The establishment and evaluation of a swine model of deinagkistrodon acutus snakebite envenomation.

OBJECTIVE: To establish a preclinical large-animal model of Deinagkistrodon acutus snakebite envenomation and evaluate its feasibility. METHODS: The venom of D. acutus (0 mg/kg, 1 mg/kg, 2 mg/kg, 5 mg/kg, or 10 mg/kg) was injected into the left biceps femoris of 11 male pigs. Then, the circumferences of the limbs were regularly measured, and changes in muscle injury biomarkers, blood parameters, coagulation function, vital organ function and injury biomarkers were regularly detected. At 24 h after venom injection, the animals were euthanized, and the pathological damage to the vital organs mentioned above was evaluated. RESULTS: The two pigs receiving 10 mg/kg and 5 mg/kg snake venom died at 8 h and 12 h after injection, respectively. The remaining pigs were equally divided into 0 mg/kg, 1 mg/kg, and 2 mg/kg snake venom groups, and all of them survived to 24 h after injection. Compared with the pigs receiving 0 mg/kg snake venom, the pigs receiving 1 mg/kg or 2 mg/kg snake venom exhibited significant abnormities, including limb swelling; increased muscle injury biomarker creatine kinase (CK) and coagulation function indicators prothrombin time and D-dimer; and decreased blood routine indicator platelet and coagulation function indicator fibrinogen. Moreover, significant abnormalities in myocardial and cerebral function and injury biomarkers in the heart, brain, liver, kidney and intestine were also observed. In particular, the abnormalities mentioned above were significantly obvious in those pigs receiving 2 mg/kg snake venom. Pathological evaluation revealed that the morphology of muscle, heart, brain, liver, kidney, and intestine in those pigs receiving 0 mg/kg snake venom was normal; however, pathological damage was observed in those pigs receiving 1 mg/kg and 2 mg/kg snake venom. Similarly, the pathological damage was more severe in those pigs receiving 2 mg/kg snake venom. CONCLUSION: The intramuscular injection of 2 mg/kg D. acutus venom seems to be an optimal dose for examining the preclinical efficacy of existing and novel therapeutics for treating D. acutus envenomation in pigs.

ACETYL-COA PRODUCTION BY OCTANOIC ACID ALLEVIATES ACUTE COMPARTMENT SYNDROME-INDUCED SKELETAL MUSCLE INJURY THROUGH REGULATING MITOPHAGY.

ABSTRACT: Background: Treatment of acute compartment syndrome (ACS)-induced skeletal muscle injury remains a challenge. Previous studies have shown that octanoic acid is a promising treatment for ACS owing to its potential ability to regulate metabolic/epigenetic pathways in ischemic injury. The present study was designed to investigate the efficacy and underlying mechanism of octanoic acid in ACS-induced skeletal muscle injury. Methods: In this study, we established a saline infusion ACS rat model. Subsequently, we assessed the protective effects of sodium octanoate (NaO, sodium salt of octanoic acid) on ACS-induced skeletal muscle injury. Afterward, the level of acetyl-coenzyme A and histone acetylation in the skeletal muscle tissue were quantified. Moreover, we investigated the activation of the AMP-activated protein kinas pathway and the occurrence of mitophagy in the skeletal muscle tissue. Lastly, we scrutinized the expression of proteins associated with mitochondrial dynamics in the skeletal muscle tissue. Results: The administration of NaO attenuated muscle inflammation, alleviating oxidative stress and muscle edema. Moreover, NaO treatment enhanced muscle blood perfusion, leading to the inhibition of apoptosis-related skeletal muscle cell death after ACS. In addition, NaO demonstrated the ability to halt skeletal muscle fibrosis and enhance the functional recovery of muscle post-ACS. Further analysis indicates that NaO treatment increases the acetyl-CoA level in muscle and the process of histone acetylation by acetyl-CoA. Lastly, we found NaO treatment exerts a stimulatory impact on the activation of the AMPK pathway, thus promoting mitophagy and improving mitochondrial dynamics. Conclusion: Our findings indicate that octanoic acid may ameliorate skeletal muscle injury induced by ACS. Its protective effects may be attributed to the promotion of acetyl-CoA synthesis and histone acetylation within the muscular tissue, as well as its activation of the AMPK-related mitophagy pathway.

Sodium octanoate alleviates cardiac and cerebral injury after traumatic cardiac arrest in a porcine model.

INTRODUCTION: Traumatic cardiac arrest (TCA) is a severe condition with a high mortality rate, and patients who survive from TCA face a poor prognosis due to post-resuscitation injury, including cardiac and cerebral injury, which remains a serious challenge. Sodium octanoate has shown protective effects against various diseases. The present study aims to investigate sodium octanoate's protective effects against cardiac and cerebral injury after TCA in a porcine model. METHODS: The study included a total of 22 male domestic pigs divided into three groups: Sham group (n = 7), TCA group (n = 7), and sodium octanoate (SO) group (n = 8). Hemorrhage was initiated via the right femoral artery by a blood pump at a rate of 2 ml·kg-1·min-1 to establish TCA model. The Sham group underwent only endotracheal intubation and arteriovenous catheterization, without experiencing the blood loss/cardiac arrest/resuscitation model. At 5 min after resuscitation, the SO group received a continuous sodium octanoate infusion while the TCA group received the same volume of saline. General indicators were monitored, and blood samples were collected at baseline and at different time points after resuscitation. At 24 h after resuscitation, pigs were sacrificed, and heart and brain were obtained for cell apoptosis detection, iron deposition staining, oxidative stress detection, and the expression of ferroptosis-related proteins (ACSL4 and GPX4). RESULTS: Sodium octanoate significantly improved mean arterial pressure, cardiac output and ejection fraction induced by TCA. Serum biomarkers of cardiac and cerebral injury were found to increase at all time points after resuscitation, while sodium octanoate significantly reduced their levels. The apoptosis rates of cardiomyocytes and cerebral cortex cells in the SO group were significantly lower than in the TCA group, along with a reduced area of iron deposition staining. The sodium octanoate also reduced oxidative stress and down-regulated ferroptosis which was indicated by protein level alteration of ACSL4 and GPX4. CONCLUSION: Our study's findings suggest that early infusion of sodium octanoate significantly alleviates post-resuscitation cardiac and cerebral injury in a porcine model of TCA, possibly through inhibition of cell apoptosis and GPX4-mediated ferroptosis. Therefore, sodium octanoate could be a potential therapeutic strategy for patients with TCA.

Extracellular vesicles derived from human ESC-MSCs target macrophage and promote anti-inflammation process, angiogenesis, and functional recovery in ACS-induced severe skeletal muscle injury.

BACKGROUND: Acute compartment syndrome (ACS) is one of the most common complications of musculoskeletal injury, leading to the necrosis and demise of skeletal muscle cells. Our previous study showed that embryonic stem cells-derived mesenchymal stem cells (ESC-MSCs) are novel therapeutics in ACS treatment. As extracellular vesicles (EVs) are rapidly gaining attention as cell-free therapeutics that have advantages over parental stem cells, the therapeutic potential and mechanisms of EVs from ESC-MSCs on ACS need to be explored. METHOD: In the present study, we examined the protective effects in the experimental ACS rat model and investigated the role of macrophages in mediating these effects. Next, we used transcriptome sequencing to explore the mechanisms by which ESC-MSC-EVs regulate macrophage polarization. Furthermore, miRNA sequencing was performed on ESC-MSC-EVs to identify miRNA candidates associated with macrophage polarization. RESULTS: We found that intravenous administration of ESC-MSC-EVs, given at the time of fasciotomy, significantly promotes the anti-inflammation process, angiogenesis, and functional recovery of muscle in ACS. The beneficial effects were associated with ESC-MSC-EVs affecting macrophage polarization by delivering various miRNAs which regulate NF-κB, JAK/STAT, and PI3K/AKT pathways. Our data further illustrate that ESC-MSC-EVs mainly modulate macrophage polarization via the miR-21/PTEN, miR-320a/PTEN, miR-423/NLRP3, miR-100/mTOR, and miR-26a/TLR3 axes. CONCLUSION: Together, our results demonstrated the beneficial effects of ESC-MSC-EVs in ACS, wherein the miRNAs present in ESC-MSC-EVs regulate the polarization of macrophages.

SULFORAPHANE ALLEVIATES POSTRESUSCITATION LUNG PYROPTOSIS POSSIBLY VIA ACTIVATING THE NRF2/HO-1 PATHWAY.

ABSTRACT: Introduction: Sulforaphane (SFN), known as the activator of the nuclear factor E2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1) pathway, has been proven to protect the lung against various pathological stimuli. The present study aimed to investigate the effect of SFN on lung injury induced by systemic ischemia reperfusion after cardiac arrest and resuscitation. Methods: After animal preparation, 24 pigs were randomly divided into sham group (n = 6), cardiopulmonary resuscitation group (CPR, n = 9), or CPR + SFN group (n = 9). The experimental model was then established by 10 min of cardiac arrest followed by 6 min of CPR. Once spontaneous circulation was achieved, a dose of 2 mg/kg of SFN diluted in 20 mL of saline was intravenously infused with a duration of 5 min. During 4 h of observation after resuscitation, extravascular lung water index (ELWI), pulmonary vascular permeability index (PVPI), and oxygenation index were regularly evaluated. At 24 h after resuscitation, lung tissues were harvested to evaluate the score of lung histopathological injury, the activity of superoxide dismutase, the contents of malondialdehyde, IL-1ß, and IL-18, and the expression levels of NOD-like receptor pyrin domain 3, cleaved caspase 1, gasdermin D (GSDMD), GSDMD N-terminal, Nrf2, and HO-1. Results: During CPR, spontaneous circulation was achieved in six and seven pigs in the CPR and CPR + SFN groups, respectively. After resuscitation, the indicators of lung injury (ELWI, PVPI, and oxygenation index) were all better in the CPR + SFN group than in the CPR group, in which the differences in ELWI and PVPI at 2, and 4 h after resuscitation were significant between the two groups. In addition, SFN significantly reduced lung injury score, improved oxidative imbalance (superoxide dismutase, malondialdehyde), decreased pyroptosis-related proinflammatory cytokines (IL-1ß, IL-18), downregulated pyroptosis-related proteins (NOD-like receptor pyrin domain 3, cleaved caspase 1, GSDMD, GSDMD N-terminal), and activated the Nrf2/HO-1 pathway when compared with the CPR group. Conclusion: SFN produced effective postresuscitation lung protection through alleviating lung pyroptosis possibly via activating the Nrf2/HO-1 pathway in pigs.

[Protective role and mechanism of tubastatin A on renal and intestinal injuries after cardiopulmonary resuscitation in swine].

OBJECTIVE: To investigate the protective effect and potential mechanism of tubastatin A (TubA), a specific inhibitor of histone deacetylase 6 (HDAC6), on renal and intestinal injuries after cardiopulmonary resuscitation (CPR) in swine. METHODS: Twenty-five healthy male white swine were divided into Sham group (n = 6), CPR model group (n = 10) and TubA intervention group (n = 9) using a random number table. The porcine model of CPR was reproduced by 9-minute cardiac arrest induced by electrical stimulation via right ventricle followed by 6-minute CPR. The animals in the Sham group only underwent the regular operation including endotracheal intubation, catheterization, and anesthetic monitoring. At 5 minutes after successful resuscitation, a dose of 4.5 mg/kg of TubA was infused via the femoral vein within 1 hour in the TubA intervention group. The same volume of normal saline was infused in the Sham and CPR model groups. Venous samples were collected before modeling and 1, 2, 4, 24 hours after resuscitation, and the levels of serum creatinine (SCr), blood urea nitrogen (BUN), intestinal fatty acid binding protein (I-FABP) and diamine oxidase (DAO) in serum were determined by enzyme-linked immunoadsordent assay (ELISA). At 24 hours after resuscitation, the upper pole of left kidney and terminal ileum were harvested to detect cell apoptosis by TdT-mediated dUTP-biotin nick end labeling (TUNEL), and the expression levels of receptor-interacting protein 3 (RIP3) and mixed lineage kinase domain-like protein (MLKL) were detected by Western blotting. RESULTS: After resuscitation, renal dysfunction and intestinal mucous injury were observed in the CPR model and TubA intervention groups when compared with the Sham group, which was indicated by significantly increased levels of SCr, BUN, I-FABP and DAO in serum. However, the serum levels of SCr and DAO starting 1 hour after resuscitation, the serum levels of BUN starting 2 hours after resuscitation, and the serum levels of I-FABP starting 4 hours after resuscitation were significantly decreased in the TubA intervention group when compared with the CPR model group [1-hour SCr (µmol/L): 87±6 vs. 122±7, 1-hour DAO (kU/L): 8.1±1.2 vs. 10.3±0.8, 2-hour BUN (mmol/L): 12.3±1.2 vs. 14.7±1.3, 4-hour I-FABP (ng/L): 661±39 vs. 751±38, all P < 0.05]. The detection of tissue samples indicated that cell apoptosis and necroptosis in the kidney and intestine at 24 hours after resuscitation were significantly greater in the CPR model and TubA intervention groups when compared with the Sham group, which were indicated by significantly increased apoptotic index and markedly elevated expression levels of RIP3 and MLKL. Nevertheless, compared with the CPR model group, renal and intestinal apoptotic indexes at 24 hours after resuscitation in the TubA intervention group were significantly decreased [renal apoptosis index: (21.4±4.6)% vs. (55.2±9.5)%, intestinal apoptosis index: (21.3±4.5)% vs. (50.9±7.0)%, both P < 0.05], and the expression levels of RIP3 and MLKL were significantly reduced [renal tissue: RIP3 protein (RIP3/GAPDH) was 1.11±0.07 vs. 1.39±0.17, MLKL protein (MLKL/GAPDH) was 1.20±0.14 vs. 1.51±0.26; intestinal tissue: RIP3 protein (RIP3/GAPDH) was 1.24±0.18 vs. 1.69±0.28, MLKL protein (MLKL/GAPDH) was 1.38±0.15 vs. 1.80±0.26, all P < 0.05]. CONCLUSIONS: TubA has the protective effect on alleviating post-resuscitation renal dysfunction and intestinal mucous injury, and its mechanism may be related to inhibition of cell apoptosis and necroptosis.

A 3D-printed microneedle extraction system integrated with patterned electrodes for minimally invasive transdermal detection.

Point-of-Care-Testing (POCT) is a convenient and timely clinical analysis method, leading the development trend of advanced biosensors. The development of POCT equipment that can achieve minimally invasive percutaneous monitoring can avoid the pain felt by the subjects and achieve in vivo and efficient measurement. Here, we reported the development of a microneedle (MN) extraction system based on patterned electrodes, which could provide convenient and minimally invasive detection of bio-analytes (including glucose, pH, and H2O2). The 3D-printed hollow MN array was used as a painless transdermal tool, while the interstitial fluid was extracted under negative-pressure conditions. The patterned electrodes could improve the electrochemical performance of the sensor, with the synergistic effect of the micropillar structure to increase the enzyme coating surface area and the nanomaterial electron layer. The patterned electrodes were placed on the back of the MN arrays for electrochemical detection. In vitro and in vivo studies showed that the MN-extraction system could detect the corresponding bio-analytes in a minimally invasive manner and it did not cause significant tissue damage. The system developed in this work will provide promising technology to expand the application of POCT for minimal tests on interstitial fluids.

ANT(9)-Ic, a Novel Chromosomally Encoded Aminoglycoside Nucleotidyltransferase from Brucella intermedia.

Aminoglycoside-modifying enzymes are among the most important mechanisms of resistance to aminoglycoside antibiotics, typically conferring high-level resistance by enzymatic drug inactivation. Previously, we isolated a multidrug-resistant Brucella intermedia strain ZJ499 from a cancer patient, and whole-genome sequencing revealed several putative novel aminoglycoside-modifying enzyme genes in this strain. Here, we report the characterization of one of them that encodes an intrinsic, chromosomal aminoglycoside nucleotidyltransferase designated ANT(9)-Ic, which shares only 33.05% to 47.44% amino acid identity with the most closely related ANT(9)-I enzymes. When expressed in Escherichia coli, ANT(9)-Ic conferred resistance only to spectinomycin and not to any other aminoglycosides tested, indicating a substrate profile typical of ANT(9)-I enzymes. Consistent with this, deletion of ant(9)-Ic in ZJ499 resulted in a specific and significant decrease in MIC of spectinomycin. Furthermore, the purified ANT(9)-Ic protein showed stringent substrate specificity for spectinomycin with a Km value of 44.83 µM and a kcat/Km of 2.8 × 104 M-1 s-1, echoing the above observations of susceptibility testing. In addition, comparative genomic analysis revealed that the genetic context of ant(9)-Ic was conserved in Brucella, with no mobile genetic elements found within its 20-kb surrounding region. Overall, our results demonstrate that ANT(9)-Ic is a novel member of the ANT(9)-I lineage, contributing to the intrinsic spectinomycin resistance of ZJ499. IMPORTANCE The emergence, evolution, and worldwide spread of antibiotic resistance present a significant global public health crisis. For aminoglycoside antibiotics, enzymatic drug modification is the most common mechanism of resistance. We identify a novel chromosomal aminoglycoside nucleotidyltransferase from B. intermedia, called ANT(9)-Ic, which shares the highest identity (47.44%) with the previously known ANT(9)-Ia and plays an important role in spectinomycin resistance of the host strain. Analysis of the genetic environment and origin of ant(9)-Ic shows that the gene and its surrounding region are widely conserved in Brucella, and no mobile elements are detected, indicating that ANT(9)-Ic may be broadly important in the natural resistance to spectinomycin of Brucella species.

Corrigendum: The combination of chest compression synchronized ventilation and aortic balloon occlusion improve the outcomes of cardiopulmonary resuscitation in swine.

[This corrects the article DOI: 10.3389/fmed.2022.1057000.].

Class 1 integrons and multiple mobile genetic elements in clinical isolates of the Klebsiella pneumoniae complex from a tertiary hospital in eastern China.

Background: The emergence of highly drug-resistant K. pneumoniae, has become a major public health challenge. In this work, we aim to investigate the diversity of species and sequence types (STs) of clinical Klebsiella isolates and to characterize the prevalence and structure of class 1 integrons. Methods: Based on the whole genome sequencing, species identification was performed by 16S rRNA gene homology and average nucleotide identity (ANI) analysis. STs were determined in accordance with the international MLST schemes for K. pneumoniae and K. variicola. Integron characterization and comparative genomic analysis were performed using various bioinformatic tools. Results: Species identification showed that the 167 isolates belonged to four species: K. pneumoniae, K. variicola subsp. variicola, K. quasipneumoniae and K. aerogenes. Thirty-six known and 5 novel STs were identified in K. pneumoniae, and 10 novel STs were identified in K. variicola subsp. variicola. Class 1 integrons were found in 57.49% (96/167) of the isolates, and a total of 169 resistance gene cassettes encoding 19 types of resistance genes, including carbapenem resistance gene (bla IPM-4) and class D ß-lactamases gene (bla OXA-1 and bla OXA-10), were identified. Among the 17 complete genomes, 29 class 1 integrons from 12 groups were found, only 1 group was encoded on chromosomes. Interestingly, one plasmid (pKP167-261) carrying two copies of approximately 19-kb IS26-Int1 complex resistance region that contains an integron and a multidrug resistance gene fragment. Conclusion: The results of this work demonstrated that the species and STs of the clinical Klebsiella isolates were more complex by the whole genome sequence analysis than by the traditional laboratory methods. Finding of the new structure of MGEs related to the resistance genes indicates the great importance of deeply exploring the molecular mechanisms of bacterial multidrug resistance.

A Nanotherapy of Octanoic Acid Ameliorates Cardiac Arrest/Cardiopulmonary Resuscitation-Induced Brain Injury via RVG29- and Neutrophil Membrane-Mediated Injury Relay Targeting.

Treatment of cardiac arrest/cardiopulmonary resuscitation (CA/CPR)-induced brain injury remains a challenging issue without viable therapeutic options. Octanoic acid (OA), a lipid oil that is mainly metabolized in the astrocytes of the brain, is a promising treatment for this type of injury owing to its potential functions against oxidative stress, apoptosis, inflammation, and ability to stabilize mitochondria. However, the application of OA is strictly limited by its short half-life and low available concentration in the target organ. Herein, based on our previous research, an OA-based nanotherapy coated with a neutrophil membrane highly expressing RVG29, RVG29-H-NPOA, was successfully constructed by computer simulation-guided supramolecular assembly of polyethylenimine and OA. The in vitro and in vivo experiments showed that RVG29-H-NPOA could target and be distributed in the injured brain focus via the relay-targeted delivery mediated by RVG29-induced blood-brain barrier (BBB) penetration and neutrophil membrane protein-induced BBB binding and injury targeting. This results in enhancements of the antioxidant, antiapoptotic, mitochondrial stability-promoting and anti-inflammatory effects of OA and exhibited systematic alleviation of astrocyte injury, neuronal damage, and inflammatory response in the brain. Due to their systematic intervention in multiple pathological processes, RVG29-H-NPOA significantly increased the 24 h survival rate of CA/CPR model rats from 40% to 100% and significantly improved their neurological functions. Thus, RVG29-H-NPOA are expected to be a promising therapeutic for the treatment of CA/CPR-induced brain injury.

Characterization and identification of a novel chromosomal class C ß-lactamase, LAQ-1, and comparative genomic analysis of a multidrug resistance plasmid in Lelliottia amnigena P13.

Introduction: Lelliottia amnigena, a bacterium usually isolated from natural environments, may cause human infections and has been suggested to be naturally resistant to second- and third-generation cephalosporins. Methods: In this study, we determined the whole-genome sequence of an isolate, L. Amnigena P13, isolated from animal farm sewage. On the basis of genome sequence analysis, susceptibility testing, molecular cloning, and enzyme kinetic parameter analysis, we identified a novel chromosome-encoded AmpC ß-lactamase, LAQ-1. Results and Discussion: bla LAQ-1 is resistant to penicillin G, ampicillin, and several first- to fourth-generation cephalosporins, such as cefazolin, cefoxitin and cefepime. The MIC levels of some ß-lactams, such as cefoxitin, cefepime, aztreonam and cefazolin, for the recombinant clone (pUCP24-bla LAQ-1/DH5α) increased by approximately 4- to 64-fold compared with those of the control strain (pUCP24/DH5α). The kinetic properties of LAQ-1, with the highest catalytic activity observed toward piperacillin, were basically the same as those of typical class C ß-lactamases, and avibactam had a strong inhibitory effect on its hydrolytic activity. The genetic background of bla LAQ-1 was relatively conserved, and no mobile genetic element (MGE) was found around it. The plasmid pP13-67 of L. amnigena P13 harbored 12 resistance genes [qnrS1, aph(6)-Id, aadA2, sul1, sul2, bla TEM-1, qacEΔ1, dfrA12, tetA and floR] related to different mobile genetic elements within an ~22 kb multidrug resistance region. The multidrug resistance region shared the highest nucleotide sequence similarities with those of the chromosomes or plasmids of different bacterial species, indicating the possibility of horizontal transfer of these resistance genes among different bacterial species.

Inverse design of a dual-mode 3-dB optical power splitter with a 445†nm bandwidth.

Optical power splitters are fundamental blocks for photonic integrated circuits. Conventional 3-dB power splitters are either constrained to single-mode regime or to the limited optical bandwidth. In this paper, an alternative design approach is proposed via combined method of topology optimizations on both analog and digital meta-structure. Based on this approach, a dual-mode power splitter is designed on silicon-on-insulator with an ultra-broad bandwidth from 1588 nm - 2033nm and an ultra-compact footprint of only 5.4 µm × 2.88 µm. The minimum feature size is 120 nm which can be compatible with silicon photonic foundry process. The simulated excess loss and crosstalk over this wavelength range for the two lowest TE modes are lower than 0.83 dB and -22 dB, respectively. To the best of our knowledge, this is a record large optical bandwidth for an integrated dual-mode 3-dB power splitter on silicon.

ALDA-1 TREATMENT ALLEVIATES LUNG INJURY AFTER CARDIAC ARREST AND RESUSCITATION IN SWINE.

ABSTRACT: Introduction: Alda-1, an aldehyde dehydrogenase 2 (ALDH2) activator, has been shown to protect the lung against a variety of diseases including regional ischemia-reperfusion injury, severe hemorrhagic shock, hyperoxia, and so on. The present study was designed to investigate the effectiveness of Alda-1 treatment in alleviating lung injury after cardiac arrest (CA) and cardiopulmonary resuscitation (CPR) in swine. Methods: A total of 24 swine were randomized into three groups: sham (n = 6), CA/CPR (n = 10), and CA/CPR + Alda-1 (n = 8). The swine model was established by 8 min of electrically induced and untreated CA, and then 8 min of manual CPR. A dose of 0.88 mg/kg of Alda-1 was intravenously injected at 5 min after CA/CPR. After CA/CPR, extravascular lung water index (ELWI), pulmonary vascular permeability index (PVPI), and oxygenation index (OI) were regularly evaluated for 4 h. At 24 h after resuscitation, lung ALDH2 activity was detected, and its injury score, apoptosis, and ferroptosis were measured. Results: After experiencing the same procedure of CA and CPR, five swine in the CA/CPR group and six swine in the CA/CPR + Alda-1 group restored spontaneous circulation. Subsequently, significantly increased ELWI and PVPI, and markedly decreased OI were observed in these two groups compared with the sham group. However, all of them were gradually improved and significantly better in the swine treated with the Alda-1 compared with the CA/CPR group. Tissue analysis indicated that lung ALDH2 activity was significantly decreased in those swine experiencing the CA/CPR procedure compared with the sham group; nevertheless, its activity was significantly greater in the CA/CPR + Alda-1 group than in the CA/CPR group. In addition, lung injury score, and its apoptosis and ferroptosis were significantly increased in the CA/CPR and CA/CPR + Alda-1 groups compared with the sham group. Likewise, Alda-1 treatment significantly decreased these pathological damages in lung tissue when compared with the CA/CPR group. Conclusions: Alda-1 treatment was effective to alleviate lung injury after CA/CPR in a swine model, in which the protective role was possibly related to the inhibition of cell apoptosis and ferroptosis. It might provide a novel therapeutic target and a feasible therapeutic drug for lung protection after CA/CPR.

Embryonic stem cell-derived mesenchymal stem cells alleviate skeletal muscle injury induced by acute compartment syndrome.

BACKGROUND: Acute compartment syndrome (ACS), a well-known complication of musculoskeletal injury, results in muscle necrosis and cell death. Embryonic stem cell-derived mesenchymal stem cells (ESC-MSCs) have been shown to be a promising therapy for ACS. However, their effectiveness and potentially protective mechanism remain unknown. The present study was designed to investigate the efficacy and underlying mechanism of ESC-MSCs in ACS-induced skeletal muscle injury. METHOD: A total of 168 male Sprague-Dawley (SD) rats underwent 2 h of intracompartmental pressure elevation by saline infusion into the anterior compartment of the left hindlimb to establish the ACS model. ESC-MSCs were differentiated from the human embryonic stem cell (ESC) line H9. A dose of 1.2 × 106 of ESC-MSCs was intravenously injected during fasciotomy. Post-ACS assessments included skeletal edema index, serum indicators, histological analysis, apoptosis, fibrosis, regeneration, and functional recovery of skeletal muscle. Then, fluorescence microscopy was used to observe the distribution of labeled ESC-MSCs in vivo, and western blotting and immunofluorescence analyses were performed to examine macrophages infiltration in skeletal muscle. Finally, we used liposomal clodronate to deplete macrophages and reassess skeletal muscle injury in response to ESC-MSC therapy. RESULT: ESC-MSCs significantly reduced systemic inflammatory responses, ACS-induced skeletal muscle edema, and cell apoptosis. In addition, ESC-MSCs inhibited skeletal muscle fibrosis and increased regeneration and functional recovery of skeletal muscle after ACS. The beneficial effects of ESC-MSCs on ACS-induced skeletal muscle injury were accompanied by a decrease in CD86-positive M1 macrophage polarization and an increase in CD206-positive M2 macrophage polarization. After depleting macrophages with liposomal clodronate, the beneficial effects of ESC-MSCs were attenuated. CONCLUSION: Our findings suggest that embryonic stem cell-derived mesenchymal stem cells infusion could effectively alleviate ACS-induced skeletal muscle injury, in which the beneficial effects were related to the regulation of macrophages polarization.

Tubastatin A Improves Post-Resuscitation Myocardial Dysfunction by Inhibiting NLRP3-Mediated Pyroptosis Through Enhancing Transcription Factor EB Signaling.

Background Myocardial dysfunction is the leading cause of early death following successful cardiopulmonary resuscitation (CPR) in people with cardiac arrest (CA), which is potentially driven by cell pyroptosis mediated by NOD-like receptor pyrin domain 3 (NLRP3) inflammasome. Recently, histone deacetylase 6 (HDAC6) inhibition was shown to exert effective myocardial protection against regional ischemia/reperfusion injury. In this study, we investigated whether tubastatin A, a specific histone deacetylase 6 inhibitor, could improve postresuscitation myocardial dysfunction through the inhibition of NLRP3-mediated cell pyroptosis and its modulation mechanism. Methods and Results Healthy male white domestic swine were used to establish the model of CA/CPR in vivo, and the H9c2 cardiomyocyte hypoxia/reoxygenation model was used to simulate the CA/CPR process in vitro. Consequently, tubastatin A inhibited NLRP3 inflammasome activation, decreased proinflammatory cytokines production and cell pyroptosis, and increased cell survival after hypoxia/reoxygenation in H9c2 cardiomyocytes in vitro. In addition, tubastatin A increased the acetylated levels of transcription factor EB and its translocation to the nucleus, and its protective effect above was partly abrogated by transcription factor EB short interfering RNA after hypoxia/reoxygenation in H9c2 cardiomyocytes. Similarly, tubastatin A promoted cardiac transcription factor EB nuclear translocation, inhibited NLRP3-mediated cell pyroptosis, and mitigated myocardial dysfunction after CA/CPR in swine. Conclusions The inhibition of histone deacetylase 6 activity by tubastatin A limited NLRP3 inflammasome activation and cell pyroptosis probably through the enhancement of transcription factor EB signaling, and therefore improved myocardial dysfunction after CA/CPR.

Alda-1, an Activator of ALDH2, Improves Postresuscitation Cardiac and Neurological Outcomes by Inhibiting Pyroptosis in Swine.

Aldehyde dehydrogenase 2 (ALDH2) has been proven to protect the heart and brain against regional ischemia/reperfusion injury, in which the protective role is related to the inhibition of pyroptosis. In the present study, we investigated whether an ALDH2 activator N-(1,3-benzodioxol-5-ylmethyl)-2,6-dichloro-benzamide (Alda-1) would improve postresuscitation cardiac and neurological outcomes in a clinically relevant swine model of cardiac arrest (CA) and resuscitation. The animal model was established by 8 min of untreated ventricular fibrillation and then 8 min of cardiopulmonary resuscitation (CPR). After restoring spontaneous circulation, the animals were randomly divided to receive either Alda-1 (0.88 mg/kg, n = 6) or saline (n = 5). Postresuscitation hemodynamic parameters, cardiac function, and cardiac and cerebral injuries were periodically measured for a total of 24 h. At 24 h postresuscitation, neurological function was evaluated, and then the animals were sacrificed, and cardiac and cerebral tissue samples were obtained for the measurements of oxidative stress, inflammation and pyroptosis. Consequently, postresuscitation cardiac and neurological dysfunction were significantly improved accompanied with significantly milder cardiac and cerebral injuries in the Alda-1 group compared with the CPR group. In addition, the increase in NLR family pyrin domain-containing 3 inflammasome expression and proinflammatory cytokine production, which indicated the occurrence of inflammatory response, were significantly less in the Alda1 group than in the CPR group. The expression level of gasdermin D used as a protein marker of pyroptosis was also significantly reduced in all resuscitated animals receiving Alda1 treatment. Moreover, the severity of oxidative stress indicated by the changes of 4-hydroxy-2-nonenal and malondialdehyde was significantly decreased in the heart and brain in all animals treated with Alda-1 compared to the CPR group. Thus, Alda-1 mitigated postresuscitation cardiac and neurological dysfunction and injuries possibly by inhibiting oxidative stress-mediated NLRP3 inflammasome activation and pyroptosis in a swine model of CA and resuscitation.

A new method using surface landmarks to locate resuscitative endovascular balloon occlusion of the aorta based on a retrospective CTA study.

BACKGROUND: Resuscitative endovascular balloon occlusion of the aorta (REBOA) can timely prevent the wounded from fatal hemorrhage. However, blind insertion of REBOA in field or emergency room may result in catheter malposition and serious complications. We aim to develop a new method based on surface landmarks to guide the accurate placement of REBOA in zone III of aorta without fluoroscopy. METHODS: A retrospective study was conducted in a university hospital, including 57 subjects who underwent computed tomography angiography (CTA) from April to December in 2019. External distances and intravascular lengths were measured by three-dimensional reconstruction of CT images, including the distances from the insertion site of femoral artery to the xiphoid process (FA-Xi), the midpoint between the xiphoid process and the umbilicus (FA-mXU), the umbilicus (FA-Ui), the midpoint of the zone III of aorta (FA-mZIII), the lowest renal artery (FA-LRA), and aortic bifurcation (FA-AB). The distal and proximal ideal margin and predicted accuracy were calculated by curvature plane reconstruction. The predicted probability of balloon positioning in zone III by different methods was compared. RESULTS: The mean age of all patients was 60 years (SD = 9.4). The average length of zone III of aorta was 9.4 cm (SD = 1.0), and the length of FA-mZIII on the right and left sides were 24.4 cm (SD = 2.1), 23.8 cm (SD = 2.1), respectively. FA-Xi was longer than FA-LRA, and FA-Ui was shorter than FA-AB (paired two-tailed test, p < 0.001). Using three methods including the optimal quartering distances, the optimal distances below the xiphoid and above the umbilicus to predict the length of REBOA catheter positioning in zone III showed no statistically significant difference. The predicted accuracy of catheter positioning in zone III on the left and right sides guided by FA-mXU were 84.2% and 86%. CONCLUSIONS: The midpoint between the xiphoid process and the umbilicus may be a new surface landmark for people of normal weight to guide rapid positioning REBOA in zone III of aorta without fluoroscopy.

The combination of chest compression synchronized ventilation and aortic balloon occlusion improve the outcomes of cardiopulmonary resuscitation in swine.

Aim: The primary mission of cardiopulmonary resuscitation (CPR) is to provide adequate blood flow and oxygen delivery for restoring spontaneous circulation from cardiac arrest (CA) events. Previously, studies demonstrated that chest compression synchronized ventilation (CCSV) improved systemic oxygen supply during CPR, and aortic balloon occlusion (ABO) augments the efficacy of external CPR by increasing blood perfusion to vital organs. However, both them failed to make a significant improvement in return of spontaneous circulation (ROSC). In this study, we investigated the effects of combined CCSV and ABO on the outcomes of CPR in swine. Methods: Thirty-one male domestic swine were subjected to 8 min of electrically induced and untreated CA followed by 8 min of CPR. CPR was performed by continuous chest compressions and mechanical ventilation. At the beginning of CPR, the animals were randomized to receive intermittent positive pressure ventilation (IPPV, n = 10), CCSV (n = 7), IPPV + ABO (n = 7), or CCSV + ABO (n = 7). During CPR, gas exchange and systemic hemodynamics were measured, and ROSC was recorded. After resuscitation, the function and injury biomarkers of vital organs including heart, brain, kidney, and intestine were evaluated. Results: During CPR, PaO2 was significantly higher accompanied by significantly greater regional cerebral oxygen saturation in the CCSV and CCSV + ABO groups than the IPPV group. Coronary perfusion pressure, end-tidal carbon dioxide, and carotid blood flow were significantly increased in the IPPV + ABO and CCSV + ABO groups compared with the IPPV group. ROSC was achieved in five of ten (IPPV), five of seven (CCSV), six of seven (IPPV + ABO), and seven of seven (CCSV + ABO) swine, with the rate of resuscitation success being significantly higher in the CCSV + ABO group than the IPPV group (P = 0.044). After resuscitation, significantly improved myocardial and neurological function, and markedly less cardiac, cerebral, renal, and intestinal injuries were observed in the CCSV + ABO group compared with the IPPV group. Conclusion: The combination of CCSV and ABO improved both ventilatory and hemodynamic efficacy during CPR, promoted ROSC, and alleviated post-resuscitation multiple organ injury in swine.

Mesenchymal Stem Cells Alleviate Post-resuscitation Cardiac and Cerebral Injuries by Inhibiting Cell Pyroptosis and Ferroptosis in a Swine Model of Cardiac Arrest.

Following cardiopulmonary resuscitation (CPR), the ensuing cardiac and cerebral injuries contribute to the poor outcome of cardiac arrest (CA) victims, in which the pathogenetic process is possibly driven by cell pyroptosis and ferroptosis. Mesenchymal stem cells (MSCs) have been shown to be a promising strategy for post-resuscitation cardiac and cerebral protection in rat, but its effectiveness in the clinically relevant swine model and the potential protective mechanism remain unknown. The present study was designed to investigate whether MSCs administration could alleviate post-resuscitation cardiac and cerebral injuries through the inhibition of cell pyroptosis and ferroptosis in swine. Twenty-four male domestic swine were randomly divided into three groups: sham, CPR, and MSC. A dose of 2.5×106/kg of MSCs derived from human embryonic stem cells was intravenously infused at 1.5, and 3 days prior to CA. The animal model was established by 8 min of CA and then 8 min of CPR. After resuscitation, cardiac, cerebral function and injury biomarkers were regularly evaluated for a total of 24 h. At 24 h post-resuscitation, pyroptosis-related proteins (NLRP3, ASC, cleaved caspase-1, GSDMD), proinflammatory cytokines (IL-1ß, IL-18), ferroptosis-related proteins (ACSL4, GPX4) and iron deposition in the heart, cortex and hippocampus were measured. Consequently, significantly greater cardiac, cerebral dysfunction and injuries after resuscitation were observed in the CPR and MSC groups compared with the sham group. However, the severity of cardiac and cerebral damage were significantly milder in the MSC group than in the CPR group. In addition, the expression levels of NLRP3, ASC, cleaved caspase-1, GSDMD and ACSL4, the contents of IL-1ß and IL-18, and the level of iron deposition were significantly higher while the expression level of GPX4 was significantly lower in the heart, cortex and hippocampus in all resuscitated animals compared with the sham group. Nevertheless, MSCs administration significantly decreased post-resuscitation cardiac, cerebral pyroptosis and ferroptosis compared to the CPR group. Our results showed that the administration of MSCs significantly alleviated post-resuscitation cardiac and cerebral injuries in swine, in which the protective effects were related to the inhibition of cell pyroptosis and ferroptosis.