remodeling is caused by various conditions and was mainly studied in pulmonary pathologies and chronic alterations of intra-thoracic pressure. We investigate the effect of the chronically increased intra-abdominal pressure (IAP) on the diaphragm by morphological and biochemical analysis. Thirty rabbits were divided into control and study groups. IAP was increased in group B to 12mmHg for 2months. The left hemidiaphragm underwent morphological, while the right underwent biochemical analysis. In H&E, all fibers were normal. ATPase analysis demonstrated that type I fibers show no differences between groups. Type ΙΙ(Α) were decreased (p = 0.016) while type ΙΙ(Β/X) fibers were increased (p = 0.025) in group B. Fibers with resistance to fatigue were decreased in group B (p = 0.024). In group B, biochemical activity for glutathione reductase (p = 0.004), glutathione peroxidase (p = 0.021), protein carbonylation (0.029), lipid peroxidation (p = 0.005), and balance of preoxidative-antioxidative factors (p = 0.006) was increased. Chronically increased IAP induces alterations to the rabbit diaphragm. Adaptation, equivalent to strenuous contraction, transforms the diaphragm to be functionally more efficient toward workload but makes it vulnerable against oxidative stress.

Development of a new experimental model of intramuscular electrical stimulation of the diaphragm in rabbits

To develop an animal model of diaphragmatic electrical stimulation able to generate an appropriate ventilatory support through the direct implantation of electrodes in the diaphragm (electroventilation). Six New Zealand female rabbits (2-3 kg) were placed on mechanical ventilation. Then, a laparotomy was performed in order to identify the motor points in each hemidiaphragm, followed by the implantation of the electrodes for diaphragmatic stimulation. We tested two types of electrodes according to the conduction of electrical stimulation: unipolar and bipolar. The electrodes were placed on different occasions in the same animals and tested with current intensities of 20, 26 and 32 mA. Each current intensity was repeated three times for 10 respiratory cycles with 1 minute interval between each cycle, and 5 minutes for new current intensity. We recorded the relationship between current intensity and inspiratory volume. The electrodes adequately stimulate the diaphragm and obtain inspired volumes using different intensity currents. The bipolar electrode generated inspiratory volumes as high as 4.5 times of baseline while the unipolar electrode reached up to 3.5 times of baseline. This model has proved to be effective for studying the performance of the diaphragm under different electrical stimulations using different set of electrodes.

Motor control of the diaphragm in anesthetized rabbits

Diaphragmatic regions are recruited in a specialized manner either as part of a central motor program during non-respiratory maneuvers, e.g. vomiting, or because of reflex responses, e.g. esophageal distension. Some studies in cats and dogs suggest that crural and costal diaphragm may be differentially activated also in response to respiratory stimuli from chemoreceptors or lung and chest wall mechanoreceptors. To verify whether this could occur also in other species, the EMG activity from the sternal, costoventral, costodorsal, and crural diaphragm was recorded in 42 anesthetized rabbits in response to various respiratory maneuvers, such as chemical stimulation, mechanical loading, lung volume and postural changes before and after vagotomy, or a non-respiratory maneuver such as esophageal distension. Regional activity was evaluated from timing of the raw EMG signal, and amplitude and shape of the moving average EMG. In all animals esophageal distension caused greater inhibition of the crural than sternal and costal diaphragm, whereas under all the other conditions differential diaphragmatic activation never occurred. These results indicate that in response to respiratory stimuli the rabbit diaphragm behaves as a single unit under the command of the central respiratory control system.

Phrenic Nerve Distribution in the Rabbit Diaphragm and Morphometric Analysis of Nerve Branches

The best method to evaluate the pathogenesis of diaphragmatic disorders is to demonstrate the distribution pattern of the phrenic nerve in the diaphragm. For this purpose the branching pattern and the microanatomic features of the phrenic nerve were observed in six rabbits. All diaphragms were stained by using Sihler's stain method. The phrenic nerve divided into three to four branches when entering the diaphragm. These branches were classified as sternal, anterolateral, posterolateral and crural. The crural branches were the thickest whereas the anterolateral branches were the thinnest. Knowledge about the distribution pattern of the phrenic nerve may be important in surgical approach to the diaphragm.

Susceptibility to cartap-induced lethal effect and diaphragmatic injury via ocular exposure in rabbits.

Cartap is extensively used to control agricultural pests. Pertinent literatures have indicated that it causes no eye irritation [D.E. Ray, Insecticides derived from plants and other organisms, in: W.J. Hayes, E.R. Laws (Eds.), Handbook of Insecticide Toxicology, Classes of Insecticides, vol. 2, Academic Press, New York, 1991, p. 611; C. Tomlin, Cartap, in: C. Tomlin (Ed.), The Insecticide Manual, 12th ed., British Crop Protection Council, Surrey, UK, 2000, p. 144]; however, the instillation of a little cartap through the eye has caused death in rabbits. The aim of this study was to determine the ocular toxicity of cartap in New Zealand White rabbits. Cartap was directly instilled into the low conjunctival sac of eyes, at doses of 0, 5, 7.5, 10 and 12.5 mg/kg body weight. The changes in the enzymes and isoenzymes of creatine kinase (CK), lactate dehydrogenase (LD), as well as pathological changes in the muscles of the heart, thigh and diaphragm were determined in the cartap-treated rabbits. Moreover, the neuromuscular effect of cartap was examined using the isolated rabbit phrenic-nerve diaphragm model. The results indicated that rabbits developed severe signs and they died within 20 min of ocular instillation. The ocular LD50 of cartap was 8.1 mg/kg body weight. Treatment with cartap increased the activities of CK and LD enzymes and their isoenzymes, CK-1, CK-2, and CK-3 in serum, and CK-3 and LD-5 in the diaphragm. Microscopically, hypercontraction bands and the rupture of myofibers of the diaphragm were observed in dead rabbits. Cartap did not affect nerve-evoked twitch but induced irreversible contracture and twitch depression on the isolated rabbit's diaphragm. These results indicate that the rabbit is susceptible to cartap toxicity; the effect of cartap caused contracture and damage to the diaphragm might play a pivotal role in respiratory paralysis and death of rabbits during intoxication.

Effects of reactive oxygen species on aspects of excitation-contraction coupling in chemically skinned rabbit diaphragm muscle fibres.

Oxidants have been suggested to enhance contractile function in unfatigued muscle. In this study we aimed to determine the effect of oxidants on "chemically skinned" diaphragm muscle fibre bundles. The sarcoplasmic reticulum and contractile proteins were exposed to superoxide anions (O2-) and hydrogen peroxide (H2O2) under controlled conditions. Application of O2-initially increased maximum Ca2+ -activated force but subsequently reduced maximum Ca2+ -activated force without altering myofilament Ca2+ sensitivity. Unlike myocardium, caffeine-induced Ca2+ release from the sarcoplasmic reticulum was also inhibited by O2- exposure in diaphragm fibre bundles. Application of H2O2 also increased maximum Ca2+ -activated force but had additional effects on resting tension (which increased to 25 % of the control maximum Ca2+ -activated force). H2O2 was without effect on myofilament Ca2+ sensitivity or caffeine-induced Ca2+ release from the sarcoplasmic reticulum. These data demonstrate that oxidants can potentiate contractile force in the diaphragm through a direct action on the contractile proteins. The potentiation of force is not sustained, however, and under these conditions the detrimental effects of O2- on Ca2+ release from the sarcoplasmic reticulum combined with the effects of oxidants on the contractile proteins will ultimately compromise excitation-contraction coupling in the diaphragm. Experimental Physiology (2001) 86.2, 161-168.

Response of the rabbit diaphragm to tendon vibration

To evaluate the potential role of diaphragmatic muscle spindles in the act of breathing, we have recorded the electromyograms of the diaphragm and the external intercostal muscle in the third interspace during high-frequency mechanical vibration (50 Hz) of the central tendon in eight anesthetized, spontaneously breathing rabbits. Vibration induced a consistent, clear-cut increase in the inspiratory activity recorded from the external intercostal, thus indicating that the mechanical stimulus applied to the diaphragm was strong enough to trigger muscle spindles at distant sites. However, vibration did not elicit any alteration in costal or crural diaphragmatic activity in any animal. Similarly, when vibration was applied during hyperventilation-induced apnea, activity was recorded in the external intercostal but not in the diaphragm. These observations support the traditional view that the diaphragm is poorly endowed with muscle spindles and that these play little or no significant role in the act of breathing.

Impaired load dependence of diaphragm relaxation during congestive heart failure in the rabbit.

The load dependence (LD) of relaxation was studied in the diaphragm of rabbits with congestive heart failure (CHF). CHF (n = 15) was induced by combined chronic volume and pressure overload. Aortic insufficiency was induced by forcing a catheter through the aortic sigmoid valves, followed 3 wk later by abdominal aortic stenosis. Six weeks after the first intervention, animals developed CHF. Sham-operated animals served as controls (C; n = 12). Diaphragm mechanics were studied in vitro on isolated strips, at 22 degrees C, in isotonic and isometric loading conditions. Contractility was lower in the CHF group, as reflected by lower total tension: 1.11 +/- 0.10 in CHF vs. 2.38 +/- 0.15 N/cm(2) in C in twitch (P < 0.001) and 2.46 +/- 0.22 in CHF vs. 4.90 +/- 0.25 N. cm(-2) in C in tetanus (P < 0.001). The index LD was used to quantify the load dependence of relaxation: LD is <1 in load-dependent muscles and tends toward 1 in load-independent muscles. LD was significantly higher in CHF than in C rabbits, in both twitch (0.99 +/- 0.01 vs. 0.75 +/- 0.03; P < 0. 001) and tetanus (0.95 +/- 0.02 vs. 0.84 +/- 0.02; P < 0.001). In the CHF rabbits' diaphragm, the fall in total tension was linearly related to the fall in load dependence of relaxation. The decrease in load dependence of relaxation in CHF animals suggests sarcoplasmic reticulum abnormalities. Impairment of the sarcoplasmic reticulum may also partly account for the decrease in contractile performance of diaphragm in CHF animals.

Further Characterization of the Type 3 Ryanodine Receptor (RyR3) Purified from Rabbit Diaphragm

We characterized type 3 ryanodine receptor (RyR3) purified from rabbit diaphragm by immunoaffinity chromatography using a specific antibody. The purified receptor was free from 12-kDa FK506-binding protein, although it retained the ability to bind 12-kDa FK506-binding protein. Negatively stained images of RyR3 show a characteristic rectangular structure that was indistinguishable from RyR1. The location of the D2 segment, which exists uniquely in the RyR1 isoform, was determined as the region around domain 9 close to the corner of the square-shaped assembly, with use of D2-directed antibody as a probe. The RyR3 homotetramer had a single class of high affinity [3H]ryanodine-binding sites with a stoichiometry of 1 mol/mol. In planar lipid bilayers, RyR3 displayed cation channel activity that was modulated by several ligands including Ca2+, Mg2+, caffeine, and ATP, which is consistent with [3H]ryanodine binding activity. RyR3 showed a slightly larger unit conductance and a longer mean open time than RyR1. Whereas RyR1 showed two classes of channel activity with distinct open probabilities (Po), RyR3 displayed a homogeneous and steeply Ca2+-dependent activity with Po approximately 1. RyR3 was more steeply affected in the channel activity by sulfhydryl-oxidizing and -reducing reagents than RyR1, suggesting that the channel activity of RyR3 may be transformed more precipitously by the redox state. This is also a likely explanation for the difference in the Ca2+ dependence of RyR3 between [3H]ryanodine binding and channel activity.

P–52 - Characterization of type 3 ryanodine receptor (RyR3) purified from rabbit diaphragm.

P–52 - Characterization of type 3 ryanodine receptor (RyR3) purified from rabbit diaphragm.

Mechanics, energetics, and crossbridge kinetics of rabbit diaphragm during congestive heart failure.

Crossbridge (CB) properties were investigated in isolated diaphragm of rabbits during congestive heart failure (CHF, n=9) induced by chronic volume and pressure overload. This model induced cardiac hypertrophy and heart failure. Controls (C) were prepared (n=14). Compared to C, peak tension in CHF fell by 57% in twitch and by 40% in tetanus; Vmax declined by 47% in twitch and by 48% in tetanus. Our study provided an analytical means of calculating from A. F. Huxley's equations the rate constants for CB attachment and detachment, CB single force (II), CB number per mm3 (m'), peak mechanical efficiency (Effmax), and turnover rate of myosin ATPase (kcat); m', II, and Effmax were lower in CHF than in C in both twitch and tetanus. The marked decline in m' and II accounted for the fall in diaphragm strength. In the overall population of C and CHF, Effmax was linearly related to II. Conversely, there was no relationship between Vmax and kcat. Dissociation between Vmax and kcat might be explained by the crucial role attributed to two apparently nonconserved surface 'loops' on the motor domain of myosin head.

Oligomerization of β-dystroglycan in rabbit diaphragm and brain as revealed by chemical crosslinking

The surface component β-dystroglycan is a member of the dystrophin–glycoprotein complex providing a trans-sarcolemmal linkage between the actin membrane cytoskeleton and the extracellular matrix component laminin- α2. Although abnormalities in this complex are involved in the pathophysiology of various neuromuscular disorders, little is known about the organization of dystrophin-associated glycoproteins in diaphragm and brain. We therefore investigated the oligomerization of β-dystroglycan and its connection with the most abundant dystrophin homologues in these two tissues. Employing detergent solubilization and alkaline extraction procedures of native membranes, it was confirmed that β-dystroglycan behaves like an integral surface molecule as predicted by its cDNA sequence. Immunoblot analysis following chemical crosslinking of native membranes showed that β-dystroglycan has a tendency to form high-molecular-mass complexes. Within these crosslinkable complexes, immuno-reactive overlaps were observed between β-dystroglycan, α-dystroglycan, laminin and 427 kDa dystrophin in diaphragm and skeletal muscle. In synaptosomes, the major brain dystrophin isoform Dp116 also exhibited an immuno-reactive overlap with members of the dystroglycan complex. These findings demonstrate that β-dystroglycan does not exist as a monomer in native membranes and imply that certain dystrophin isoforms and dystrophin-associated components interact with this surface protein in diaphragm and brain as has been previously shown for skeletal and heart muscle.

Homogeneidad celular en las diversas porciones del diafragma

The diaphragm is the main inspiratory muscle. It is composed of two parts, the costal and crural, with both anatomical and functional differences. The general morphometric characteristics of the diaphragm have been described in various species but homogeneity throughout the muscle has not been adequately studied. The aim of this study was to evaluate the fiber phenotype of various parts of the diaphragm. The entire diaphragm muscles of five New Zealand rabbits were removed and each was divided into quarters. The specimens were processed for morphometry (hematoxyllineosin stains, NADH-TR and ATPase at pH levels of 4.2, 4.6 and 9.4). For each portion we measured percent and size of fibers, expressing the latter as minimum diameter (Dm), measured area (Ar) and calculated area (Ac). Left and right diaphragm hemispheres (20 portions examined) were similar for fiber percentages and sizes. For left and right halves, respectively 50 +/- 2 and 51 +/- 4% of fibers were type I; type I Dm measurements were 38 +/- 5 and 41 +/- 4 microns; type I Ar values were 1798 +/- 481 and 2030 +/- 390 micron 2; type I Ac values were 1181 +/- 360 and 1321 +/- 382 micron 2; type II Dm values were 46 +/- 4 and 46 +/- 5 microns; type II Ar values were 2466 +/- 388 micron 2 and 2539 +/- 456 micron 2; type II Ac data were 1642 +/- 255 and 1655 +/- 382 micron 2. We likewise found no differences between costal and crural portions of the muscle (n = 20). For costal and crural portions, respectively, 50 +/- 3 and 50 +/- 2% of fibers were type I; type I Dm sizes were 39 +/- 5 and 40 +/- 4 microns; type I Ar measurements were 1859 +/- 521 and 1964 +/- 365 micron 2; type I Ac figures were 1231 +/- 317 and 1266 +/- 288 micron 2; type II Dm were 47 +/- 4 and 44 +/- 3 microns; type II Ar were 2563 +/- 481 and 2430 +/- 331 micron 2; type II Ac were 1729 +/- 373 and 1557 +/- 212 micron 2. Type II fibers, however, were somewhat larger than type I fibers in all portions (p = 0.001). New Zealand rabbit diaphragm muscle has similar percentages of slow and rapid contraction fibers. The size is not different from that observed in other species of mammals of similar size. Fiber type proportions are similar throughout the muscle, with more type II fibers present in all areas. The morphometric characters, therefore, suggest an homogeneous throughout the diaphragm, suggesting homogeneous response of the muscle to usual loads, and also suggesting the possibility of proposing longitudinal morphometric studies using this species as a model.

Some aspects of functional morphology of extracellular matrix in lymph capillary endothelium

Argyrophilic portion of endothelial glycocalyx in lymph capillaries from the central tendon of rabbit diaphragm was examined. It is shown that optical density along the perimeter of endotheliocyte is unequal and depends on the cell shape (scalloped, fusiform, or irregular). Aseptic inflammation is characterized by a marked increase in high-density zones and expansion of argyrophilic glycocalyx. It is proposed to consider argyrophilic glycocalyx as an independent structural and functional unit of the endothelium.

Characterization of Type 3 Ryanodine Receptor (RyR3) of Sarcoplasmic Reticulum from Rabbit Skeletal Muscles

We investigated type 3 isoform (RyR3) of ryanodine receptor in rabbit skeletal muscles using an antibody specific for RyR3. By Western blot analysis and by immunoprecipitation, a single polypeptide for RyR3 was detected in sarcoplasmic reticulum vesicles from rabbit diaphragm but not in those from back muscle. The molecular mass was slightly smaller than that of RyR1, the major isoform in skeletal muscles. Each of RyR1 and RyR3 formed a homotetramer in rabbit diaphragm. RyR3 had a single class of [3H]ryanodine binding sites of high affinity (KD = 1.6 nM). From the Bmax of the binding, the content of RyR3 was estimated to be only 0.6% of RyR1 in rabbit diaphragm. -3H-Ryanodine binding to RyR3 was biphasically dependent on Ca2+, as is true of RyR1, and was stimulated further by adenine nucleotide, caffeine, or high salt concentration. Procaine and ruthenium red inhibited the binding. RyR3 was more resistant to Mg2+ inhibition than RyR1. Interestingly, RyR3 showed about a 7-fold lower Ca2+ sensitivity for activation than RyR1. Comparison with the counterparts in bullfrog skeletal muscles indicates that the Ca2+ sensitivities of RyR3 homologs are similar to each other, whereas those of RyR1 homologs are species-specific.

IN VIVO DIAPHRAGMATIC LENGTHENING CONTRACTIONS AND THE RELATIONSHIP TO FORCE PRODUCTION IN THE RABBIT 1639

Lengthening contractions (CL) are known to cause injury in limb skeletal muscle. It is unknown whether the diaphragm (DPM), the primary muscle of inspiration, could be induced to undergo CL in vivo and whether DPM injury occurs as a result. We hypothesized that the DPM could be stimulated to undergo lengthening contractions at physiologic frequencies in vivo and that DPM muscle would decrease following CL, indicating injury. We studied 4 anesthetized (Ketamine/Xylazine), supine, intubated, and mechanically ventilated female New Zealand White rabbits (3.15 ± 0.99 kg). Pressure of the airway opening (Pao) was measured as an indicator of force generating capacity of the DPM. Changes in midcostal diaphragm fiber length were measured via surgical implantation of sonomicrometer crystals (1mm diam). CL were induced by superimposing bilateral transvenous phrenic stimulations of the DPM at 20 Hz (duty cycle: 30 sec on/30 sec off) on mechanical ventilation(tidal volume = 20 ml, frequency = 25/min) for ten minutes. Changes in force production were evaluated by measuring frequency-pressure curves prior to, 5 minutes post CL, and 30 minutes post CL. Mean airway opening pressures were 100.12 ± 3.26% and 116 ± 4.68% of control values(N.S.) at 5 and 30 minutes post CL respectively. Under these experimental conditions, there is no CL induced force decrement indicative of damage to the intact rabbit diaphragm.