Hypothermic Machine Preservation in Liver Transplantation
Hypothermic Machine Preservation in Liver Transplantation
Hypothermic machine perfusion (HMP) is in its infancy in clinical liver transplantation. Potential benefits include diminished preservation injury (PI) and improved graft function. Molecular data to date has been limited to extrapolation of animal studies. We analyzed liver tissue and serum collected during our Phase 1 trial of liver HMP. Grafts preserved with HMP were compared to static cold stored (SCS) transplant controls. Reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry and transmission electron microscopy (TEM) were performed on liver biopsies. Expression of inflammatory cytokines, adhesion molecules and chemokines, oxidation markers, apoptosis and acute phase proteins and the levels of CD68 positive macrophages in tissue sections were evaluated. RT-PCR of reperfusion biopsy samples in the SCS group showed high expression of inflammatory cytokines, adhesion molecules and chemokines, oxidative markers and acute phase proteins. This upregulation was significantly attenuated in livers that were preserved by HMP. Immunofluorescence showed larger numbers of CD68 positive macrophages in the SCS group when compared to the HMP group. TEM samples also revealed ultrastructural damage in the SCS group that was not seen in the HMP group. HMP significantly reduced proinflammatory cytokine expression, relieving the downstream activation of adhesion molecules and migration of leukocytes, including neutrophils and macrophages when compared to SCS controls.
Orthotopic liver transplantation (OLT) remains the most effective therapy for end-stage liver disease and is limited only by a persistent shortage of suitable donor organs. This has prompted the increased use of extended criteria donor (ECD) livers, which exceed traditional limits for steatosis and/or donor age, or are procured via donation after cardiac death (DCD). These livers are more likely to have early allograft dysfunction (EAD) or primary nonfunction (PNF) posttransplant due sensitivity to preservation-based ischemia/reperfusion injury (IRI). Static cold storage (SCS), currently the only technique in clinical use for liver preservation, has been unchanged for two decades. As the need to access ECD livers increases, a demand for more effective preservation becomes a necessity.
Hypothermic machine perfusion (HMP) has recently seen a renewal of interest within the field of kidney transplantation, with a major multicenter clinical trial showing benefits of HMP over SCS. With the success of HMP within the kidney space, transplant surgeons have begun to explore the viability of this technique for liver transplantation. We have recently completed a phase 1 clinical trial with standard criteria donor (SCD) livers that resulted in decreased EAD, less biliary complications, lower serum injury markers and shorter hospital stays in the HMP group. A range of risk factors (donor age, graft quality, steatosis, warm ischemia time [WIT], cold ischemia time [CIT] and virology) can affect patient outcomes as well as the potential development of preservation injury (PI) leading to EAD or PNF. Though our data suggest that HMP results in better clinical outcomes, what remains unclear is the exact mechanisms behind this phenomenon. During the course of this trial, we determined levels of specific inflammatory markers within a small subset of patients and found significant differences in the expression of markers such as TNF-α, IL-8 and MCP-1. This preliminary analysis has generated further interest in other molecular mechanisms possibly involved in the clinical improvements seen after HMP; namely, chemotaxis/adhesion, acute-phase protein induction and oxidative damage.
We have recently completed the first clinical trial with the use of HMP for preservation of livers with excellent results. Therefore, we sought to characterize the tissue expression of common and uncommon cytokines that become activated in inflammatory, chemotactic and oxygen-mediated pathways during the time course of liver preservation to explore the molecular mechanistic benefits of HMP in human liver transplantation.
Abstract and Introduction
Abstract
Hypothermic machine perfusion (HMP) is in its infancy in clinical liver transplantation. Potential benefits include diminished preservation injury (PI) and improved graft function. Molecular data to date has been limited to extrapolation of animal studies. We analyzed liver tissue and serum collected during our Phase 1 trial of liver HMP. Grafts preserved with HMP were compared to static cold stored (SCS) transplant controls. Reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry and transmission electron microscopy (TEM) were performed on liver biopsies. Expression of inflammatory cytokines, adhesion molecules and chemokines, oxidation markers, apoptosis and acute phase proteins and the levels of CD68 positive macrophages in tissue sections were evaluated. RT-PCR of reperfusion biopsy samples in the SCS group showed high expression of inflammatory cytokines, adhesion molecules and chemokines, oxidative markers and acute phase proteins. This upregulation was significantly attenuated in livers that were preserved by HMP. Immunofluorescence showed larger numbers of CD68 positive macrophages in the SCS group when compared to the HMP group. TEM samples also revealed ultrastructural damage in the SCS group that was not seen in the HMP group. HMP significantly reduced proinflammatory cytokine expression, relieving the downstream activation of adhesion molecules and migration of leukocytes, including neutrophils and macrophages when compared to SCS controls.
Introduction
Orthotopic liver transplantation (OLT) remains the most effective therapy for end-stage liver disease and is limited only by a persistent shortage of suitable donor organs. This has prompted the increased use of extended criteria donor (ECD) livers, which exceed traditional limits for steatosis and/or donor age, or are procured via donation after cardiac death (DCD). These livers are more likely to have early allograft dysfunction (EAD) or primary nonfunction (PNF) posttransplant due sensitivity to preservation-based ischemia/reperfusion injury (IRI). Static cold storage (SCS), currently the only technique in clinical use for liver preservation, has been unchanged for two decades. As the need to access ECD livers increases, a demand for more effective preservation becomes a necessity.
Hypothermic machine perfusion (HMP) has recently seen a renewal of interest within the field of kidney transplantation, with a major multicenter clinical trial showing benefits of HMP over SCS. With the success of HMP within the kidney space, transplant surgeons have begun to explore the viability of this technique for liver transplantation. We have recently completed a phase 1 clinical trial with standard criteria donor (SCD) livers that resulted in decreased EAD, less biliary complications, lower serum injury markers and shorter hospital stays in the HMP group. A range of risk factors (donor age, graft quality, steatosis, warm ischemia time [WIT], cold ischemia time [CIT] and virology) can affect patient outcomes as well as the potential development of preservation injury (PI) leading to EAD or PNF. Though our data suggest that HMP results in better clinical outcomes, what remains unclear is the exact mechanisms behind this phenomenon. During the course of this trial, we determined levels of specific inflammatory markers within a small subset of patients and found significant differences in the expression of markers such as TNF-α, IL-8 and MCP-1. This preliminary analysis has generated further interest in other molecular mechanisms possibly involved in the clinical improvements seen after HMP; namely, chemotaxis/adhesion, acute-phase protein induction and oxidative damage.
We have recently completed the first clinical trial with the use of HMP for preservation of livers with excellent results. Therefore, we sought to characterize the tissue expression of common and uncommon cytokines that become activated in inflammatory, chemotactic and oxygen-mediated pathways during the time course of liver preservation to explore the molecular mechanistic benefits of HMP in human liver transplantation.
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