Early Influences of Microbiota on White Matter Development in Germ-Free Piglets

Abnormalities in the prefrontal cortex (PFC), as well as the underlying white matter (WM) tracts, lie at the intersection of many neurodevelopmental disorders. The influence of microorganisms on brain development has recently been brought into the clinical and research spotlight as alterations in commensal microbiota are implicated in such disorders, including autism spectrum disorders, schizophrenia, depression, and anxiety via the gut-brain axis. In addition, gut dysbiosis is common in preterm birth patients who often display diffuse WM injury and delayed WM maturation in critical tracts including those within the PFC and corpus callosum. Microbial colonization of the gut aligns with ongoing postnatal processes of oligodendrogenesis and the peak of brain myelination in humans; however, the influence of microbiota on gyral WM development remains elusive. Here, we develop and validate a neonatal germ-free swine model to address these issues, as piglets share key similarities in WM volume, developmental trajectories, and distribution to humans. We find significant region-specific reductions, and sexually dimorphic trends, in WM volume, oligodendrogenesis, and mature oligodendrocyte numbers in germ-free piglets during a key postnatal epoch of myelination. Our findings indicate that microbiota plays a critical role in promoting WM development during early life when the brain is vulnerable to environmental insults that can result in an array of disabilities manifesting later in life.

Regeneration of Subcutaneous Cartilage in a Swine Model Using Autologous Auricular Chondrocytes and Electrospun Nanofiber Membranes Under Conditions of Varying Gelatin/PCL Ratios

The scarcity of ideal biocompatible scaffolds makes the regeneration of cartilage in the subcutaneous environment of large animals difficult. We have previously reported the successful regeneration of good-quality cartilage in a nude mouse model using the electrospun gelatin/polycaprolactone (GT/PCL) nanofiber membranes. The GT/PCL ratios were varied to generate different sets of membranes to conduct the experiments. However, it is unknown whether these GT/PCL membranes can support the process of cartilage regeneration in an immunocompetent large animal model. We seeded swine auricular chondrocytes onto different GT/PCL nanofiber membranes (GT:PCL = 30:70, 50:50, and 70:30) under the sandwich cell-seeding mode. Prior to subcutaneously implanting the samples into an autologous host, they were cultured in vitro over a period of 2 weeks. The results revealed that the nanofiber membranes with different GT/PCL ratios could support the process of subcutaneous cartilage regeneration in an autologous swine model. The maximum extent of homogeneity in the cartilage tissues was achieved when the G5P5 (GT: PC = 50: 50) group was used for the regeneration of cartilage. The formed homogeneous cartilage tissues were characterized by the maximum cartilage formation ratio. The extents of the ingrowth of the fibrous tissues realized and the extents of infiltration of inflammatory cells achieved were found to be the minimum in this case. Quantitative analyses were conducted to determine the wet weight, cartilage-specific extracellular matrix content, and Young’s modulus. The results indicated that the optimal extent of cartilage formation was observed in the G5P5 group. These results indicated that the GT/PCL nanofiber membranes could serve as a potential scaffold for supporting subcutaneous cartilage regeneration under clinical settings. An optimum GT/PCL ratio can promote cartilage formation.

Transient Activation of Hedgehog Signaling Inhibits Cellular Senescence and Inflammation in Radiated Swine Salivary Glands through Preserving Resident Macrophages

Salivary gland function is commonly and irreversibly damaged by radiation therapy for head and neck cancer. This damage greatly decreases the patient’s quality of life and is difficult to remedy. Previously, we found that the transient activation of Hedgehog signaling alleviated salivary hypofunction after radiation in both mouse and pig models through the inhibition of radiation-induced cellular senescence that is mediated by resident macrophages in mouse submandibular glands. Here we report that in swine parotid glands sharing many features with humans, the Hedgehog receptor PTCH1 is mainly expressed in macrophages, and levels of PTCH1 and multiple macrophage markers are significantly decreased by radiation but recovered by transient Hedgehog activation. These parotid macrophages mainly express the M2 macrophage marker ARG1, while radiation promotes expression of pro-inflammatory cytokine that is reversed by transient Hedgehog activation. Hedgehog activation likely preserves parotid macrophages after radiation through inhibition of P53 signaling and consequent cellular senescence. Consistently, VEGF, an essential anti-senescence cytokine downstream of Hedgehog signaling, is significantly decreased by radiation but recovered by transient Hedgehog activation. These findings indicate that in the clinically-relevant swine model, transient Hedgehog activation restores the function of irradiated salivary glands through the recovery of resident macrophages and the consequent inhibition of cellular senescence and inflammation.

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-1β, 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-1β 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.

Genome-wide expression of the residual lung reacting to experimental Pneumonectomy

Background Acute or chronic irreversible respiratory failure may occur in patients undergoing pneumonectomy. Aim of this study was to determine transcriptome expression changes after experimental pneumonectomy in swine model. Experimental left pneumonectomy was performed in five pigs under general anaesthesia. Both the resected and the remaining lung, after 60 post-operative completely uneventful days, underwent genome-wide bulk RNA-Sequencing (RNA-Seq). Results Histological analysis showed dilation of air spaces and rupture of interalveolar septa. In addition, mild inflammation, no fibrosis, radial stretch of the bronchus, strong enlargement of airspaces and thinning of the blood supply were observed. Bioinformatic analyses of bulk RNA-Seq data identified 553 Differentially Expressed Genes (DEGs) at adjusted P-value below 0.001, between pre- and post-pneumonectomy. The top 10 up-regulated DEGs were Edn1, Areg, Havcr2, Gadd45g, Depp1, Cldn4, Atf3, Myc, Gadd45b, Socs3; the top 10 down-regulated DEGs were Obscn, Cdkn2b, ENSSSCG00000015738, Prrt2, Amer1, Flrt3, Efnb2, Tox3, Znf793, Znf365. Leveraging digital cytometry tools, no difference in cellular abundance was found between the two experimental groups, while the analysis of cell type-specific gene expression patterns highlighted a striking predominance of macrophage-specific genes among the DEGs. DAVID-based gene ontology analysis showed a significant enrichment of “Extrinsic apoptotic signaling pathway” (FDR q = 7.60 × 10− 3) and “Response to insulin” (FDR q = 7.60 × 10− 3) genes, along with an enrichment of genes involved as “Negative regulators of DDX58/IFIH1 signaling” (FDR q = 7.50 × 10− 4) found by querying the REACTOME pathway database. Gene network analyses indicated a general dysregulation of gene inter-connections. Conclusion This translational genomics study highlighted the existence both of individual genes, mostly dysregulated in certain cellular populations (e.g., macrophages), and gene-networks involved in pulmonary reaction after left pneumonectomy. Their involvement in lung homeostasis is largely supported by previous studies, carried out both in humans and in other animal models (under homeostatic or disease-related conditions), that adopted candidate-gene approaches. Overall, the present findings represent a preliminary assessment for future, more focused, studies on compensatory lung adaptation, pulmonary regeneration and functional reload.

545 Cardiac protection by pirfenidone after myocardial infarction: a bioinformatic analysis

Aims Left ventricular (LV) remodelling after myocardial infarction (MI) is promoted by an intense fibrotic response, which could be targeted by an anti-fibrotic drug such as pirfenidone. Methods and results We explored the relationship between protein modulation by pirfenidone and post-MI remodelling, based on publicly available molecular information and transcriptomic data from a swine model of MI. We also compared the effects of pirfenidone and angiotensin-converting enzyme inhibitors/angiotensin receptor blockers (ACEi/ARB), mineralocorticoid receptor blockers (MRA) and beta-blockers. We identified six causative motives of post-MI remodelling (cardiomyocyte cell death, impaired myocyte contractility, extracellular matrix remodelling and fibrosis, hypertrophy, renin–angiotensin–aldosterone system activation, and inflammation), 4 pirfenidone targets and 21 bioflags (indirect effectors). When considering both targets and bioflags, pirfenidone showed a broad relationship encompassing all six motives. p38γ-MAPK12 blockade inhibits cardiomyocyte apoptosis, cardiomyocyte hypertrophy and inflammation. Furthermore, pirfenidone can modulate extracellular matrix remodelling and cardiac fibrosis by targeting the TGFβ1-SMAD2/3 pathway and other effector proteins such as matrix metalloproteases 2 and 14, PDGFA/B, and IGF1, which promote myocardial fibrosis, cardiomyocyte hypertrophy and impaired contractility. All the gold standard drugs were found to be important for specific clinical motives, but pirfenidone had a more widespread action on the molecular pathways active in the post-MI setting. Conclusions A bioinformatic approach allowed to identify several possible mechanisms of action of pirfenidone with beneficial effects in the post-MI LV remodelling, and suggests additional effects over guideline-recommended therapies. These findings support clinical studies evaluating the beneficial effects of pirfenidone in patients with MI.

Intramuscular uptake of tranexamic acid during haemorrhagic shock in a swine model

Background Tranexamic acid (TXA) reduce mortality in bleeding trauma patients, with greater effect if administered early. Serum concentrations above 10 µg/mL are considered sufficient to inhibit fibrinolysis. Normally administered intravenously (i.v.), TXA can also be administered intramuscularly (i.m.). This could be advantageous in low resource and military settings, if sufficient serum concentrations can be reached in shocked patients with reduced muscular blood perfusion. Accordingly, we aimed to: (1) Determine the impact of shock on the pharmacokinetics of i.m. TXA, and (2) Compare the pharmacokinetics of i.v. versus i.m. TXA in ongoing shock. Materials and methods In a prospective experimental study, N = 18 Norwegian landrace pigs (40–50 kg), utilised in a surgical course in haemostatic emergency surgery, were subjected to various abdominal and thoracic trauma. After 1 h of surgery the animals were given 15 mg/kg TXA either i.v. or i.m. A control group without injury, or surgery, received intramuscular TXA. Blood samples were drawn at 0, 5, 15, 25, 35, 45, 60 and 85 min. The samples were centrifuged and analysed with liquid chromatography–tandem mass spectrometry (LC–MS/MS) for TXA serum-concentrations. Results In shocked pigs, i.m. administration resulted in a mean maximum serum concentration (Cmax) of 20.9 µg/mL, and i.v. administration a Cmax of 48.1 µg/mL. Cmax occurred 15 min after i.m. administration and 5 min after i.v. administration. In non-shocked swine, i.m. administration resulted in a Cmax of 36.9 µg/mL after 15 min. In all groups, mean TXA serum concentrations stayed above 10 µg/mL from administration to end of experiments. Conclusions I.m. administration of TXA in shocked pigs provides serum concentrations associated with inhibition of fibrinolysis. It may be an alternative to i.v. and intraosseous administration during stabilisation and transport of trauma patients to advanced medical care.