Mechanical Ventilation Animals Research Articles

Transvenous stimulation yields exposure-dependent protection from ventilator-induced diaphragm atrophy.

Mechanical ventilation (MV)-induced diaphragmatic atrophy can contribute to weaning difficulties. A temporary transvenous diaphragm neurostimulation (TTDN) device that elicits diaphragm contractions has previously been shown to mitigate atrophy during MV in a preclinical model; however, its effects on different myofiber types remain unknown. It is important to examine these effects, as each myofiber type plays a role in the range of diaphragmatic movements to ensure successful liberation from MV. Eighteen pigs were assigned to one of three ventilation conditions for 50 hours: MV-Only and TTDN contracting the diaphragm every other breath or every breath synchronously with MV (TTDN50% + MV and TTDN100% + MV, respectively). Six pigs were assigned to a never-ventilated, never-paced (NV-NP) group. Diaphragm biopsies were fiber-typed, and myofiber cross-sectional areas were measured and normalized to subject weight. There were effect differences based on TTDN exposure. The TTDN100% + MV group showed less atrophy in Type 2A and 2X myofibers than the TTDN50% + MV group, relative to the NV-NP group. The TTDN50% + MV animals showed less MV-induced atrophy in type 1 myofibers than TTDN100% + MV animals. Additionally, there were no significant differences in proportions of myofiber types between each condition. TTDN applied synchronously with MV for 50 hours mitigates MV-induced atrophy in all myofiber types, with no evidence of stimulation-induced myofiber-type shift. At this stimulation profile, enhanced protection for type 1 myofibers and type 2 myofibers was seen when diaphragm contractions occurred every other breath and every breath, respectively.NEW & NOTEWORTHY This research adds to our current understanding of applying temporary transvenous diaphragmatic neurostimulation (TTDN) synchronously with mechanical ventilation by examining its diaphragm-myofiber effects. We observed that using this therapy for 50 hours with mechanical ventilation not only mitigated ventilator-induced atrophy on all myofiber types with dose effects, it also did not invoke alterations in diaphragm myofiber type proportions. These findings suggest that applying TTDN with mechanical ventilation at different doses represents its broad spectrum use and viability as a diaphragm protective strategy.

Epidermal growth factor receptor inhibition with Gefitinib does not alter lung responses to mechanical ventilation in fetal, preterm lambs.

BackgroundEpidermal growth factor receptor (EGFR) is important for airway branching and lung maturation. Mechanical ventilation of preterm lambs causes increases in EGFR and EGFR ligand mRNA in the lung. Abnormal EGFR signaling may contribute to the development of bronchopulmonary dysplasia.HypothesisInhibition of EGFR signaling will decrease airway epithelial cell proliferation and lung inflammation caused by mechanical ventilation in preterm, fetal sheep.MethodsFollowing exposure of the fetal head and chest at 123±1 day gestational age and with placental circulation intact, fetal lambs (n = 4-6/group) were randomized to either: 1) Gefitinib 15 mg IV and 1 mg intra-tracheal or 2) saline IV and IT. Lambs were further assigned to 15 minutes of either: a) Injurious mechanical ventilation (MV) or b) Continuous positive airway pressure (CPAP) 5 cmH2O. After the 15 minute intervention, the animals were returned to the uterus and delivered after i) 6 or ii) 24 hours in utero.ResultsMV caused lung injury and inflammation, increased lung mRNA for cytokines and EGFR ligands, caused airway epithelial cell proliferation, and decreased airway epithelial phosphorylated ERK1/2. Responses to MV were unchanged by Gefitinib. Gefitinib altered expression of EGFR mRNA in the lung and liver of both CPAP and MV animals. Gefitinib decreased the liver SAA3 mRNA response to MV at 6 hours. There were no differences in markers of lung injury or inflammation between CPAP animals receiving Gefitinib or saline.ConclusionInhibition of the EGFR pathway did not alter acute lung inflammation or injury from mechanical ventilation in preterm sheep.

Mechanical Ventilation Alters the Development of Staphylococcus aureus Pneumonia in Rabbit.

Ventilator-associated pneumonia (VAP) is common during mechanical ventilation (MV). Beside obvious deleterious effects on muco-ciliary clearance, MV could adversely shift the host immune response towards a pro-inflammatory pattern through toll-like receptor (TLRs) up-regulation. We tested this hypothesis in a rabbit model of Staphylococcus aureus VAP. Pneumonia was caused by airway challenge with S. aureus, in either spontaneously breathing (SB) or MV rabbits (n = 13 and 17, respectively). Pneumonia assessment regarding pulmonary and systemic bacterial burden, as well as inflammatory response was done 8 and 24 hours after S. aureus challenge. In addition, ex vivo stimulations of whole blood taken from SB or MV rabbits (n = 7 and 5, respectively) with TLR2 agonist or heat-killed S. aureus were performed. Data were expressed as mean±standard deviation. After 8 hours of infection, lung injury was more severe in MV animals (1.40±0.33 versus [vs] 2.40±0.55, p = 0.007), along with greater bacterial concentrations (6.13±0.63 vs. 4.96±1.31 colony forming units/gram, p = 0.002). Interleukin (IL)-8 and tumor necrosis factor (TNF)-αserum concentrations reached higher levels in MV animals (p = 0.010). Whole blood obtained from MV animals released larger amounts of cytokines if stimulated with TLR2 agonist or heat-killed S. aureus (e.g., TNF-α: 1656±166 vs. 1005±89; p = 0.014). Moreover, MV induced TLR2 overexpression in both lung and spleen tissue. MV hastened tissue injury, impaired lung bacterial clearance, and promoted a systemic inflammatory response, maybe through TLR2 overexpression.

Cervical spinal cord injury exacerbates ventilator-induced diaphragm dysfunction.

Cervical spinal cord injury (SCI) can dramatically impair diaphragm muscle function and often necessitates mechanical ventilation (MV) to maintain adequate pulmonary gas exchange. MV is a life-saving intervention. However, prolonged MV results in atrophy and impaired function of the diaphragm. Since cervical SCI can also trigger diaphragm atrophy, it may create preconditions that exacerbate ventilator-induced diaphragm dysfunction (VIDD). Currently, no drug therapy or clinical standard of care exists to prevent or minimize diaphragm dysfunction following SCI. Therefore, we first tested the hypothesis that initiating MV acutely after cervical SCI will exacerbate VIDD and enhance proteolytic activation in the diaphragm to a greater extent than either condition alone. Rats underwent controlled MV for 12 h following acute (∼24 h) cervical spinal hemisection injury at C2 (SCI). Diaphragm tissue was then harvested for comprehensive functional and molecular analyses. Second, we determined if antioxidant therapy could mitigate MV-induced diaphragm dysfunction after cervical SCI. In these experiments, SCI rats received antioxidant (Trolox, a vitamin E analog) or saline treatment prior to initiating MV. Our results demonstrate that compared with either condition alone, the combination of SCI and MV resulted in increased diaphragm atrophy, contractile dysfunction, and expression of atrophy-related genes, including MuRF1. Importantly, administration of the antioxidant Trolox attenuated proteolytic activation, fiber atrophy, and contractile dysfunction in the diaphragms of SCI + MV animals. These findings provide evidence that cervical SCI greatly exacerbates VIDD, but antioxidant therapy with Trolox can preserve diaphragm contractile function following acute SCI.

Response of Alpha-actinin Isoforms to Mechanical Ventilation-induced Diaphragmatic Atrophy

Alpha-actinins are actin-binding proteins and important structural components of the Z-line. In mammalian skeletal muscle, the two isoforms are expressed, α-actinin-2 and α-actinin-3. α-actinin-2 is expressed in all skeletal muscles fibers, whereas α-actinin-3 expression is restricted to the fast fibers only, thus, α-actinin-3 is assumed to be associated with fast muscle fiber function. We previously examined α-actinin-3 level in the soleus muscle by using hindlimb unloading. This type of disuse induces the atrophy particularly in slow muscle fibers. In contrast, fast fibers are also atrophied by diaphragmatic inactivity, indicate that response of α-actinin-3 to atrophic stimuli with mechanical ventilation (MV) may differ from results of locomotor muscle disuse. PURPOSE: To examine the expression levels of α-actinin isoforms in rat diaphragm after MV. METHODS: Male Wistar rats were randomly divided into a control (n=5) or a 12h of MV (n=5) group. Mechanically ventilated animals were anesthetized, tracheostomized, and ventilated with 21% O2. Animals in the control group were acutely anesthetized but were not exposed to MV. After the treatment, diaphragms were removed, frozen with lipid nitrogen and stored at -80°C. Western blotting analysis was performed to Myosin heavy chain (MyHC) composition and expression of α-actinin isoforms. RESULTS: MV resulted in a decrease (14.0%, p < 0.01) in the cross-sectional area of diaphragm. MyHC composition did not differ between MV (I: 21.4, IIa: 19.3, IId/x: 49.2, IIb: 10.4%) and control (I: 22.2, IIa: 18.3, IId/x: 48.3, IIb: 11.2%) animals. Furthermore, there was no significant difference in expression levels of α-actinin isoforms between MV and control groups. CONCLUSION: The expression levels of α-actinin isoforms in rat diaphragm are not changed by the 12-h MV.

Mechanical ventilation induces diaphragmatic mitochondrial dysfunction and increased oxidant production

Mechanical ventilation (MV) is a life-saving intervention used in patients with acute respiratory failure. Unfortunately, prolonged MV results in diaphragmatic weakness, which is an important contributor to the failure to wean patients from MV. Our laboratory has previously shown that reactive oxygen species (ROS) play a critical role in mediating diaphragmatic weakness after MV. However, the pathways responsible for MV-induced diaphragmatic ROS production remain unknown. These experiments tested the hypothesis that prolonged MV results in an increase in mitochondrial ROS release, mitochondrial oxidative damage, and mitochondrial dysfunction. To test this hypothesis, adult (3–4 months of age) female Sprague–Dawley rats were assigned to either a control or a 12-h MV group. After treatment, diaphragms were removed and mitochondria were isolated for subsequent respiratory and biochemical measurements. Compared to control, prolonged MV resulted in a lower respiratory control ratio in diaphragmatic mitochondria. Furthermore, diaphragmatic mitochondria from MV animals released higher rates of ROS in both State 3 and State 4 respiration. Prolonged MV was also associated with diaphragmatic mitochondrial oxidative damage as indicated by increased lipid peroxidation and protein oxidation. Finally, our data also reveal that the activities of the electron transport chain complexes II, III, and IV are depressed in mitochondria isolated from diaphragms of MV animals. In conclusion, these results are consistent with the concept that diaphragmatic inactivity promotes an increase in mitochondrial ROS emission, mitochondrial oxidative damage, and mitochondrial respiratory dysfunction.

Mechanical Ventilation Down-Regulates Surfactant Protein A and Keratinocyte Growth Factor Expression in Premature Rabbits

Surfactant-associated proteins (SP-A, SP-B, and SP-C) are critical for the endogenous function of surfactant. Keratinocyte growth factor (KGF) and vascular endothelial growth factor (VEGF) are key regulators of lung development. The objective of this study was to evaluate the effects of early mechanical ventilation on the expression of these important regulatory proteins in a preterm rabbit model. Premature fetuses were delivered at 29 d of gestation and randomized to necropsy at birth, i.e. no ventilation (NV), spontaneous breathing (SB), or mechanical ventilation (MV) for 16 h. MV animals were further randomized to treatment with dexamethasone (dex). Our findings showed that SB rabbits increased their expression of SP-A mRNA and protein after birth compared with NV controls. MV significantly attenuated this response in the absence of dex. Exposure to dex elevated SP-B mRNA expression in both SB and MV rabbits. KGF protein levels were markedly increased in SB animals compared with MV counterparts. VEGF levels were similar in SB and MV animals, but were significantly increased compared with NV controls. These data suggest that MV alters surfactant-associated protein and growth factor expression, which may contribute to injury in the developing lung.

Comparison of lung injury after normal or small volume optimized resuscitation in a model of hemorrhagic shock

To compare lung injury induced by a hemorrhagic shock resuscitated with normal saline or with small volumes of a hypertonic/hyperoncotic solution. Randomized, controlled, laboratory study in an animal research laboratory. Nineteen pigs (43 +/- 4 kg). After anesthesia and mechanical ventilation animals were bled to induce a 2-h deep shock and resuscitated for 2 h using normal saline (NS, 2 ml/kg per minute, n = 7) or the association of 7.2% NaCl with 6% hydroxyethylstarch 200/0.5 (HSHES, 4 ml/kg in 10 min followed by 0.2 ml/kg per minute, n = 7) to reach cardiac index and mixed venous oxygen saturation goals. Lungs were removed 6[Symbol: see text]h after the initiation of hemorrhage. Five animals were used as controls without hemorrhage. Resuscitation goals were achieved using 90 +/- 17 ml/kg NS or 6.8 +/- 1.9 ml/kg HSHES. Lung injury was noted in both hemorrhage groups but was not influenced by the type of resuscitation. Extravascular lung water was measured at 9.6 +/- 1.8 ml/kg in the NS group, 9.2 +/- 1.6 ml/kg in the HSHES, group and 6.4 +/- 1 m/kg in the control group. The degree of histological alveolar membrane focal thickening and interstitial neutrophil infiltration were significantly more pronounced in the hemorrhage groups with no difference between the two types of fluid loading. Finally, pulmonary levels of IL-8 were higher after hemorrhage regardless of the type of resuscitation. When included in an optimized and goal directed resuscitation, the use of normal saline or a small volume of hypertonic/hyperoncotic solution does not result in a different early hemorrhage-induced lung injury.

Mechanical ventilation promotes redox status alterations in the diaphragm

Oxidative stress is an important mediator of diaphragm muscle atrophy and contractile dysfunction during prolonged periods of controlled mechanical ventilation (MV). To date, specific details related to the impact of MV on diaphragmatic redox status remain unknown. To fill this void, we tested the hypothesis that MV-induced diaphragmatic oxidative stress is the consequence of both an elevation in intracellular oxidant production in conjunction with a decrease in the antioxidant buffering capacity. Adult rats were assigned to one of two experimental groups: 1) control or 2) 12 h of MV. Compared with controls, diaphragms from MV animals demonstrated increased oxidant production, diminished total antioxidant capacity, and decreased glutathione levels. Heme oxygenase-1 (HO-1) mRNA and protein levels increased (23.0- and 5.1-fold, respectively) following MV. Thioredoxin reductase-1 and manganese superoxide dismutase mRNA levels were also increased in the diaphragm following MV (2.4- and 1.6-fold, respectively), although no change was detected in the levels of either protein. Furthermore, copper-zinc superoxide dismutase and glutathione peroxidase mRNA were not altered following MV, although protein content decreased -1.3- and -1.7-fold, respectively. We conclude that MV promotes increased oxidant production and impairment of key antioxidant defenses in the diaphragm; collectively, these changes contribute to the MV-induced oxidative stress in this key inspiratory muscle.

Reloading the Diaphragm Following Mechanical Ventilation Does Not Promote Injury

Mechanical ventilation (MV) is used clinically to treat patients who are incapable of maintaining adequate alveolar ventilation. Prolonged MV is associated with diaphragmatic atrophy and a decrement in maximal specific force production (P(O)). Collectively, these alterations may predispose the diaphragm to injury on the return to spontaneous breathing (ie, reloading). Therefore, these experiments tested the hypothesis that reloading the diaphragm following MV exacerbates MV-induced diaphragmatic contractile dysfunction, while causing muscle fiber membrane damage and inflammation. To test this postulate, Sprague-Dawley rats were randomly assigned to the following groups: (1) control; (2) 24 h of controlled MV; and (3) 24 h of controlled MV followed by 2 h of anesthetized spontaneous breathing. Controls were anesthetized in the short term but were not exposed to MV, whereas MV animals were anesthetized, tracheostomized, and ventilated. Reloaded animals remained under anesthesia, but were removed from MV and returned to spontaneous breathing for 2 h. Compared to the situation with control animals, MV resulted in a 26% decrement in diaphragmatic specific P(O) without muscle fiber membrane damage, as measured by an increase in membrane permeability (using the procion orange technique). Further, there were no increases in neutrophil or macrophage influx. Two hours of reloading did not exacerbate MV-induced diaphragmatic contractile dysfunction or cause fiber membrane damage, but increased neutrophil infiltration, myeloperoxidase activity, and muscle edema. We conclude that the return to spontaneous breathing following 24 h of controlled MV does not exacerbate MV-induced diaphragm contractile dysfunction or result in fiber membrane damage, but increases neutrophil infiltration.

Mechanical ventilation induces alterations of the ubiquitin-proteasome pathway in the diaphragm

Prolonged mechanical ventilation (MV) results in diaphragmatic atrophy due, in part, to an increase in proteolysis. These experiments tested the hypothesis that MV-induced diaphragmatic proteolysis is accompanied by increased expression of key components of the ubiquitin-proteasome pathway (UPP). To test this postulate, we investigated the effect of prolonged MV on UPP components and determined the trypsin-like and peptidylglutamyl peptide hydrolyzing activities of the 20S proteasome. Adult Sprague-Dawley rats were assigned to either control or 12-h MV groups (n=7/group). MV animals were anesthetized, tracheostomized, and ventilated with room air for 12 h. Animals in the control group were acutely anesthetized but not exposed to MV. Compared with controls, MV animals demonstrated increased diaphragmatic mRNA levels of two ubiquitin ligases, muscle atrophy F-box (+8.3-fold) and muscle ring finger 1 (+19.0-fold). However, MV did not alter mRNA levels of 14-kDa ubiquitin-conjugating enzyme, polyubiquitin, proteasome-activating complex PA28, or 20S alpha-subunit 7. Protein levels of 14-kDa ubiquitin-conjugating enzyme and proteasome-activating complex PA28 were not altered following MV, but 20S alpha-subunit 7 levels declined (-17.7%). MV increased diaphragmatic trypsin-like activity (+31%) but did not alter peptidylglutamyl peptide hydrolyzing activity. Finally, compared with controls, MV increased ubiquitin-protein conjugates in both the myofibrillar (+24.9%) and cytosolic (+54.7%) fractions of the diaphragm. These results are consistent with the hypothesis that prolonged MV increases diaphragmatic levels of key components within the UPP and that increases in 20S proteasome activity contribute to MV-induced diaphragmatic proteolysis and atrophy.

Cumulative Effects of Aging and Mechanical Ventilation on In Vitro Diaphragm Function

Unloading the diaphragm, via mechanical ventilation (MV), results in significant diaphragmatic atrophy, contractile dysfunction, and oxidative stress in young adult animals. Since aging increases skeletal muscle susceptibility to atrophy and injury, we tested the hypothesis that MV-induced diaphragmatic contractile dysfunction would be exacerbated in aging rats. Fisher 344/Brown Norway hybrid rats (4 months old [young] and 30 months old [old]) were assigned to either control or MV groups. MV rats were anesthetized, tracheostomized, and ventilated with 21% O(2) for 12 h. Arterial BP, pH, and blood gas homeostasis were maintained in the MV animals throughout the experimental period. Animals in the control group were acutely anesthetized, and the diaphragms were immediately removed. Muscle strips from the mid-costal diaphragm were removed from each experimental animal, and contractile properties were studied in vitro. Compared to young control animals, aging (old control animals) was associated with a 13% decrease in maximal isometric tension (24.5 N/cm(2) vs 21.3 N/cm(2)). Although, MV induced similar relative losses (24%) in diaphragmatic isometric tension in both young and old animals receiving MV, the combined effects of aging and MV resulted in a 34% decrement in diaphragmatic isometric tension compared to young control animals (24.5 N/cm(2) vs 16.1 N/cm(2)). These data do not support the hypothesis that aging exacerbates the relative MV-induced impairment in diaphragmatic isometric tension. Nonetheless, the additive effects of aging and MV have dramatic effects on diaphragmatic force reserve. This could exacerbate weaning difficulties in older individuals receiving MV.

Conventional mechanical ventilation of healthy lungs induced pro-inflammatory cytokine gene transcription.

We investigated the potential inflammatory reaction induced by mechanical ventilation (MV) using 10 ml/kg tidal volume and no positive end-expiratory pressure (PEEP) in control (C, n = 8), spontaneously breathing (SB, n = 12) and mechanically ventilated (MV, n = 12) rabbits with normal lungs. After 6 h (MV and SB groups) or immediately (C group), lungs were removed for measurement of wet-to-dry (W/D) weight ratio and for bronchoalveolar lavage (BAL). Pulmonary mechanics were also studied. MV animals developed a modest but significant (P < 0.01) impairment of arterial blood oxygenation and had higher W/D lung weight ratio than C ones. In MV group, BAL macrophage count was greater (P < 0.05) than in SB one. MV induced an upregulation of MCP-1, TNF-alpha, and IL-1beta gene transcription (mRNAs), without significant elevation of the corresponding protein cytokines in the BAL supernatant, except for MCP-1 (P < 0.05). These data suggest that MV, even using moderate tidal volume, elicits a pro-inflammatory stimulus to the lungs.

THE CUMULATIVE EFFECTS OF AGING AND DIAPHRAGM UNLOADING ON IN VITRO DIAPHRAGMATIC FUNCTION

Evidence indicates that aged locomotor muscle suffers exaggerated atrophy during periods of unloading. It is also established that aging is associated with a reduction in maximal specific tension (Po) in both locomotor muscles and the diaphragm. Further, we have demonstrated that unloading the diaphragm, via mechanical ventilation (MV), results in significant diaphragmatic atrophy and contractile dysfunction in young adult animals. It is currently unkown if unlloading the diaphragm results in greater contractile dysfunction in old animals compared to young adults. PURPOSE: We tested the hypothesis that compared to young adult animals, aging would exacerbate MV-induced diaphragmatic contractile dysfunction. METHODS: Fisher Brown Norway Cross rats (4 mo. (young) and 30 mo. (old)) were assigned to either the control group (CON) or the mechanical ventilation group (MV). MV rats were anesthetized, tracheostomized, and ventilated with 21% O2 for 12 hours. MV animals were fed enterally with a gastric catheter and arterial blood pressure, pH, and blood gas homeostasis were maintained throughout the experimental period. Animals in the CON group were acutely anesthetized but were not exposed to MV. Muscle strips from the mid-costal diaphragm were removed from each experimental animal and contractile properties were studied in vitro to determine the effects of MV on diaphragmatic Po. RESULTS: Compared to young CON, aging (old CON) was associated with a 20% decrease in Po (26.4 N/cm2 vs. 20.7 N/cm2). MV induced similar relative losses (−25%) in diaphragmatic Po in both young and old MV animals (19.7 N/cm2 vs. 15.5 N/cm2). Further, compared to young CON, the combined effects of aging and MV resulted in a 40% decrement in diaphragmatic Po. CONCLUSIONS: Unloading the diaphragm by MV results in a significant decrease in diaphragmatic Po in both young and old animals. However, these data do not support the hypothesis that aging exacerbates the relative MV-induced impairment in diaphragmatic Po. Nonetheless, the additive effects of aging and MV may place constraints on the diaphragmatic force reserve during periods of increased loads and could lead to respiratory failure. National Institutes of Health (R01 HL62361) awarded to S.K. Powers

Mechanical ventilation results in progressive contractile dysfunction in the diaphragm.

These experiments tested the hypothesis that a relatively short duration of controlled mechanical ventilation (MV) will impair diaphragmatic maximal specific force generation (specific P(o)) and that this force deficit will be exacerbated with increased time on the ventilator. To test this postulate, adult Sprague-Dawley rats were randomly divided into one of six experimental groups: 1) control (n = 12); 2) 12 h of MV (n = 4); 3) 18 h of MV (n = 4); 4) 18 h of anesthesia and spontaneous breathing (n = 4); 5) 24 h of MV (n = 7); and 6) 24 h of anesthesia and spontaneous breathing (n = 4). MV animals were anesthetized, tracheostomized, and ventilated with room air. Animals in the control group were acutely anesthetized but were not exposed to MV. Animals in two spontaneous breathing groups were anesthetized and breathed spontaneously for either 18 or 24 h. No differences (P > 0.05) existed in diaphragmatic specific P(o) between control and the two spontaneous breathing groups. In contrast, compared with control, all durations of MV resulted in a reduction (P < 0.05) in diaphragmatic specific tension at stimulation frequencies ranging from 15 to 160 Hz. Furthermore, the MV-induced decrease in diaphragmatic specific P(o) was time dependent, with specific P(o) being approximately 18 and approximately 46% lower (P < 0.05) in animals mechanically ventilated for 12 and 24 h, respectively. These data support the hypothesis that relatively short-term MV impairs diaphragmatic contractile function and that the magnitude of MV-induced force deficit increases with time on the ventilator.