A two-hit model of sepsis plus hyperoxia causes lung permeability and inflammation

Abstract

Mouse models of acute lung injury (ALI) have been instrumental for studies of the biologic underpinnings of lung inflammation and permeability, but murine models of sepsis generate minimal lung injury. Our goal was to create a murine sepsis model of ALI that reflects the inflammation, lung edema, histologic abnormalities and physiologic dysfunction that characterize ALI. Using a cecal slurry (CS) model of polymicrobial abdominal sepsis and exposure to hyperoxia (95%), we systematically varied timing and dose of the CS injection, fluids and antibiotics and dose of hyperoxia. We found that CS alone had a high mortality rate that was improved with the addition of antibiotics and fluids. Despite this, we did not see evidence of ALI as measured by bronchoalveolar lavage (BAL) cell count, total protein, CXCL-1 or by lung wet:dry weight ratio. Addition of hyperoxia (95% FiO2) to CS immediately after CS injection increased BAL cell counts and CXCL-1 and lung wet:dry weight ratio but was associated with 40% mortality. Splitting the hyperoxia treatment into two 12 hour exposures (0-12 hours24-36 hours) after CS injection increased survival to 75% and caused significant lung injury compared to CS alone as measured by increased BAL total cell count (92500 vs 240000, p=0.0004), BAL protein (71 vs 103 ug/ml, p=0.0030, and lung wet:dry weight ratio (4.5 vs 5.5 p=0.0005), and compared to sham as measured by increased BAL CXCL-1 (20 vs 2372 pg/ml, p<0.0001), and histologic lung injury score (1.9 vs 4.2, p=0.0077). Additionally, our final model showed evidence of lung epithelial (increased BAL and plasma RAGE) and endothelial (increased Syndecan-1 and sulfated glycosaminoglycans) injury. In conclusion, we have developed a clinically relevant mouse model of sepsis-induced ALI using IP injection of CS, antibiotics and fluids, and hyperoxia. This clinically relevant model can be used for future studies of sepsis-induced ALI.

Blocking P2X7 receptor with AZ 10606120 exacerbates vascular hyperpermeability and inflammation in murine polymicrobial sepsis

“…Of note, only one animal in the CS group did not survive during this study. As in our previous studies (Bastarache et al, 2021 ; Meegan et al, 2020 ), mortality is not increased at 24 h in this model; therefore, a limitation of this study is that it does not address the effects of AZ106 administration on mortality, an important area for future study.…”

“…Of note, only one animal in the CS group did not survive during this study. As in our previous studies (Bastarache et al, 2021;Meegan et al, 2020), mortality is not increased at 24 h in this model; therefore, a limitation of this study is that it does not address the effects of AZ106 administration on mortality, an important area for future study. Though several groups have shown benefits of P2X7R blockade in sepsis using P2X7R null mice in the cecal ligation and puncture (CLP) model (Santana et al, 2015;Savio et al, 2017), or P2X7R antagonists in a mouse CLP model (Wu et al, 2017) or rat fecal slurry model (Arulkumaran et al, 2018), our mouse model of cecal slurry-induced sepsis and the use of a different antagonist compound may have contributed to the differences in our results of P2X7R antagonism compared to other reports.…”

“…First, injections of CS are more consistent and reproducible than surgical models such as CLP and avoid the need for survival surgery. CS injections can also be titrated to induce desired illness severity, and the CS model produces organ-specific vascular hyperpermeability and clinically relevant end-organ dysfunction, as we and others have previously demonstrated (Bastarache et al, 2021;Meegan et al, 2020;Wynn et al, 2007Wynn et al, , 2008. Regarding the use of AZ106 over other P2X7R inhibition strategies, we opted for a pharmacological antagonist over a knockout mouse model to enhance the potential for therapeutic translatability.…”

Advances in Rodent Experimental Models of Sepsis

“…In a recent study, to test the effect of resolvin D2 at different stages of sepsis, a two-hit model was employed-an initial CLP surgery followed by a secondary lung infection with Pseudomonas aeruginosa-and it was found that resolvin D2 promoted mechanisms of host defense [84]. To establish a murine model of nonpulmonary sepsis, researchers used CS as the initial insult followed by hyperoxia [85]. In another study, mice subjected to CLP surgery coupled with a secondary P. aeurginosa treatment showed increased susceptibility to infection after the initial sepsis event [86].…”

Inflammatory Responses to Polymicrobial Intra-Abdominal Sepsis Are Highly Variable but Strongly Correlated to Enterobacteriaceae Outgrowth

“…However, the composition of cecal slurries derived for use in animal modeling will reflect the profound differences in the murine gut microbiome among individuals, vendors, and shipments (17)(18)(19)(20) (much like those differences seen in humans) (5). Although these differences can be controlled by using a single preparation for a given set of experiments, the degree to which these preparations vary in biological effect and impact generalizability is unknown (12,21). Furthermore, focusing on a particular preparation and set of microbiota limits the potential to study the role of pathogens in the intrinsic heterogeneity of human sepsis.…”