Ischemic Preconditioning of Liver in Cadaver Donors
Condition: Liver Cirrhosis; Liver Disease
MeSH terms: Liver Diseases; Liver Cirrhosis; Cadaver
Study type: Interventional

Summary: 
      The long-term goals of this proposal are to develop clinical protocols of donor
      preconditioning to improve liver graft function and ameliorate complications of poor graft
      function after liver transplantation. Achievement of these objectives would improve liver
      recipient outcomes, increase utilization of livers and alleviate the current critical
      shortage of livers for transplantation. More stringent liver donor selection intended to
      decrease the complications of poor graft function conflicts directly with efforts to maximize
      the use of donor livers. Ischemic preconditioning (IPC) of liver attenuates hepatic ischemia
      reperfusion injury (IRI) in animals. Preliminary data show hepatic IPC effectively decreases
      IRI following hepatic resection in humans.

      The specific aims of this project are: AIM 1: To test the hypothesis that 10 minutes of
      hepatic ischemic preconditioning in deceased donors would improve liver graft function and
      decrease injury in the early post transplant period. AIM 2: To test the hypothesis that
      ischemic preconditioning of deceased donor livers would decrease systemic inflammatory
      response in liver recipients in the early post transplant period. AIM 3: To examine whether
      ischemic preconditioning of deceased donor livers decreases early post transplant pulmonary
      edema and acute rejection and shortens hospital stay.
    

Detailed description: 
      Specific Aims

        1. To test the hypothesis that 10 minutes of hepatic ischemic preconditioning in deceased
           donors would improve liver graft function and decrease injury in the early post
           transplant period.

           To accomplish this aim, we will compare International Normalized Ratios of prothrombin
           time (INR/PT) and serum aspartate (AST) and alanine aminotransferase (ALT) and total
           bilirubin (TB) levels immediately post transplant and on day 1, 3, 7, 14 and 30 and
           injury score of reperfusion liver biopsies in recipients of livers from IPC and No IPC
           donors.

        2. To test the hypothesis that ischemic preconditioning of deceased donor livers would
           decrease the systemic inflammatory response in liver recipients in the early post
           transplant period.

           To accomplish this aim, we will compare plasma levels of cytokines tumor necrosis
           factor-alpha (TNF-alpha), interleukins-6, 8 and 10 (IL-6, 8 and 10) and soluble
           L-selectin, expression levels of adhesion molecules (CD11beta/CD18 and L-selectin) and
           oxidative burst of neutrophils, platelet P-selectin and platelet-neutrophil complexes
           (PNC) in the peripheral blood 3, 12, 24 and 48 hours following reperfusion in recipients
           of livers from IPC and No IPC donors.

        3. To examine whether ischemic preconditioning of deceased donor livers decreases early
           post transplant pulmonary edema and acute rejection and shortens hospital stay.

      To accomplish this aim, we will compare interstitial and alveolar edema in chest radiographs
      done after transplant and on post operative days 1, 2 and 3; biopsy confirmed acute rejection
      within 30 days post transplant, and number of days to discharge after transplantation in
      recipients of livers from IPC and No IPC donors.

      Research Design A prospective, randomized, and single masked (liver recipients) study will be
      conducted in one liver transplant center over a period of two years. Deceased liver donors
      will be randomized in equal proportions to one of two organ recovery procedures: 1). IPC or
      2). No IPC. Allocation will be stratified by age < 50 or age >= 50 to facilitate examination
      of the effects of age on IPC. As the trial will enroll donors over a two-year period,
      treatment assignments will be balanced over blocks of length 12, 16 and 20, where the length
      will be selected at random, to control for time varying factors. Recovered livers are
      transplanted into recipients > 18 years of age. All other aspects of donor management, organ
      recovery and preservation, recipient selection, graft implantation and post transplant
      management including immunosuppression will be according to standard practice.

      II. Research Methods

        1. Liver Recovery and IPC: Donors are positioned supine, mechanically ventilated and
           administered 100% oxygen and a muscle relaxant. The abdomen and chest are opened in the
           midline. Briefly, round and falciform ligaments are divided and the gallbladder is
           incised and irrigated. The gastroduodenal artery is ligated and junction of splenic and
           superior mesenteric veins or, when pancreas is procured, the inferior mesenteric vein is
           prepared for canulation. The supraceliac aorta is exposed just below the diaphragm. The
           infrarenal abdominal aorta is isolated for canulation. No mobilization of the kidneys is
           performed before organ perfusion. Thoracic organs are mobilized either simultaneously or
           before mobilization of abdominal organs. Five hundred units/kg of heparin is given
           intravenously and ascending thoracic and infrarenal abdominal aorta are canulated.
           Another canula is inserted either into splenic vein or inferior mesenteric vein. The
           supraceliac abdominal aorta is clamped and the inferior vena cava is vented in the
           pericardium. UW solution (40C) is infused into abdominal aorta and portal vein (2 liters
           each). The abdominal cavity is packed with ice slush. Following completion of organ
           perfusion heart, lungs, liver and pancreas and kidneys are removed in sequence. Livers
           are placed in plastic bags containing a liter of UW solution (40C) and packed in ice
           until the time of transplantation.

           Ischemic preconditioning is performed soon after opening the abdomen by clamping the
           hepatic hilum with a vascular clamp for five minutes, which is repeated again after five
           minutes of reperfusion. During IPC the peritoneal cavity is inspected for bleeding,
           congestion and edema of the pancreas and small intestine.

        2. Graft Implantation: Recipient hepatectomy is done in a standard manner with caval
           preservation and without utilization of venovenous bypass. The liver graft is implanted
           in a piggyback manner88. The allograft is reperfused after completion of the
           suprahepatic caval and portal vein anastomoses. The graft is flushed with approximately
           300 ml of recipient blood, which is vented via the donor's infrahepatic vena cava, after
           which this structure is ligated. Arterial and bile duct anastomoses are completed after
           graft reperfusion.

        3. Liver Biopsy and Reperfusion Injury: A wedge and a needle biopsy of the right lobe is
           performed immediately upon opening the donor's abdomen and before IPC. Another wedge and
           needle biopsy is done 90 min after recipient reperfusion. Liver biopsy after
           transplantation is performed only when clinically indicated. Formalin fixed specimens
           are embedded in paraffin, sectioned five microns thin and stained with hematoxylin and
           eosin. The amount of fat in donor biopsies is graded semi-quantitatively as follows89;
           No fat = grade 0, 1-15% = grade I, 16-30% = grade II, 31-45% = grade III, > 45% = grade
           IV. Reperfusion injury is graded (0-10 in increasing severity) by examining 30 high
           power fields (600x) of each post perfusion biopsy. Apoptotic cells are enumerated and
           hepatocyte swelling, zone 3 hemorrhage and necrosis are assigned a semi-quantitative
           score90. The reperfusion scores in the proposed study are expected to range 0.5 to 5
           based on our previous work36. The pathologist is masked regarding the group assignment
           of all biopsies.

        4. Immunosuppression and Acute Rejection: All patients receive tacrolimus and steroids for
           immunosuppression. In patients with hepatorenal syndrome tacrolimus use is delayed for
           several days and may be used at a much lower dose than the standard use. Pharmacological
           immunosuppression will be quantified by determining the mean dose of steroid per day,
           the mean of the weekly median tacrolimus level for the first five weeks and the
           proportion of recipients in each group that might receive other agents such as
           mycophenolate and IL-2 receptor antibody. Immunosuppresion caused by recipient's liver
           failure will be assessed by their model for end stage liver disease (MELD) score at the
           time of transplantation. Following transplantation liver biopsies are performed only
           when clinically indicated. Acute cellular rejection is diagnosed when at least two of
           the three following criteria are present: portal mononulclear infiltration, bile duct
           inflammation/damage and subendothelial inflammation of the portal venules and/or
           terminal hepatic venules. Severity of the rejection is graded based on the rejection
           activity index as follows: 0-2 = no rejection, 3 = borderline, 4-5 = mild, 6-7 =
           moderate and 8-9 = severe91. Mild rejection is treated initially with escalation of
           prograf dose alone or in combination with steroids. Steroid resistant rejection, upon
           confirmation with a biopsy is treated with muromonab (OKT3).

        5. Plasma TNF-alpha, IL-6 and 8, and soluble L-selectin: Plasma is separated from systemic
           venous blood taken 3, 12, 24, 48 hours after reperfusion from recipients of livers with
           and without IPC and samples are stored at -700C. TNF-alpha, IL-6 and IL-8 and soluble
           L-selectin levels are quantitated with commercially available enzyme-linked
           immunosorbent assay kits (R & D Systems, Minneapolis, MN) in duplicates. The assays are
           performed according to manufacturer's instructions.

        6. Neutrophil CD11beta/CD18 and L-selectin, Platelet P-selectin and Platelet-Neutrophil
           Complexes and Oxidative burst of Neutrophils in peripheral blood: Systemic venous blood
           is collected into heparinized (10 U/ml) syringes 3, 12, 24 and 48 hours after
           reperfusion from recipients of livers with and without IPC. Fifty microL of blood are
           added to saturating concentrations of fluorescein isothiocyante (FITC), or phycoerythrin
           (PE) conjugated monoclonal antibodies (Dako Corp, Carpenteria, CA) to CD 11beta/CD18, CD
           62 L (L-selectin), CD 42 (platelet von Willebrand factor receptor), and CD 62P
           (P-selectin). Following 10 min of incubation at room temperature, red cells are lysed by
           the addition of 200 microL of FACSlyse (Becton Dickinson, Franklin Lakes, NJ). After 10
           min of incubation 250 microL of 0.2% formaldehyde in PBS is added. Samples are analyzed
           in a FasCalibur flow cytometry (Becton Dickinson, Fair Lakes, NJ). Fluorescence of FITC
           is measured at 515 nm and PE at 580 nm. Neutrophils are distinguished by their readily
           identifiable characteristics on forward and side-scatter plot. A minimum of 5000
           neutrophil events are counted. Events staining positive for both CD 11beta and CD 42 are
           considered to be PNC as described by Peters et al72. To minimize the risk of identifying
           individual platelets and neutrophils as PNC flow rate is adjusted to result in < 4500
           events/sec. Results are considered positive if the fluorescence intensity exceeds 98% of
           isotype matched control antibodies. Results are expressed as median fluorescence
           intensity (MFI). Oxidative burst is measured by utilization of oxidation of
           dihydrorhodamine to florescent rhodamine by neutrophil generated reactive oxygen species
           as described by Vowells et al92. Briefly, 300 microL of heparinized whole blood is added
           to 4 ml of red cell lysis buffer (370C). After five minutes of incubation the pellet is
           resuspended in HBSS containing 5% fetal calf serum. 1.8 microL of DHR in DMSO is added
           and incubated at room temperature for 10 min followed by flow cytometric analysis.
           Stimulation with PMA (1microg/ml) is used as a positive control.

        7. Pulmonary Edema: Chest radiographs are performed in an antero-posterior projection with
           the patient in semi-erect position upon completion of the transplant and on
           postoperative days 1, 2 and 3. Pulmonary edema is assessed by the presence or absence of
           interstitial edema and alveolar edema as per accepted criteria93. The radiologist is
           masked regarding group assignment of subjects.
    

Intervention name(s): Ischemic Preconditioning
Intervention type(s): Procedure
Intervention description(s): Experimental: Hepatic Ischemic Preconditioning blood flow to the liver will be cut off by hilar clamping for ten minutes followed by release of the clamp prior to removal of the liver from the donor.
Allocation: Randomized
Intervention model: Single Group Assignment
Masking: Single (Participant)
Primary purpose: Treatment
Keywords: Ischemia/reperfusion of the liver; Ischemic Preconditioning; Cadaver organ donor