An ex vivo porcine spleen perfusion as a model of bacterial sepsis

Abstract

An ex vivo, porcine spleen perfusion model was established to study the early events occurring in the spleen prior to the onset of bacterial sepsis, using organs retrieved from animals slaughtered for food production. Porcine spleens were harvested from adult pigs and connected to a normothermic extracorporeal perfusion circuit. A constant perfusion of heparinized blood was performed for 6 hours. After injection of Streptococcus pneumoniae to the circuit serial samples of both blood and spleen biopsies were collected and analysed. Functionality of the perfused organs was assessed by monitoring the blood-gas parameters, flow rate and filtering capability of the organ. Interestingly, we observed full clearance of bacteria from the blood and an increase in bacterial counts in the spleen. Classical histology and immunohistochemistry on biopsies also confirmed no major damages in the organ architecture and changes in the immune cell distribution, other than the presence of clusters of pneumococci. A time-course study confirmed that each focus of infection derived from the replication of single pneumococcal cells within splenic macrophages. The model proposed - in line with the 3Rs principles - has utility in the replacement of experimental animals in infection research. Murine models are prevalently used to study pneumococcal infections, but are often not predictive for humans due to substantial differences in the immune systems of the two species. This model is designed to overcome these limitations, since porcine immunology and splenic architecture in particular, closely resemble those of humans.

Splenic macrophages as the source of bacteraemia during pneumococcal pneumonia

“…Normothermic ex vivo human spleen perfusion was performed as previously described [10] . Following spleen removal, the splenic artery was cannulated and flushed with Soltran preservative solution (Baxter, Deerfield, IL, USA) and transported to the lab on ice.…”

“…For human spleen perfusion experiments, we determined samples sizes using pilot data from our published, porcine spleen perfusion model [ 9 , 10 ]. Briefly, for a statistical power of 80% and a significance level of 0.05, and a predicted effect size of a 20% increase or decrease in CFU/G in the tissue, a sample size of 4 organs was calculated.…”

“…Administration of an anti-CD169 monoclonal antibody completely prevented disease in mice, as did macrolides but not beta-lactams [9] . We demonstrated that in porcine spleens, which have a microarchitecture similar to the human organ, pneumococci replicated in CD169+ macrophages during ex vivo perfusion [ 9 , 10 ]. Involvement of CD169+ macrophages provided an explanation for the efficacy of macrolides in preventing late onset sepsis because they, unlike beta-lactams, accumulate within macrophages [11] .…”

“…Direct extrapolation of our previous findings to human disease management is confounded because the architecture of the human spleen displays significant differences to that of other animals [ 10 , 12 ]. In particular, the human spleen contains three types of tissue macrophages including the red pulp macrophages (CD68+CD163+CD169-) sheath and follicle associated macrophages (CD68+CD163-CD169-), and the peri-arteriolar sheath macrophages (CD68+CD163-CD169++) [ 10 , 12 ]. To address this issue, we obtained evidence from a combination of ex vivo human spleen infections, a non-human primate and murine pneumonia models [13] , on the role of the spleen in IPD.…”

Procurement and ex-situ perfusion of isolated slaughterhouse-derived livers as a model of donors after circulatory death

“…Consequently, technical refinement of critical steps (organ procurement, blood collection, and perfusion system) was pursued. Particular attention was paid to organ iel et al, 2018;Chung et al, 2019). Unfortunately, among the many studies on DCD and machine perfusion published over the last 20 years, only few have used non-dedicated experimental animals (Grosse-Siestrup et al, 2001, 2002a.…”