Non-acidotic hypercapnia limits atrophy and loss of specific force in rat diaphragm after 5 days of controlled mechanical ventilation in parallel with increased local inflammation

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Addinsall, Lars Larsson This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4082716/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Controlled Mechanical Ventilation (CMV) is associated with Ventilator Induced Lung Injury (VILI) and Ventilator Induced Diaphragm Dysfunction (VIDD). VIDD delays weaning from the respirator and increases the risk of further complications and health care costs, which are disproportionately increased with increasing duration of mechanical ventilation. Hypercapnia is frequently observed and tolerated as “permissive hypercapnia” during lung protective MV strategies. The systemic effects of hypercapnia are well known and considered potentially protective for diaphragm muscle in acute and short-time experimental MV studies. However, hypercapnia is commonly associated with acidosis, affecting immunity and inflammation pathways. Methods This study aims to determine the potential of hypercapnia in the absence of acidosis on diaphragm muscle structure and function in a well-established clinically relevant experimental ICU model, not limited by early mortality. The effects of hypercapnia at physiological pH on diaphragm single fibers cross sectional area (CSA) and specific force (maximum force normalized to CSA) were investigated. Results Non-Acidotic Hypercapnia (NAH) reduced body mass loss, diaphragm muscle fiber atrophy and loss of specific force, in parallel with an increased gene expression of proinflammatory cytokines (TNF-α and IL-1β) and of the MuRF-1 atrogene. In the diaphragm, TNF-α gene expression was significantly increased in NAH rats compared with 5 days normocapnic and controls, while IL-1β showed an increasing trend. In the lung lysates, IL-1β gene expression was significantly increased in 5 days normocapnic rats compared with the controls, while gene expression of TNF-α was increased in the NAH rats compared with controls. In NAH rats the increase was not significant. The gene expression of mitochondrial factors TFAM (regulator of mitochondrial gene expression), MFN2 (involved in mitochondrial fusion, quality control and cell metabolism), PARKIN (involved in mitochondrial quality control and mitophagy), ULK-1 (activator of mitophagy) was analyzed. NAH reversed, significantly the decreased gene expression of ULK 1 observed in the 5 days normocapnic rats. Conclusions These results suggest that non-acidotic hypercapnia limits the development of VIDD, irrespective of amplified local muscle inflammation. Therefore, we suggest its clinical role may be complementary to the known anti-inflammatory effects of hypercapnic acidosis (HCA), which has preventive VIDD effects as well. Critical Care VIDD hypercapnia diaphragm mechanical ventilation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background The loss of function of the diaphragm muscle resulting from Mechanical Ventilation (MV) in Intensive Care Unit (ICU) patients has been termed Ventilator Induced Diaphragmatic Dysfunction (VIDD) in analogy with the often-concomitant Ventilator Induced Lung Injury (VILI). VIDD is characterized by a rapid and progressive loss of diaphragm muscle mass and function. Hypercapnia is a frequent condition in mechanically ventilated ICU patients and accepted as “permissive hypercapnia”, typically in Acute Respiratory Distress Syndrome (ARDS), during lung protective mechanical ventilation strategies [ 1 ]. Hypercapnia reduces mortality in patients randomized to receive high tidal volume (TV, 12ml/Kg) and no additional protective effect of permissive hypercapnia was found in patients ventilated with low TV (6ml/Kg) suggesting positive effects of CO 2 per se and not exclusively related to reduced strain and stress on pulmonary tissues during lung protectives MV strategies. On the other hand, the classical “mainstream” meta-analysis on the prognosis of ARDS patients, have attributed the protective effects, almost exclusively to low TV-, while considering hypercapnia as simply a side effect that must be accepted [ 2 ]. According to the data from experimental rat and piglet models, hypercapnia can attenuate VIDD progression and severity. This suggests the possibility to implement MV strategies protecting both lungs and diaphragm respectively against VILI and VIDD, like in ARDS patients with “baby lung” where alveolar stress and strain should be minimized. Shellekens and coworkers [ 3 ], reported a protective effect of hypercapnia after 18 hours of Controlled MV (CMV), measured as an improved force-generating capacity (specific force) in diaphragm fibers compared with normocapnic mechanically ventilated rats for the same duration. However, these rats were subjected to hypercapnic in combination with acidosis (HCA) and it remains unclear if these potential benefits are associated with hypercapnia per se, or influenced by the associated acidosis. Moreover, in MV piglets, HCA preserved the trans-diaphragmatic pressure (Pdi) following 72 hours of CMV [ 4 ]. However, this study lacked exploration of molecular mechanisms underlying the improvements [ 4 ]. Combined, these studies showed the potential therapeutic benefit of hypercapnia during MV, yet these observations were limited to animals exposed to a short period of CMV, which lacks clinical translation to the condition experienced by ICU patients and may be confounded by respiratory acidosis. The effects of non-acidotic hypercapnia on diaphragm muscle size and function were therefore investigated within a clinically relevant timeframe, 5 days, and compared with normocapnic 5 days mechanically ventilated and sham-operated controls. Specific interest is focused on the effects of inflammation, protein degradation pathways and mitochondrial factors linked to muscle atrophy and dysfunction. Indeed, CMV per se is associated with mechanical perturbations of both pulmonary and diaphragmatic tissues leading to inflammatory responses [ 5 ] and to a derangement of energy metabolism and consequent energy crisis [ 6 , 7 ]. Materials and Methods Animals and ICU Model Adult female Sprague Dawley rats were included in the study and divided into three groups: a sham-operated control group (n = 5), a 5-day mechanically ventilated non-acidotic hypercapnia experimental group (5D + NAH group, n = 5, with EtCO 2 maintained between 70 and 80 mmHg)), and 5-day mechanically ventilated normocapnic group (5D, n = 5, with EtCO 2 maintained between 37 and 45 mmHg. All the mechanically ventilated rats were exposed to deep sedation, controlled CMV and neuromuscular blockade for 5 days with gas concentration of 30–35% O2, 3% CO2 and the rest N 2 . In both mechanically ventilated groups the EtCO 2 (indirect monitoring of PaCo 2 ) was maintained in the desired range by adjusting the FiCO 2 , while maintaining constant the TV and consequently the stress and strain on the lungs, which are known sources of release of inflammatory factors. We induced hypercapnia 2–3 hours after the start of CMV allowing the renal buffering to keep the blood pH in the normal range and at the same time avoiding sodium bicarbonate administration, that may worsen the intracellular acidosis and therefore having a negative impact on many physiological processes including alveolar healing Cortez-Puentez et al. 2019), Masterson et al 2020. All aspects of this study were approved by the Karolinska Institute ethical committee (N263/14). The experimental model used in this study is not limited by early mortality and is a modification of the model originally developed by Dworkin and co-workers [ 8 – 10 ] optimized to be minimally invasive for the study of the effects of the ICU condition on skeletal muscle structure and function [ 11 , 12 ]. The model is not limited by early mortality and the longest duration a rat has been ventilated in this model is 96 days [ 8 ]. All experimental animals were maintained in fluid and nutritional balance throughout the duration of the experimental procedures by introducing: 1) intra-arterial solution (0.6 ml/h) containing 21 ml H 2 O, 24 ml 0.5 N lactated Ringers, 0.84 g oxacillin Na, 0.65 mg alpha-cobrotoxin, 0.3 mg vitamin K (Synkavite), and 20 mg K + (as KCl); 2) an intravenous solution (0.6 ml/h) containing 26 ml H 2 O, 16 ml 0.5 N lactated Ringers, 20% glucose (Baxter, Deerfield, IL, USA), and 0.32 g oxacillin Na for the initial 24, then 8.5% Travasol amino acids and 20% Intralipid (Kabi, Sweden) were added subsequently to provide adequate nutrients [ 8 – 10 ]. Body temperature, peripheral perfusion, and oxygen saturation were monitored and maintained in the physiological range by an infrared paw probe (MouseSTAT, Kent Scientific corp., USA). Controls were anesthetized with isoflurane, maintained with spontaneous breathing, and sacrificed within 2 hours of the initial isoflurane anesthesia and surgery. During surgery or any possible irritating manipulation, the Minimum Alveolar Concentration (MAC) was kept > 1.5% in order to maintain: 1) high-voltage slow-wave activity of the electroencephalogram (EEG); 2) mean arterial pressure 90–100 mmHg and heart rate below 400 beats/min; and 3) no evident EEG, blood pressure, or heart rate responses to surgical manipulation. Isoflurane was delivered into the inspiratory gas stream by a precision mass-flow controller. Post-operative Isoflurane was gradually lowered and maintained around MAC of < 0.5% during the remaining experimental period, according to the analgosedation needs and hemodynamic conditions. Rats were ventilated through a coaxial tracheal cannula at 72 breaths/min with an inspiratory and expiratory ratio of 1:2 and a tidal volume of 6-8ml/kg, constant during the experiments and equal in both normocapnic an hypercapnic groups in order to avoid potential additional variables related to different stress and strain on the lungs. Gas concentrations were delivered by a precision volumetric respirator. Airway pressure was monitored continuously in order to maintain normocapnic (end-tidal CO 2 = 37–45 mmHg) and normoxic (SpO 2 > 90%) conditions. The hypercapnia (End tidal Co2, EtCO 2 between 70 and 80 mmHg) was induced and maintained by increasing the FiCO 2 while keeping constant the TV and consequently unvaried the stress and strain on the lungs and diaphragm, which are therefore comparable with normocapnic group. Intermittent hyperinflations (6 per hour at 19–20 cmH 2 O) over a constant positive end-expiratory pressure (PEEP = 1.5 cm H 2 O) were set to prevent alveolar de-recruitment and consequent atelectasis. Post-synaptic neuromuscular blockade was induced on the first day (intra-arterial 150 µg α-Cobratoxin) and maintained by continuous infusion (187 µg/day). CMV started after neuromuscular blockade induction. Urination was maintained above 1 ml/h. In no case did animals show any clinical signs of infection or septicemia. Muscle Tissue, Membrane Permeabilization, and Single Muscle Fibre Contractile Measurements Following ICU conditions, deeply sedated rats were euthanized by heart removal and weighed. Diaphragm muscle bundles were dissected from the mid-costal-appositional zone. All muscle bundles were dissected free and tied with surgical silk to glass capillary tubes at 110% length. Bundles were then treated with skinning solution (relaxing solution containing glycerol; 1:1) for 24 hours at 4°C before being transferred to -20°C. Following which, muscle bundles were subjected to increasing concentrations of sucrose solution (0.5-2 M), snap-frozen in liquid nitrogen-chilled propane, and maintained at -140°C [ 13 ]. Prior to contractile assessment, bundles were subjected to decreasing concentrations of sucrose solution and kept in a skinning solution at − 20°C. Single muscle fibers are carefully dissected from the muscle bundle by surgical forceps and placed between two connectors. One connector leads to a force transducer (model 400A, Aurora Scientific, Canada) and the other to a lever arm system (model 308B, Aurora Scientific). The fibers were attached to the connectors as previously described [ 14 , 15 ]. The apparatus was mounted on the stage of an inverted microscope (model IX70; Olympus, Sweden). While submerged in the relaxing solution, the fiber sarcomere length was set to 2.65–2.75 µm. The diameter of the fiber segment, prior to the mechanical experiment, was measured at a magnification of x32 with an image analysis system. Fiber depth was measured by recording the vertical displacement of the microscope nosepiece while focusing on the top and bottom surfaces of the fiber. The focusing control of the microscope was used as a micrometer. Fiber CSA was calculated from the diameter and depth, assuming an elliptical circumference, and was corrected for the 20% swelling which is known to occur during skinning [ 15 ]. For the mechanical recordings, relaxing and activating solutions contained 4mM Mg-ATP, 1mM free Mg 2+ , 20mM imidazole, 7mM EGTA, 14.5mM creatine phosphate, and KCl to adjust the ionic strength to 180 mM and pH 7.0. The concentrations of free Ca 2+ were 10 − 9 M (relaxing solution) and 10 − 4.5 M (activating solution), expressed as pCa 2+ (-log [Ca 2+ ]). Apparent stability constants for Ca 2+ -EGTA were corrected for temperature (15°C) and ionic strength (180 mM). The custom-made computer program was used to calculate the concentration of each metal, ligand, and metal-ligand complex [ 16 ]. Immediately preceding each activation, the fiber was immersed for 15 seconds in a solution with a reduced Ca 2+ -EGTA buffering capacity. This solution is identical to the relaxing solution except that the EGTA concentration is reduced to 0.5 mM, which results in a more rapid attainment of steady force during subsequent activation. Force was measured by slack-test procedure [ 17 ]. This was calculated as the difference between the maximal steady-state isometric force in activating solution and the resting force measured in the same segment while in the relaxing solution. Maximal force production was normalized to CSA (P 0 /CSA), obtaining the specific force (SF). A strict acceptance criterion was applied for contractile measurements. First, the sarcomere length was checked during the experiment, using a high-speed video analysis system (model 901A HVSL, Aurora Scientific). A muscle fiber was accepted and included in the analyses: (i) if the sarcomere length of a single muscle fiber changed < 0.10 µm between relaxation and maximum activation, (ii) if maximal force changed < 10% from first to seventh activation [ 14 , 15 ]. Real Time Quantitative PCR (qPCR) Approximately 10mg diaphragm and lung tissue were homogenized bacterial lysis plus Tris/glycine buffer before total cellular RNA was extracted and purified using a RelinaPrep RNA Miniprep kit (Promega, Australia). A GoScript reverse transcription mix (Promega) was used to reverse transcribe 0.5 µg of total RNA. qPCR was performed using GoTaq qPCR master mix (Promega) and oligonucleotide primers for the genes of interest. Ct values were normalized to the geometric average of three housekeeping genes (beta-actin, alpha-tubulin, and GAPDH) and expressed in arbitrary units. Statistics Statistical analysis was performed using the software Graphpad Prism. One-way ANOVA with Tukey’s post hoc test was used to compare treatment groups. p < 0.05 was considered statistically significant. Data are presented as mean ± SEM. Results In vivo observations The survival rate after 5 days of normocapnic ICU conditions (Et CO 2 between 37–45 mmHg) was 67%, while under non-acidotic hypercapnic conditions all animals survived the 5 day treatment period. Before the induction of hypercapnia there were no significant differences between the two groups with regard to the body weight, mean arterial pressure (MAP), peak inspiratory airways P (PIP aw ), dynamic lung compliance (C D ) (C D = V T /(PIP aw -PEEP), (PEEP, positive end expiratory pressure), SpO 2 , peripheral perfusion and urine output. 8–10 hours after the induction of hypercapnia (EtCO 2 between 70 and 80 mmHg), the daily average of heart rate and peripheral perfusion started to be significantly increased compared to normocapnic rats, while PIP aw was significantly lower in hypercapnic rats (p < 0,05) (Table 1 ). This is in accordance with the already known effects of CO 2 [ 18 ]. Moreover, MAP did not differ between the two groups. It is well known that hypercapnia increases the cardiac index, but at the same time decreases the peripheral vascular resistances, resulting in no net effect on MAP (Table 1 ). The physiological variability of the C D in function of respiratory rate (RR) during CMV was ruled out because the RR was constant at 72/min. Throughout the duration of the experiment the cardiac pump function and the urine production were normal, which are indirect indicators of a non-acidotic condition (Table 1 ). This is a result of renal pH compensation, which was confirmed with blood gas analysis after 5 days (Table 1 ). Table 1 MAP, mean arterial pressure; PIP aw peak inspiratory airways pressure PIP, peak inspiratory pressure, DC RS dynamic compliance of the respiratory system All the values are averages of the daily values. Normocapnic animals (NC); hypercapnic animals (HC). HR (beats/min) Base line NC DAY 1 NC DAY 2 NC DAY 3 NC DAY 4 NC DAY 5 HC DAY 1 HC DAY 2 HC DAY 3 HC DAY 4 HC DAY 5 MAP (mmHg) 120 ± 5 125 ± 5 115 ± 3 118 ± 7 114 ± 4 110 ± 7 127 ± 6 121 ± 8 115 ± 4 105 ± 5 110 ± 3 PIP aw (mmHg) 8.5 ± 1 8,8 ± 1.5 8.7 ± 1.2 8.8 ± 1.1 8.5 ± 2.1 10.2± 6.5 ± 0.9 6.9 ± 0.7 7.2 ± 0.4 6.7 ± 0.5 7.7 ± 0.6 DC RS (ml/mmHg) 0.25 ± 0.03 0.28 ± 0.04 0.28 ± 0.03 0.29 ± 0.02 0.24 ± 0.02 0.38 ± 0.02 0.36 ± 0,03 0.34 ± 0.02 0.37 ± 0.04 0.32 ± 0.03 Urine output (ml/h) 0,8 ml/h 1.4 1,6 1,5 1,6 0,9 I,5 1,6 1,9 1,8 SpO 2 (%) 96% 96 97 97 98 98 99 98 97 97 98 Peripheral perfusion (%) 0.1 0.2 0.09 0.19 0,22 0.24 0,3 0.32 0,28 0,42 0,39 Contractile function at single muscle fiber level A total of 240 diaphragm fibers (an average of 16 per animal) met the acceptance criteria and were included in the study. Our results show that after 5 days of normocapnic ICU conditions, the CSA of diaphragm fibers decreased by 47% compared with controls (p < 0.001). Non – acidotic hypercapnic treatment preserved muscle CSA to 87% of control value (p < 0.05) (Fig. 1 A). Specific force (SF), defined by the absolute force normalized to CSA, was decreased by 48% of controls following normocapnic ICU conditions (p < 0.001). Non-acidotic hypercapnia increased SF by 21% when compared with 5 days normocapnic rats yet remained 27% less than control (p < 0.05; Fig. 1 B). Inflammatory markers In the diaphragm, a significant upregulation of TNF-α gene expression was observed in rats exposed to 5 days CMV under NAH conditions compared with controls and 5 days normocapnic rats, while IL-1β showed a non-statistically significant increasing trend in the in the two groups of mechanically ventilated rats compared with the controls (Fig. 2 ). In the lung lysates, the gene expression of IL-1β was significantly increased in 5 days normocapnic group compared with the controls, while in NAH rats was an increasing trend. The gene expression of TNF-α was significantly higher in NAH rats compared with the controls. (Fig. 3 ). Markers of protein degradation Our group has previously observed temporal changes o proteolytic pathways in diaphragms of MV rats. Previously, the E3 ubiquitin ligase MuRF-1 was upregulated after 6 hours exposure to CMV, which remain elevated during the 14 day observation period [ 11 ]. In the results observed here, CMV tended to increase MuRF-1 gene expression following 5 days CMV (p = 0.054). NAH increased MuRF-1 gene expression compared to control (p < 0.01), while the gene expression of Atrogin-1 did not change (Fig. 4 ). Mitochondrial response During muscle inactivity, mitochondrial functions are altered resulting in a disrupted energy metabolism, activation of proteolytic pathways and downregulation of muscle protein synthesis [ 6 , 19 ]. Four factors involved in different aspects of mitochondrial functions and homeostasis were measured at the gene level: TFAM (a regulator of mitochondrial gene expression), MFN2 (involved in mitochondrial fusion, quality control and cell metabolism), PARKIN (involved in mitochondrial quality control and mitophagy), ULK-1 (an activator of mitophagy). Five days of normocapnic CMV resulted in decreased gene expression of all these mitochondrial factors, significantly in the case of MFN2 and PARKIN and a clear trend in the case of ULK1. Non acidotic hypercapnia, at the same duration, significantly reversed the gene expression of ULK1 (p < 0.05; Fig. 5 ). Discussion The major observations from this study focusing on the effects of non-acidotic hypercapnia on diaphragm structure and function in rats exposed to 5 days CMV were: 1. Non-acidotic hypercapnia increased the survival rate of rats subjected to critical care conditions, 2 . Non-acidotic hypercapnia reduced significantly the loss of body mass, the diaphragm muscle fiber atrophy and loss of function (specific force) compared with normocapnic rats after 5 days of CMV. 3. Non-acidotic hypercapnia increased TNF-α gene expression in the diaphragm and in the lung compared with normocapnic CMV and control conditions. 4. Non-acidotic hypercapnia significantly reversed ULK-1 expression induced by normocapnic CMV conditions. 5. In accordance with previous studies, the muscle atrophy in response to 5 days CMV was associated with the upregulation of the atrogene MuRF-1 and this was significantly elevated in non -acidotic hypercapnic animals discussed here (Fig. 4 ). Hypercapnia has many effects on cardiovascular, respiratory and immune systems. HCA has anti-inflammatory effects, including attenuation of lung neutrophils recruitment, reduction of pulmonary and systemic cytokines concentrations, of cell apoptosis and Oxygen- and Nitrogen-derived free radical injury [ 20 , 21 ]. Moreover, HCA, inhibits endotoxin-induced neutrophil adherence to pulmonary endothelial cells by inhibiting inflammatory cascades, which results in a decrease in lactate dehydrogenase release and neutrophil adherence to lipopolysaccharide-activated human pulmonary artery endothelial cells, thereby suppressing the acute immune response [ 21 ]. These anti-inflammatory responses are involved, for instance, in the acute phases of severe pneumonia, and are attributed to the main cause of death after COVID-19 infection. On the other hand the anti-inflammatory effects of HCA limit the exaggerated acute immune response, but can worsen the severity of prolonged bacterial pneumonia by reducing bacterial killing [ 22 ]. Moreover, there are data against buffering hypercapnic acidosis, since it worsens the lung healing after injury increasing susceptibility to prolonged sepsis [ 23 ]. However, when hypercapnia is associated with normal blood pH (NAH), a pro-inflammatory response is expected. This response it is not necessarily negative yet can be dependent on clinical context. Therefore, in future studies it is important to understand which effects are due to CO 2 and which to hypercapnic acidosis and use the HCA or NAH according to the specific phase of the disease state. Indeed, cases of infection, sepsis, and the associated Systemic Inflammatory Response Syndrome (SIRS), should be distinguished from its opposing, and potentially subsequent, Compensatory Anti-inflammatory Response Syndrome (CARS). In particular, it is clinically relevant to understand if the effects on the diaphragm are due to the CO 2 per se or to the associated acidosis and to measure the already known pro-inflammatory effects of NAH on atrophy and dysfunction caused by ICU conditions. In the present study we hypothesized that non-acidotic hypercapnia, would limit the loss of mass and function without anti-inflammatory effects or triggering a pro-inflammatory response, as our results showed. Results from the current study demonstrate the protective effects of non-acidotic hypercapnia on diaphragm fiber size and function after long-term CMV, irrespective of the associated pro-inflammatory responses. Non-acidotic hypercapnia is forwarded as a possible complementary clinical tool to be used after the acute phases of infections and sepsis, when it is necessary to support the inflammatory responses and the immune response, instead of limiting them, like HCA does. Therefore, hypercapnia might be a promising therapeutic option with different effects and applications in function of the associated blood pH. While HCA would be more suitable in the acute phase of infective and inflammatory processes, non-acidotic hypercapnia might be more suitable in the following subacute states, when healing, mainly of the lung tissue, and immune processes should be sustained. Thus, hypercapnia will lead to a protective effect on both diaphragm and lung, but type of hypercapnia may need to be adjusted to the phase of the critical disease. Conclusions Our results suggest that NAH limits the development of VIDD and the loss of body mass, irrespective of amplified local muscle inflammation. Therefore, we suggest the clinical use of NAH in mechanically ventilated patients alternatively to HCA, depending on the clinical context. Indeed, the two conditions have opposite effects on inflammatory responses, but the same protective effects on diaphragm. Declarations Ethics approval and consent to participate: • All aspects of this study were approved by the Karolinska Institute ethical committee (N263/14). Consent for publication: • Not applicable. Competing interests: • The authors declare that they have no competing interests. Funding: This study was supported by grants from the Swedish Medical Research Council and Stockholm City Council (Alf 20150423, 20170133), ESICM, Viron MMI and Karolinska Institutet to LL. Author Contribution The experimental work was performed in the research laboratory of Lars Larsson in the Department of Physiology and Pharmacology, Karolinska Institutet. NC designed the study, was responsible for the in vivo part of the experiments, performed the single muscle cell contractile measurements and wrote the paper with the contribution of ABA and LL. ABA participate to in vivo experiments, performed the gene expression analyses and contributed in writing the ms. LL provided the funds and the experimental setups, participate to in vivo part of the experiments and did the final dissection. Acknowledgements: • The authors thank Yvette Hedström, Ya Wen and Meishan Li for their precious help in the monitoring of the in vivo phases of the study and in the preparation of the samples for subsequent analyses. Availability of data and materials: The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. References Contreras M, Masterson C, Laffey JG: Permissive hypercapnia: what to remember. Curr Opin Anaesthesiol 2015, 28(1):26–37. Gendreau S, Geri G, Pham T, Vieillard-Baron A, Mekontso Dessap A: The role of acute hypercapnia on mortality and short-term physiology in patients mechanically ventilated for ARDS: a systematic review and meta-analysis. Intensive Care Med 2022, 48(5):517–534. 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Coakley RJ, Taggart C, Greene C, McElvaney NG, O'Neill SJ: Ambient pCO2 modulates intracellular pH, intracellular oxidant generation, and interleukin-8 secretion in human neutrophils. J Leukoc Biol 2002, 71(4):603–610. Curley G, Laffey JG, Kavanagh BP: Bench-to-bedside review: carbon dioxide. Crit Care 2010, 14(2):220. O'Croinin DF, Nichol AD, Hopkins N, Boylan J, O'Brien S, O'Connor C, Laffey JG, McLoughlin P: Sustained hypercapnic acidosis during pulmonary infection increases bacterial load and worsens lung injury. Crit Care Med 2008, 36(7):2128–2135. Coakley RJ, Taggart C, McElvaney NG, O'Neill SJ: Cytosolic pH and the inflammatory microenvironment modulate cell death in human neutrophils after phagocytosis. Blood 2002, 100(9):3383–3391. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4082716","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":279781571,"identity":"edca91b7-ff06-46ab-a258-0981b354b297","order_by":0,"name":"Nicola Cacciani","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6UlEQVRIiWNgGAWjYDACZiB+AGUfYGCQkJNgJkZLApIWY8JaGJC0gEDiDEKqzdmZHz5IqGGQ528//PDAzx0W6TPbGZg/fMCjxbKZzdgg4RiD4YwzaQYHe89I5M5mZmCTxGeVwWEeNokENqDbbvAwHOBtk8idB9TCzENQyz+GBHmgloN/2yTS5ZgZmD//IaQlsY0hwQCo5TDQlgRpYIhJ4/O+wWGgXxL7JAw3Av1yWLZNwnBmM2ObZA8+LecPP3zw4ZuNvNzxw48/vm2rk5c4f/jwhx/4rIEACWQOYwNhDaNgFIyCUTAK8AIA4qBE735lWa4AAAAASUVORK5CYII=","orcid":"","institution":"Karolinska Institute","correspondingAuthor":true,"prefix":"","firstName":"Nicola","middleName":"","lastName":"Cacciani","suffix":""},{"id":279781573,"identity":"02a3c2e2-bba0-44ed-8734-930d649fadf4","order_by":1,"name":"Alex B. Addinsall","email":"","orcid":"","institution":"Karolinska Institute","correspondingAuthor":false,"prefix":"","firstName":"Alex","middleName":"B.","lastName":"Addinsall","suffix":""},{"id":279781575,"identity":"ce3390ea-7cd0-43a1-a6d2-f6274bd47685","order_by":2,"name":"Lars Larsson","email":"","orcid":"","institution":"Karolinska Institute","correspondingAuthor":false,"prefix":"","firstName":"Lars","middleName":"","lastName":"Larsson","suffix":""}],"badges":[],"createdAt":"2024-03-12 10:06:55","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4082716/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4082716/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":52788743,"identity":"f26cdb1b-a190-4502-8bf9-1a0cb5271a7e","added_by":"auto","created_at":"2024-03-15 19:38:46","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":28251,"visible":true,"origin":"","legend":"\u003cp\u003eSingle diaphragm muscle fibers cross sectional area measured at fixed sarcomere length (A) and specific force (B) in control sham operated rats (CNT), mechanically ventilated for 5 days in normocapnic conditions (5 Day), and mechanically ventilated for 5 days in hypercapnic non-acidotic conditions (5 Day + NAH). Values are represented as means ± SEM.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4082716/v1/7570211e603c59ef42dec42c.jpeg"},{"id":52788742,"identity":"254c8580-34e6-4ee7-8c85-5a8e915b0dee","added_by":"auto","created_at":"2024-03-15 19:38:46","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":27962,"visible":true,"origin":"","legend":"\u003cp\u003eGene expression levels in the diaphragm muscle lysates of IL-1 beta and TNF-α quantified with quantitative PCR in control sham operated rats (CNT), mechanically ventilated for 5 days in normocapnic conditions (5 Day), and mechanically ventilated for 5 days in hypercapnic non-acidotic conditions (5 Day + NAH). Values are represented as mean-centered and sigma normalized.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4082716/v1/917ddde7aa046a943c2ea3c7.jpeg"},{"id":52788741,"identity":"9a612809-b57c-4387-a674-7bd14f936048","added_by":"auto","created_at":"2024-03-15 19:38:46","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":39071,"visible":true,"origin":"","legend":"\u003cp\u003eGene expression levels in the lung lysates of IL-1 beta and TNF-α quantified with quantitative PCR in control sham operated rats (CNT), mechanically ventilated for 5 days in normocapnic conditions (5 Day), and mechanically ventilated for 5 days in hypercapnic non-acidotic conditions (5 Day + NAH). Values are represented as mean-centered and sigma normalized.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4082716/v1/5bf17ebfba8831ac44983d81.jpeg"},{"id":52788744,"identity":"ab114c2e-badb-4ea9-98af-1a1710ab5fb2","added_by":"auto","created_at":"2024-03-15 19:38:46","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":29968,"visible":true,"origin":"","legend":"\u003cp\u003eGene and protein expression levels in the diaphragm muscle lysates of Atrogin-1 and Murf-1 quantified with quantitative PCR in control sham operated rats (CNT), mechanically ventilated for 5 days in normocapnic conditions (5 Day), and mechanically ventilated for 5 days in hypercapnic non-acidotic conditions (5 Day + NAH). Values are represented as mean-centered and sigma normalized.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4082716/v1/4643e7362fe9ce476060dc24.jpeg"},{"id":52788745,"identity":"9fadd202-52c1-4084-9a2e-0e45e049e9ca","added_by":"auto","created_at":"2024-03-15 19:38:47","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":46119,"visible":true,"origin":"","legend":"\u003cp\u003eGene expression levels in the diaphragm muscle lysates of mitochondrial factors, Tfam, Mfn2, PARKIN, ULK1 quantified with quantitative PCR in control sham operated rats (CNT), mechanically ventilated for 5 days in normocapnic conditions (5 Day), and mechanically ventilated for 5 days in hypercapnic non-acidotic conditions (5 Day + NAH). Values are represented as mean-centered and sigma normalized.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4082716/v1/be3da3adeff516464966e9c8.jpeg"},{"id":52834381,"identity":"b7458c81-5a4c-46ef-a682-3e568806083f","added_by":"auto","created_at":"2024-03-17 09:29:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":505445,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4082716/v1/c08ec45a-7552-4c35-a704-7f36a3a935a5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Non-acidotic hypercapnia limits atrophy and loss of specific force in rat diaphragm after 5 days of controlled mechanical ventilation in parallel with increased local inflammation","fulltext":[{"header":"Background","content":"\u003cp\u003eThe loss of function of the diaphragm muscle resulting from Mechanical Ventilation (MV) in Intensive Care Unit (ICU) patients has been termed Ventilator Induced Diaphragmatic Dysfunction (VIDD) in analogy with the often-concomitant Ventilator Induced Lung Injury (VILI). VIDD is characterized by a rapid and progressive loss of diaphragm muscle mass and function. Hypercapnia is a frequent condition in mechanically ventilated ICU patients and accepted as \u0026ldquo;permissive hypercapnia\u0026rdquo;, typically in Acute Respiratory Distress Syndrome (ARDS), during lung protective mechanical ventilation strategies [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Hypercapnia reduces mortality in patients randomized to receive high tidal volume (TV, 12ml/Kg) and no additional protective effect of permissive hypercapnia was found in patients ventilated with low TV (6ml/Kg) suggesting positive effects of CO\u003csub\u003e2\u003c/sub\u003e per se and not exclusively related to reduced strain and stress on pulmonary tissues during lung protectives MV strategies. On the other hand, the classical \u0026ldquo;mainstream\u0026rdquo; meta-analysis on the prognosis of ARDS patients, have attributed the protective effects, almost exclusively to low TV-, while considering hypercapnia as simply a side effect that must be accepted [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. According to the data from experimental rat and piglet models, hypercapnia can attenuate VIDD progression and severity. This suggests the possibility to implement MV strategies protecting both lungs and diaphragm respectively against VILI and VIDD, like in ARDS patients with \u0026ldquo;baby lung\u0026rdquo; where alveolar stress and strain should be minimized. Shellekens and coworkers [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], reported a protective effect of hypercapnia after 18 hours of Controlled MV (CMV), measured as an improved force-generating capacity (specific force) in diaphragm fibers compared with normocapnic mechanically ventilated rats for the same duration. However, these rats were subjected to hypercapnic in combination with acidosis (HCA) and it remains unclear if these potential benefits are associated with hypercapnia per se, or influenced by the associated acidosis. Moreover, in MV piglets, HCA preserved the trans-diaphragmatic pressure (Pdi) following 72 hours of CMV [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, this study lacked exploration of molecular mechanisms underlying the improvements [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Combined, these studies showed the potential therapeutic benefit of hypercapnia during MV, yet these observations were limited to animals exposed to a short period of CMV, which lacks clinical translation to the condition experienced by ICU patients and may be confounded by respiratory acidosis.\u003c/p\u003e \u003cp\u003eThe effects of non-acidotic hypercapnia on diaphragm muscle size and function were therefore investigated within a clinically relevant timeframe, 5 days, and compared with normocapnic 5 days mechanically ventilated and sham-operated controls. Specific interest is focused on the effects of inflammation, protein degradation pathways and mitochondrial factors linked to muscle atrophy and dysfunction. Indeed, CMV per se is associated with mechanical perturbations of both pulmonary and diaphragmatic tissues leading to inflammatory responses [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] and to a derangement of energy metabolism and consequent energy crisis [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals and ICU Model\u003c/h2\u003e \u003cp\u003eAdult female Sprague Dawley rats were included in the study and divided into three groups: a sham-operated control group (n\u0026thinsp;=\u0026thinsp;5), a 5-day mechanically ventilated non-acidotic hypercapnia experimental group (5D\u0026thinsp;+\u0026thinsp;NAH group, n\u0026thinsp;=\u0026thinsp;5, with EtCO\u003csub\u003e2\u003c/sub\u003e maintained between 70 and 80 mmHg)), and 5-day mechanically ventilated normocapnic group (5D, n\u0026thinsp;=\u0026thinsp;5, with EtCO\u003csub\u003e2\u003c/sub\u003e maintained between 37 and 45 mmHg. All the mechanically ventilated rats were exposed to deep sedation, controlled CMV and neuromuscular blockade for 5 days with gas concentration of 30\u0026ndash;35% O2, 3% CO2 and the rest N\u003csub\u003e2\u003c/sub\u003e. In both mechanically ventilated groups the EtCO\u003csub\u003e2\u003c/sub\u003e (indirect monitoring of PaCo\u003csub\u003e2\u003c/sub\u003e) was maintained in the desired range by adjusting the FiCO\u003csub\u003e2\u003c/sub\u003e, while maintaining constant the TV and consequently the stress and strain on the lungs, which are known sources of release of inflammatory factors. We induced hypercapnia 2\u0026ndash;3 hours after the start of CMV allowing the renal buffering to keep the blood pH in the normal range and at the same time avoiding sodium bicarbonate administration, that may worsen the intracellular acidosis and therefore having a negative impact on many physiological processes including alveolar healing Cortez-Puentez et al. 2019), Masterson et al 2020. All aspects of this study were approved by the Karolinska Institute ethical committee (N263/14).\u003c/p\u003e \u003cp\u003eThe experimental model used in this study is not limited by early mortality and is a modification of the model originally developed by Dworkin and co-workers [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] optimized to be minimally invasive for the study of the effects of the ICU condition on skeletal muscle structure and function [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The model is not limited by early mortality and the longest duration a rat has been ventilated in this model is 96 days [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAll experimental animals were maintained in fluid and nutritional balance throughout the duration of the experimental procedures by introducing: 1) intra-arterial solution (0.6 ml/h) containing 21 ml H\u003csub\u003e2\u003c/sub\u003eO, 24 ml 0.5 N lactated Ringers, 0.84 g oxacillin Na, 0.65 mg alpha-cobrotoxin, 0.3 mg vitamin K (Synkavite), and 20 mg K\u003csup\u003e+\u003c/sup\u003e (as KCl); 2) an intravenous solution (0.6 ml/h) containing 26 ml H\u003csub\u003e2\u003c/sub\u003eO, 16 ml 0.5 N lactated Ringers, 20% glucose (Baxter, Deerfield, IL, USA), and 0.32 g oxacillin Na for the initial 24, then 8.5% Travasol amino acids and 20% Intralipid (Kabi, Sweden) were added subsequently to provide adequate nutrients [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Body temperature, peripheral perfusion, and oxygen saturation were monitored and maintained in the physiological range by an infrared paw probe (MouseSTAT, Kent Scientific corp., USA). Controls were anesthetized with isoflurane, maintained with spontaneous breathing, and sacrificed within 2 hours of the initial isoflurane anesthesia and surgery.\u003c/p\u003e \u003cp\u003eDuring surgery or any possible irritating manipulation, the Minimum Alveolar Concentration (MAC) was kept\u0026thinsp;\u0026gt;\u0026thinsp;1.5% in order to maintain: 1) high-voltage slow-wave activity of the electroencephalogram (EEG); 2) mean arterial pressure 90\u0026ndash;100 mmHg and heart rate below 400 beats/min; and 3) no evident EEG, blood pressure, or heart rate responses to surgical manipulation. Isoflurane was delivered into the inspiratory gas stream by a precision mass-flow controller. Post-operative Isoflurane was gradually lowered and maintained around MAC of \u0026lt;\u0026thinsp;0.5% during the remaining experimental period, according to the analgosedation needs and hemodynamic conditions. Rats were ventilated through a coaxial tracheal cannula at 72 breaths/min with an inspiratory and expiratory ratio of 1:2 and a tidal volume of 6-8ml/kg, constant during the experiments and equal in both normocapnic an hypercapnic groups in order to avoid potential additional variables related to different stress and strain on the lungs. Gas concentrations were delivered by a precision volumetric respirator. Airway pressure was monitored continuously in order to maintain normocapnic (end-tidal CO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;37\u0026ndash;45 mmHg) and normoxic (SpO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;\u0026gt;\u0026thinsp;90%) conditions. The hypercapnia (End tidal Co2, EtCO\u003csub\u003e2\u003c/sub\u003e between 70 and 80 mmHg) was induced and maintained by increasing the FiCO\u003csub\u003e2\u003c/sub\u003e while keeping constant the TV and consequently unvaried the stress and strain on the lungs and diaphragm, which are therefore comparable with normocapnic group. Intermittent hyperinflations (6 per hour at 19\u0026ndash;20 cmH\u003csub\u003e2\u003c/sub\u003eO) over a constant positive end-expiratory pressure (PEEP\u0026thinsp;=\u0026thinsp;1.5 cm H\u003csub\u003e2\u003c/sub\u003eO) were set to prevent alveolar de-recruitment and consequent atelectasis. Post-synaptic neuromuscular blockade was induced on the first day (intra-arterial 150 \u0026micro;g α-Cobratoxin) and maintained by continuous infusion (187 \u0026micro;g/day). CMV started after neuromuscular blockade induction. Urination was maintained above 1 ml/h. In no case did animals show any clinical signs of infection or septicemia.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMuscle Tissue, Membrane Permeabilization, and Single Muscle Fibre Contractile Measurements\u003c/h2\u003e \u003cp\u003eFollowing ICU conditions, deeply sedated rats were euthanized by heart removal and weighed. Diaphragm muscle bundles were dissected from the mid-costal-appositional zone. All muscle bundles were dissected free and tied with surgical silk to glass capillary tubes at 110% length. Bundles were then treated with skinning solution (relaxing solution containing glycerol; 1:1) for 24 hours at 4\u0026deg;C before being transferred to -20\u0026deg;C. Following which, muscle bundles were subjected to increasing concentrations of sucrose solution (0.5-2 M), snap-frozen in liquid nitrogen-chilled propane, and maintained at -140\u0026deg;C [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Prior to contractile assessment, bundles were subjected to decreasing concentrations of sucrose solution and kept in a skinning solution at \u0026minus;\u0026thinsp;20\u0026deg;C. Single muscle fibers are carefully dissected from the muscle bundle by surgical forceps and placed between two connectors. One connector leads to a force transducer (model 400A, Aurora Scientific, Canada) and the other to a lever arm system (model 308B, Aurora Scientific). The fibers were attached to the connectors as previously described [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The apparatus was mounted on the stage of an inverted microscope (model IX70; Olympus, Sweden). While submerged in the relaxing solution, the fiber sarcomere length was set to 2.65\u0026ndash;2.75 \u0026micro;m. The diameter of the fiber segment, prior to the mechanical experiment, was measured at a magnification of x32 with an image analysis system. Fiber depth was measured by recording the vertical displacement of the microscope nosepiece while focusing on the top and bottom surfaces of the fiber. The focusing control of the microscope was used as a micrometer. Fiber CSA was calculated from the diameter and depth, assuming an elliptical circumference, and was corrected for the 20% swelling which is known to occur during skinning [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor the mechanical recordings, relaxing and activating solutions contained 4mM Mg-ATP, 1mM free Mg\u003csup\u003e2+\u003c/sup\u003e, 20mM imidazole, 7mM EGTA, 14.5mM creatine phosphate, and KCl to adjust the ionic strength to 180 mM and pH 7.0. The concentrations of free Ca\u003csup\u003e2+\u003c/sup\u003e were 10\u003csup\u003e\u0026minus;\u0026thinsp;9\u003c/sup\u003e M (relaxing solution) and 10\u003csup\u003e\u0026minus;\u0026thinsp;4.5\u003c/sup\u003e M (activating solution), expressed as pCa\u003csup\u003e2+\u003c/sup\u003e (-log [Ca\u003csup\u003e2+\u003c/sup\u003e]). Apparent stability constants for Ca\u003csup\u003e2+\u003c/sup\u003e-EGTA were corrected for temperature (15\u0026deg;C) and ionic strength (180 mM). The custom-made computer program was used to calculate the concentration of each metal, ligand, and metal-ligand complex [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Immediately preceding each activation, the fiber was immersed for 15 seconds in a solution with a reduced Ca\u003csup\u003e2+\u003c/sup\u003e-EGTA buffering capacity. This solution is identical to the relaxing solution except that the EGTA concentration is reduced to 0.5 mM, which results in a more rapid attainment of steady force during subsequent activation.\u003c/p\u003e \u003cp\u003eForce was measured by slack-test procedure [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. This was calculated as the difference between the maximal steady-state isometric force in activating solution and the resting force measured in the same segment while in the relaxing solution. Maximal force production was normalized to CSA (P\u003csub\u003e0\u003c/sub\u003e/CSA), obtaining the specific force (SF). A strict acceptance criterion was applied for contractile measurements. First, the sarcomere length was checked during the experiment, using a high-speed video analysis system (model 901A HVSL, Aurora Scientific). A muscle fiber was accepted and included in the analyses: (i) if the sarcomere length of a single muscle fiber changed\u0026thinsp;\u0026lt;\u0026thinsp;0.10 \u0026micro;m between relaxation and maximum activation, (ii) if maximal force changed\u0026thinsp;\u0026lt;\u0026thinsp;10% from first to seventh activation [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eReal Time Quantitative PCR (qPCR)\u003c/h2\u003e \u003cp\u003eApproximately 10mg diaphragm and lung tissue were homogenized bacterial lysis plus Tris/glycine buffer before total cellular RNA was extracted and purified using a RelinaPrep RNA Miniprep kit (Promega, Australia). A GoScript reverse transcription mix (Promega) was used to reverse transcribe 0.5 \u0026micro;g of total RNA. qPCR was performed using GoTaq qPCR master mix (Promega) and oligonucleotide primers for the genes of interest. Ct values were normalized to the geometric average of three housekeeping genes (beta-actin, alpha-tubulin, and GAPDH) and expressed in arbitrary units.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using the software Graphpad Prism. One-way ANOVA with Tukey\u0026rsquo;s post hoc test was used to compare treatment groups. p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eIn vivo observations\u003c/h2\u003e \u003cp\u003eThe survival rate after 5 days of normocapnic ICU conditions (Et CO\u003csub\u003e2\u003c/sub\u003e between 37\u0026ndash;45 mmHg) was 67%, while under non-acidotic hypercapnic conditions all animals survived the 5 day treatment period. Before the induction of hypercapnia there were no significant differences between the two groups with regard to the body weight, mean arterial pressure (MAP), peak inspiratory airways P (PIP\u003csub\u003eaw\u003c/sub\u003e), dynamic lung compliance (C\u003csub\u003eD\u003c/sub\u003e) (C\u003csub\u003eD\u003c/sub\u003e= V\u003csub\u003eT\u003c/sub\u003e/(PIP\u003csub\u003eaw\u003c/sub\u003e-PEEP), (PEEP, positive end expiratory pressure), SpO\u003csub\u003e2\u003c/sub\u003e, peripheral perfusion and urine output. 8\u0026ndash;10 hours after the induction of hypercapnia (EtCO\u003csub\u003e2\u003c/sub\u003e between 70 and 80 mmHg), the daily average of heart rate and peripheral perfusion started to be significantly increased compared to normocapnic rats, while PIP\u003csub\u003eaw\u003c/sub\u003e was significantly lower in hypercapnic rats (p\u0026thinsp;\u0026lt;\u0026thinsp;0,05) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This is in accordance with the already known effects of CO\u003csub\u003e2\u003c/sub\u003e [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Moreover, MAP did not differ between the two groups. It is well known that hypercapnia increases the cardiac index, but at the same time decreases the peripheral vascular resistances, resulting in no net effect on MAP (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The physiological variability of the C\u003csub\u003eD\u003c/sub\u003e in function of respiratory rate (RR) during CMV was ruled out because the RR was constant at 72/min. Throughout the duration of the experiment the cardiac pump function and the urine production were normal, which are indirect indicators of a non-acidotic condition (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This is a result of renal pH compensation, which was confirmed with blood gas analysis after 5 days (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMAP, mean arterial pressure; PIP\u003csub\u003eaw\u003c/sub\u003e peak inspiratory airways pressure PIP, peak inspiratory pressure, DC\u003csub\u003eRS\u003c/sub\u003e dynamic compliance of the respiratory system All the values are averages of the daily values. Normocapnic animals (NC); hypercapnic animals (HC).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"12\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHR (beats/min)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBase\u003c/p\u003e \u003cp\u003eline\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNC\u003c/p\u003e \u003cp\u003eDAY 1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNC\u003c/p\u003e \u003cp\u003eDAY 2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNC\u003c/p\u003e \u003cp\u003eDAY 3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNC\u003c/p\u003e \u003cp\u003eDAY 4\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNC\u003c/p\u003e \u003cp\u003eDAY 5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eHC\u003c/p\u003e \u003cp\u003eDAY 1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eHC\u003c/p\u003e \u003cp\u003eDAY 2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eHC\u003c/p\u003e \u003cp\u003eDAY 3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eHC\u003c/p\u003e \u003cp\u003eDAY 4\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003eHC\u003c/p\u003e \u003cp\u003eDAY 5\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMAP (mmHg)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e120\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e125\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e115\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e118\u0026thinsp;\u0026plusmn;\u0026thinsp;7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e114\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e110\u0026thinsp;\u0026plusmn;\u0026thinsp;7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e127\u0026thinsp;\u0026plusmn;\u0026thinsp;6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e121\u0026thinsp;\u0026plusmn;\u0026thinsp;8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e115\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e105\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e110\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePIP\u003c/b\u003e\u003csub\u003e\u003cb\u003eaw\u003c/b\u003e\u003c/sub\u003e \u003cb\u003e(mmHg)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8,8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e10.2\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e6.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e6.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e7.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e6.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e7.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDC\u003c/b\u003e\u003csub\u003e\u003cb\u003eRS\u003c/b\u003e\u003c/sub\u003e \u003cb\u003e(ml/mmHg)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0,03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eUrine output (ml/h)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0,8 ml/h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1,6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1,5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1,6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0,9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eI,5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1,6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1,9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e1,8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSpO\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e \u003cb\u003e(%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e96%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e98\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePeripheral perfusion (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0,3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0,28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0,42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0,39\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eContractile function at single muscle fiber level\u003c/h3\u003e\n\u003cp\u003eA total of 240 diaphragm fibers (an average of 16 per animal) met the acceptance criteria and were included in the study. Our results show that after 5 days of normocapnic ICU conditions, the CSA of diaphragm fibers decreased by 47% compared with controls (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Non \u0026ndash; acidotic hypercapnic treatment preserved muscle CSA to 87% of control value (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Specific force (SF), defined by the absolute force normalized to CSA, was decreased by 48% of controls following normocapnic ICU conditions (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Non-acidotic hypercapnia increased SF by 21% when compared with 5 days normocapnic rats yet remained 27% less than control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eInflammatory markers\u003c/h2\u003e \u003cp\u003eIn the diaphragm, a significant upregulation of TNF-α gene expression was observed in rats exposed to 5 days CMV under NAH conditions compared with controls and 5 days normocapnic rats, while IL-1β showed a non-statistically significant increasing trend in the in the two groups of mechanically ventilated rats compared with the controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In the lung lysates, the gene expression of IL-1β was significantly increased in 5 days normocapnic group compared with the controls, while in NAH rats was an increasing trend. The gene expression of TNF-α was significantly higher in NAH rats compared with the controls. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMarkers of protein degradation\u003c/h2\u003e \u003cp\u003eOur group has previously observed temporal changes o proteolytic pathways in diaphragms of MV rats. Previously, the E3 ubiquitin ligase MuRF-1 was upregulated after 6 hours exposure to CMV, which remain elevated during the 14 day observation period [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In the results observed here, CMV tended to increase MuRF-1 gene expression following 5 days CMV (p\u0026thinsp;=\u0026thinsp;0.054). NAH increased MuRF-1 gene expression compared to control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), while the gene expression of Atrogin-1 did not change (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial response\u003c/h2\u003e \u003cp\u003eDuring muscle inactivity, mitochondrial functions are altered resulting in a disrupted energy metabolism, activation of proteolytic pathways and downregulation of muscle protein synthesis [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Four factors involved in different aspects of mitochondrial functions and homeostasis were measured at the gene level: TFAM (a regulator of mitochondrial gene expression), MFN2 (involved in mitochondrial fusion, quality control and cell metabolism), PARKIN (involved in mitochondrial quality control and mitophagy), ULK-1 (an activator of mitophagy). Five days of normocapnic CMV resulted in decreased gene expression of all these mitochondrial factors, significantly in the case of MFN2 and PARKIN and a clear trend in the case of ULK1. Non acidotic hypercapnia, at the same duration, significantly reversed the gene expression of ULK1 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe major observations from this study focusing on the effects of non-acidotic hypercapnia on diaphragm structure and function in rats exposed to 5 days CMV were: \u003cb\u003e1.\u003c/b\u003e Non-acidotic hypercapnia increased the survival rate of rats subjected to critical care conditions, \u003cb\u003e2\u003c/b\u003e. Non-acidotic hypercapnia reduced significantly the loss of body mass, the diaphragm muscle fiber atrophy and loss of function (specific force) compared with normocapnic rats after 5 days of CMV. \u003cb\u003e3.\u003c/b\u003e Non-acidotic hypercapnia increased TNF-α gene expression in the diaphragm and in the lung compared with normocapnic CMV and control conditions. \u003cb\u003e4.\u003c/b\u003e Non-acidotic hypercapnia significantly reversed ULK-1 expression induced by normocapnic CMV conditions. \u003cb\u003e5.\u003c/b\u003e In accordance with previous studies, the muscle atrophy in response to 5 days CMV was associated with the upregulation of the atrogene MuRF-1 and this was significantly elevated in non -acidotic hypercapnic animals discussed here (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHypercapnia has many effects on cardiovascular, respiratory and immune systems. HCA has anti-inflammatory effects, including attenuation of lung neutrophils recruitment, reduction of pulmonary and systemic cytokines concentrations, of cell apoptosis and Oxygen- and Nitrogen-derived free radical injury [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Moreover, HCA, inhibits endotoxin-induced neutrophil adherence to pulmonary endothelial cells by inhibiting inflammatory cascades, which results in a decrease in lactate dehydrogenase release and neutrophil adherence to lipopolysaccharide-activated human pulmonary artery endothelial cells, thereby suppressing the acute immune response [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. These anti-inflammatory responses are involved, for instance, in the acute phases of severe pneumonia, and are attributed to the main cause of death after COVID-19 infection. On the other hand the anti-inflammatory effects of HCA limit the exaggerated acute immune response, but can worsen the severity of prolonged bacterial pneumonia by reducing bacterial killing [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Moreover, there are data against buffering hypercapnic acidosis, since it worsens the lung healing after injury increasing susceptibility to prolonged sepsis [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. However, when hypercapnia is associated with normal blood pH (NAH), a pro-inflammatory response is expected. This response it is not necessarily negative yet can be dependent on clinical context. Therefore, in future studies it is important to understand which effects are due to CO\u003csub\u003e2\u003c/sub\u003e and which to hypercapnic acidosis and use the HCA or NAH according to the specific phase of the disease state. Indeed, cases of infection, sepsis, and the associated Systemic Inflammatory Response Syndrome (SIRS), should be distinguished from its opposing, and potentially subsequent, Compensatory Anti-inflammatory Response Syndrome (CARS). In particular, it is clinically relevant to understand if the effects on the diaphragm are due to the CO\u003csub\u003e2\u003c/sub\u003e per se or to the associated acidosis and to measure the already known pro-inflammatory effects of NAH on atrophy and dysfunction caused by ICU conditions.\u003c/p\u003e \u003cp\u003eIn the present study we hypothesized that non-acidotic hypercapnia, would limit the loss of mass and function without anti-inflammatory effects or triggering a pro-inflammatory response, as our results showed. Results from the current study demonstrate the protective effects of non-acidotic hypercapnia on diaphragm fiber size and function after long-term CMV, irrespective of the associated pro-inflammatory responses. Non-acidotic hypercapnia is forwarded as a possible complementary clinical tool to be used after the acute phases of infections and sepsis, when it is necessary to support the inflammatory responses and the immune response, instead of limiting them, like HCA does. Therefore, hypercapnia might be a promising therapeutic option with different effects and applications in function of the associated blood pH. While HCA would be more suitable in the acute phase of infective and inflammatory processes, non-acidotic hypercapnia might be more suitable in the following subacute states, when healing, mainly of the lung tissue, and immune processes should be sustained. Thus, hypercapnia will lead to a protective effect on both diaphragm and lung, but type of hypercapnia may need to be adjusted to the phase of the critical disease.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur results suggest that NAH limits the development of VIDD and the loss of body mass, irrespective of amplified local muscle inflammation. Therefore, we suggest the clinical use of NAH in mechanically ventilated patients alternatively to HCA, depending on the clinical context. Indeed, the two conditions have opposite effects on inflammatory responses, but the same protective effects on diaphragm.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate:\u003c/strong\u003e \u003cp\u003e\u0026bull; All aspects of this study were approved by the Karolinska Institute ethical committee (N263/14).\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication:\u003c/strong\u003e \u003cp\u003e\u0026bull; Not applicable.\u003ch2\u003eCompeting interests:\u003c/h2\u003e \u003cp\u003e\u0026bull; The authors declare that they have no competing interests.\u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis study was supported by grants from the Swedish Medical Research Council and Stockholm City Council (Alf 20150423, 20170133), ESICM, Viron MMI and Karolinska Institutet to LL.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eThe experimental work was performed in the research laboratory of Lars Larsson in the Department of Physiology and Pharmacology, Karolinska Institutet. NC designed the study, was responsible for the in vivo part of the experiments, performed the single muscle cell contractile measurements and wrote the paper with the contribution of ABA and LL. ABA participate to in vivo experiments, performed the gene expression analyses and contributed in writing the ms. LL provided the funds and the experimental setups, participate to in vivo part of the experiments and did the final dissection.\u003c/p\u003e\u003ch2\u003eAcknowledgements:\u003c/h2\u003e \u003cp\u003e\u0026bull; The authors thank Yvette Hedstr\u0026ouml;m, Ya Wen and Meishan Li for their precious help in the monitoring of the in vivo phases of the study and in the preparation of the samples for subsequent analyses.\u003c/p\u003e\u003ch2\u003eAvailability of data and materials:\u003c/h2\u003e \u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eContreras M, Masterson C, Laffey JG: Permissive hypercapnia: what to remember. Curr Opin Anaesthesiol 2015, 28(1):26\u0026ndash;37.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGendreau S, Geri G, Pham T, Vieillard-Baron A, Mekontso Dessap A: The role of acute hypercapnia on mortality and short-term physiology in patients mechanically ventilated for ARDS: a systematic review and meta-analysis. Intensive Care Med 2022, 48(5):517\u0026ndash;534.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchellekens WJ, van Hees HW, Kox M, Linkels M, Acuna GL, Dekhuijzen PN, Scheffer GJ, van der Hoeven JG, Heunks LM: Hypercapnia attenuates ventilator-induced diaphragm atrophy and modulates dysfunction. Crit Care 2014, 18(1):R28.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJung B, Sebbane M, Le Goff C, Rossel N, Chanques G, Futier E, Constantin JM, Matecki S, Jaber S: Moderate and prolonged hypercapnic acidosis may protect against ventilator-induced diaphragmatic dysfunction in healthy piglet: an in vivo study. Crit Care 2013, 17(1):R15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKeskinidou C, Vassiliou AG, Dimopoulou I, Kotanidou A, Orfanos SE: Mechanistic Understanding of Lung Inflammation: Recent Advances and Emerging Techniques. J Inflamm Res 2022, 15:3501\u0026ndash;3546.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePicard M, Jung B, Liang F, Azuelos I, Hussain S, Goldberg P, Godin R, Danialou G, Chaturvedi R, Rygiel K \u003cem\u003eet al\u003c/em\u003e: Mitochondrial dysfunction and lipid accumulation in the human diaphragm during mechanical ventilation. Am J Respir Crit Care Med 2012, 186(11):1140\u0026ndash;1149.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSalah H, Li M, Cacciani N, Gastaldello S, Ogilvie H, Akkad H, Namuduri AV, Morbidoni V, Artemenko KA, Balogh G \u003cem\u003eet al\u003c/em\u003e: The chaperone co-inducer BGP-15 alleviates ventilation-induced diaphragm dysfunction. Sci Transl Med 2016, 8(350):350ra103.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDworkin BR, Dworkin S: Learning of physiological responses: I. Habituation, sensitization, and classical conditioning. Behav Neurosci 1990, 104(2):298\u0026ndash;319.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDworkin BR, Dworkin S: Baroreflexes of the rat. III. Open-loop gain and electroencephalographic arousal. Am J Physiol Regul Integr Comp Physiol 2004, 286(3):R597-605.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDworkin R, Modin G, Kunz S, Rich R, Zak O, Sande M: Comparative efficacies of ciprofloxacin, pefloxacin, and vancomycin in combination with rifampin in a rat model of methicillin-resistant Staphylococcus aureus chronic osteomyelitis. Antimicrob Agents Chemother 1990, 34(6):1014\u0026ndash;1016.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCorpeno R, Dworkin B, Cacciani N, Salah H, Bergman HM, Ravara B, Vitadello M, Gorza L, Gustafson AM, Hedstrom Y \u003cem\u003eet al\u003c/em\u003e: Time course analysis of mechanical ventilation-induced diaphragm contractile muscle dysfunction in the rat. J Physiol 2014, 592(17):3859\u0026ndash;3880.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOchala J, Gustafson AM, Diez ML, Renaud G, Li M, Aare S, Qaisar R, Banduseela VC, Hedstrom Y, Tang X \u003cem\u003eet al\u003c/em\u003e: Preferential skeletal muscle myosin loss in response to mechanical silencing in a novel rat intensive care unit model: underlying mechanisms. J Physiol 2011, 589(Pt 8):2007\u0026ndash;2026.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFrontera WR, Larsson L: Contractile studies of single human skeletal muscle fibers: a comparison of different muscles, permeabilization procedures, and storage techniques. Muscle Nerve 1997, 20(8):948\u0026ndash;952.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLarsson L, Moss RL: Maximum velocity of shortening in relation to myosin isoform composition in single fibres from human skeletal muscles. J Physiol 1993, 472:595\u0026ndash;614.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoss RL: Sarcomere length-tension relations of frog skinned muscle fibres during calcium activation at short lengths. J Physiol 1979, 292:177\u0026ndash;192.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFabiato A: Computer programs for calculating total from specified free or free from specified total ionic concentrations in aqueous solutions containing multiple metals and ligands. Methods Enzymol 1988, 157:378\u0026ndash;417.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEdman KA: The velocity of unloaded shortening and its relation to sarcomere length and isometric force in vertebrate muscle fibres. J Physiol 1979, 291:143\u0026ndash;159.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMasterson C, Horie S, McCarthy SD, Gonzalez H, Byrnes D, Brady J, Fandino J, Laffey JG, O'Toole D: Hypercapnia in the critically ill: insights from the bench to the bedside. Interface Focus 2021, 11(2):20200032.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHyatt H, Deminice R, Yoshihara T, Powers SK: Mitochondrial dysfunction induces muscle atrophy during prolonged inactivity: A review of the causes and effects. Arch Biochem Biophys 2019, 662:49\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCoakley RJ, Taggart C, Greene C, McElvaney NG, O'Neill SJ: Ambient pCO2 modulates intracellular pH, intracellular oxidant generation, and interleukin-8 secretion in human neutrophils. J Leukoc Biol 2002, 71(4):603\u0026ndash;610.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCurley G, Laffey JG, Kavanagh BP: Bench-to-bedside review: carbon dioxide. Crit Care 2010, 14(2):220.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eO'Croinin DF, Nichol AD, Hopkins N, Boylan J, O'Brien S, O'Connor C, Laffey JG, McLoughlin P: Sustained hypercapnic acidosis during pulmonary infection increases bacterial load and worsens lung injury. Crit Care Med 2008, 36(7):2128\u0026ndash;2135.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCoakley RJ, Taggart C, McElvaney NG, O'Neill SJ: Cytosolic pH and the inflammatory microenvironment modulate cell death in human neutrophils after phagocytosis. Blood 2002, 100(9):3383\u0026ndash;3391.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Critical Care, VIDD, hypercapnia, diaphragm, mechanical ventilation","lastPublishedDoi":"10.21203/rs.3.rs-4082716/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4082716/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eControlled Mechanical Ventilation (CMV) is associated with Ventilator Induced Lung Injury (VILI) and Ventilator Induced Diaphragm Dysfunction (VIDD). VIDD delays weaning from the respirator and increases the risk of further complications and health care costs, which are disproportionately increased with increasing duration of mechanical ventilation. Hypercapnia is frequently observed and tolerated as \u0026ldquo;permissive hypercapnia\u0026rdquo; during lung protective MV strategies. The systemic effects of hypercapnia are well known and considered potentially protective for diaphragm muscle in acute and short-time experimental MV studies. However, hypercapnia is commonly associated with acidosis, affecting immunity and inflammation pathways.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThis study aims to determine the potential of hypercapnia in the absence of acidosis on diaphragm muscle structure and function in a well-established clinically relevant experimental ICU model, not limited by early mortality. The effects of hypercapnia at physiological pH on diaphragm single fibers cross sectional area (CSA) and specific force (maximum force normalized to CSA) were investigated.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eNon-Acidotic Hypercapnia (NAH) reduced body mass loss, diaphragm muscle fiber atrophy and loss of specific force, in parallel with an increased gene expression of proinflammatory cytokines (TNF-α and IL-1β) and of the MuRF-1 atrogene. In the diaphragm, TNF-α gene expression was significantly increased in NAH rats compared with 5 days normocapnic and controls, while IL-1β showed an increasing trend. In the lung lysates, IL-1β gene expression was significantly increased in 5 days normocapnic rats compared with the controls, while gene expression of TNF-α was increased in the NAH rats compared with controls. In NAH rats the increase was not significant. The gene expression of mitochondrial factors TFAM (regulator of mitochondrial gene expression), MFN2 (involved in mitochondrial fusion, quality control and cell metabolism), PARKIN (involved in mitochondrial quality control and mitophagy), ULK-1 (activator of mitophagy) was analyzed. NAH reversed, significantly the decreased gene expression of ULK 1 observed in the 5 days normocapnic rats.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThese results suggest that non-acidotic hypercapnia limits the development of VIDD, irrespective of amplified local muscle inflammation. Therefore, we suggest its clinical role may be complementary to the known anti-inflammatory effects of hypercapnic acidosis (HCA), which has preventive VIDD effects as well.\u003c/p\u003e","manuscriptTitle":"Non-acidotic hypercapnia limits atrophy and loss of specific force in rat diaphragm after 5 days of controlled mechanical ventilation in parallel with increased local inflammation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-15 19:38:42","doi":"10.21203/rs.3.rs-4082716/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"533132ed-7591-4738-b4d2-470cf6777a0e","owner":[],"postedDate":"March 15th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-03-17T09:29:39+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-15 19:38:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4082716","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4082716","identity":"rs-4082716","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}