The scarcity of deceased donor organs that match patients awaiting liver transplantation has led to the development of different strategies to increase the size of the organ pool. The use of living donor organs, split livers, and organs from increasingly older donors has been successfully incorporated into practice. In contrast, organs with evidence of steatosis (>30%), particularly macrovesicular steatosis, continue to be rejected by most transplant centers (1). The rate of primary nonfunction is significantly more frequent when steatotic livers are used compared with normal livers. There are multiple pathological conditions that can lead to steatosis, including alcoholism, noninsulin-dependent diabetes, and dyslipidemias, but obesity is the most common cause (1).
The obese Zucker rat is frequently used as a model for nonalcoholic fatty liver disease. Obesity, by itself, is associated with insulin resistance, augmented oxidative stress, and impaired energy metabolism.
A significantly decreased energy state is one of the hallmarks of ischemia. The preservation of adenosine triphosphate (ATP) during ischemia and its recovery during reperfusion may partially determine the cell viability and organ function after transplantation (2). Modification of the preservation solution and pharmacologic manipulation have been used to maintain ATP levels and facilitate ATP regeneration after reperfusion (2).
Ischemic preconditioning (IP) (i.e., periods of short, nonlethal ischemia and subsequent reperfusion before sustained ischemia), was originally shown to be effective in the heart (3–5) and subsequently in the liver (6–8). It has been only recently demonstrated that IP may protect livers from obese rats during warm ischemia and transplantation (9,10). However, in the IP rat liver transplantation study, details about cellular high energy metabolism and animal survival were not available (10).
The present study consists of two separate parts. First, we assessed energy balance and metabolic alterations in grafts from lean and obese rats during cold ischemia using quantitative magnetic resonance spectroscopy (MRS). We hypothesized that IP in both obese and lean rats is associated with a preserved energy balance and a reduction of ischemia/reperfusion (I/R)-induced metabolic alterations. In the second part of the study, we assessed 24-h survival after the transplantation of liver grafts from obese rats with or without IP. We hypothesized that IP of donor grafts from obese rats before cold ischemia improves recipient survival after orthotopic liver transplantation.
All animal experiments were performed at the University of California at San Francisco with approval by the UCSF Committee on Animal Research. Animal care was in agreement with the National Institute of Health guidelines for ethical research.
Obese Zucker (fa/fa) rats are frequently used as an experimental model for nutritionally induced obesity (11). Obese Zucker rats lack cerebral leptin receptors, which results in an increased food intake, insulin resistance, and decreased energy expenditure.
The study consists of two protocols. Initially, a cold ischemia protocol was performed to determine the effects of IP on cellular metabolism in liver grafts from lean and obese donors. Cellular metabolism was assessed by quantitative MRS in preconditioned as well as non-preconditioned tissue harvested at 4, 6, and 8 h of ischemia at 4°C. This protocol demonstrated that the beneficial effects of IP on liver grafts from obese donors were most pronounced after 4 h of cold ischemia. The subsequent transplantation protocol investigated 24-h survival after transplantation of grafts from obese rats with and without IP preceding 4 h of preservation at 4°C.
Animals were randomized to one of the study groups. All surgical procedures were performed by the same individual (T.L.).
Four inbred age-matched male lean and four male obese (fa/fa) Zucker rats were anesthetized with isoflurane (Abbott Laboratories, North Chicago, IL) using a Summit rodent anesthesia machine (Summit Medical Equipment CO, OR). The liver was exposed through a midline incision and fully mobilized. The right lateral lobe of the liver was exposed to 10 min of ischemia followed by 10 min of reperfusion. This IP protocol has been shown to be protective in different experimental protocols (6,11,12). IP was achieved by clamping the vascular supply to the right lateral lobe. The remaining liver (left and median lobes) served as the control (non-IP). Subsequently, the abdominal aorta was dissected free at the bifurcation of the iliac arteries and clamped. The aorta was then catheterized with a 22-gauge Angiocath (BD Infusion Therap Inc., Sandy, UT) and the liver was subsequently flushed in a standard fashion at 4°C with 10 mL of University of Wisconsin (UW) solution (Via Span, Du Pont Pharma, Wilmington, DE) containing 10 IU/mL heparin. This typically removes most of the blood from the liver.
The liver was quickly removed, perfused with UW solution, and subsequently placed in cold UW solution. Tissue samples weighing approximately 100 mg were taken from the right lateral lobe and control at the following time points: immediately after being flushed with UW (T = 0) and after 4, 6, and 8 h of cold ischemia (T = 4, T = 6, T = 8, respectively). Obtained tissue samples were immediately flash-frozen in liquid nitrogen and stored at −80°C for further analysis by MRS.
At T = 0, tissue was also obtained for histological analysis. Tissues were fixed in 10% formalin and then embedded in paraffin for light microscopy. Sections were cut at 5 μ and stained with hematoxylin and eosin (H&E) for histological examination at magnification 100× and 200×. Samples were reviewed by a blinded pathologist who was not made aware of the source of the samples.
The flash-frozen liver tissues (100 mg each) were extracted using 12% perchloric acid (PCA) according to a protocol developed in our laboratory (14).
All 1H-MRS experiments were performed as described previously (15). In brief, all one-dimensional MR spectra of liver PCA aqueous and lipid extracts were recorded on a Bruker 500 DRX spectrometer and processed using XWINNMR and 1D-WINNMR software (Bruker Biospin Inc., Fremont, CA). A standard Bruker water presaturation sequence (“zgpr”) was used. An external standard substance, trimethylsilyl propionic-2,2,3,3,-d4 acid (TMSP, 0.5 and 1 mmol/L in D2O for water-soluble and lipid extracts, correspondingly) was used as a chemical shift reference (0 ppm). The absolute concentrations of single metabolites (μmol/g) were then referred to the TMSP integral and calculated.
All PCA extracts were analyzed by 31P-MRS immediately after 1H-MRS and the addition of 100 mmol/L EDTA (for complexation of divalent ions bounded at ATP). Phosphor spectra were obtained on a Bruker 300 MHz Avance spectrometer using a composite pulse decoupling sequence. An external standard methyl diphosphoric acid (2.3 mmol/L D2O) in a thin capillary was placed into the NMR tube and served as a chemical shift reference (18.6 ppm) as well as allowing for phosphor metabolite quantification. The absolute concentrations of ATP, adenosine diphosphate (ADP), nicotinamide adenine dinucleotide (NAD), phosphomonoesters, phosphodiesters, and sugar phosphates were calculated.
Obtained tissue and blood from animals were coded by one investigator (T.L.). The analyses of tissue samples were performed in a blinded fashion by two of the investigators (N.S. and D.K.).
In each group, 8 orthotopic liver transplantations were performed using 16 obese male Zucker rats as donors and 16 lean Zucker rats as recipients. Anesthetic management was the same as described above. After midline laparotomy, the portal vein and hepatic artery of the donor rats were dissected free. In the IP group (n = 8), both vessels were occluded for 10 min followed by 10 min of reperfusion after removal of the bulldog clamp. The subsequent treatment in the IP group and the control group (non-IP, n = 8) was identical. The left lateral lobes of the livers were removed before harvesting, thus reducing the liver graft to 70% of original size. Reduction of the liver to 70% was necessary to implant the larger liver of obese animals into lean recipients. The organs were then harvested identically as described in the cold ischemia part of the study.
Based on our findings in the cold ischemia study, the liver grafts were stored for 4 h in UW solution at 4°C. Subsequently, the grafts were implanted into the recipient rats applying Kamada and Calne's (13) technique without hepatic artery reconstruction. Heparin was not used other than during flushing of the organ.
At the end of the procedure, animals received 10 mL saline intraperitoneally. After the transplantation procedure, animals were allowed to recover. Animals were continuously monitored for 2 h and then every 2 h for additional 4 h.
Animals were then monitored as regulated by the Committee of Animal Care, University of San Francisco. Assessment included signs of poor clinical conditions such as lethargy, ruffled fur, guarding on abdominal palpation, lack of grooming, and decreased food intake. Animals that appeared to do poorly were killed. Immunosuppression was not necessary in this rat model. Surviving animals were killed at the end of the 24-h observation period.
All data are presented as mean ± sd. The primary end-point was to compare the impact of IP on cellular metabolism in obese rats. An unpaired two-tailed Student's t-test was used to compare results between IP and non-IP rats.
Subsequently, to investigate differences between obese and lean rats, we used lean non-IP rats for comparison of all study groups. Hence, analysis of variance with post-test Dunnett's multiple comparison was used to compare groups at a given time point to non-IP lean rats. Survival was determined by Fisher's exact test. P values < 0.05 were considered to be statistically significant.
At baseline obese (fa/fa) Zucker rats were heavier than age-matched lean controls (Table 1). Standard histological evaluation revealed only minimal to no changes consistent with hepatic steatosis in livers from obese rats (Fig. 1).
Nevertheless, livers from obese rats demonstrated a profoundly different cellular metabolic profile at baseline compared with lean controls (non-IP left lobe, Table 1). Livers from obese rats were associated with increased total fatty acid (including triglycerides) concentrations (Table 2). Also, livers from obese rats had significantly increased concentrations of lactate (Table 2). No significant differences in hepatic carbohydrates (glucose, glycogen) or sugar phosphates were seen with a trend towards increased levels in obese livers (Table 1). Most importantly, livers from obese rats had a decreased energy state as indicated by ATP concentrations as well as by decreased energy balance [ATP/ADP] (Table 2). In addition, NAD concentrations were significantly decreased in livers from obese rats (Table 2). A slight, although not statistically significant, decrease in hepatic glutathione was seen in livers from obese rats.
At baseline (T0), IP of the right lateral lobe significantly reduced hepatic ATP concentrations and ATP/ADP ratios in livers from obese rats as well as in lean livers (IP right lateral lobe). Hepatic energy balances in the right lateral IP lobe of lean livers were: 2.07 ± 0.24 (versus 2.68 ± 0.27 in non-IP lean, P < 0.01); in livers from obese rats: 1.23 ± 0.22 (versus 1.98 ± 0.26 in non-IP obese, P < 0.002). No other significant changes in hepatic metabolism were observed between IP and non-IP at baseline.
Although IP livers started with a lower energy balance at the baseline, they (from both lean and obese rats) experienced a smaller decrease of ATP and ATP/ADP ratios after 4 h of cold preservation (Fig. 2). Interestingly, IP of livers from obese grafts preserved the hepatic energy balance at the levels of non-IP lean livers: 0.65 ± 0.04 (IP obese) compared to 0.66 ± 0.05 (non-IP lean).
The groups with the highest and lowest energy balance at T4 were IP grafts from lean rats and non-IP grafts from obese rats (0.84 ± 0.06 and 0.50 ± 0.06, respectively, all P < 0.01 versus non-IP lean controls).
IP in lean livers demonstrated a beneficial effect on hepatic energy balance up to 8 h of cold preservation (Fig. 2). In contrast, this protective effect was lost in livers from obese rats at this time point. After 8 h of ischemia, only IP lean livers demonstrated preserved ATP/ADP ratios (0.54 ± 0.17) as opposed to no differences between non-IP lean (0.26 ± 0.06), non-IP obese (0.23 ± 0.04), and IP obese (0.28 ± 0.03) groups (Fig. 2).
As expected during ischemia, the lactate concentrations increased significantly from T0 in all groups with continuing cold ischemia (Fig. 3). Preconditioned lean livers accumulated less lactate than nonpreconditioned livers at all cold ischemia time points: T4: 6.71 ± 0.35 (IP lean) versus 9.59 ± 2.13 μmol/g (non-IP lean, P < 0.05); T6: 9.64 ± 0.82 versus 12.01 ± 1.11 μmol/g (P < 0.01); T8: 9.66 ± 0.48 versus 10.97 ± 0.49 μmol/g (P < 0.01). In livers from obese rats the preconditioning effects on lactate levels were detectable only at T4, 4 h of cold ischemia: 7.93 ± 0.95 (IP obese) versus 13.35 ± 3.18 μmol/g (non-IP obese, P < 0.01). At 6 h and 8 h of cold ischemia in livers of obese rats, IP no longer ameliorated the increase in lactate concentrations (Fig. 3). No other statistically significant metabolic changes were found among hepatic metabolites.
Lean recipients were older than donors in order to be of a comparable size to obese donors. Preservation time and anhepatic time were not different between IP obese and non IP obese donor groups (Table 2).
Seven of the recipient animals in the IP group were in good clinical condition at the time of death. One animal died spontaneously during the 24 h observation period. All recipient animals in the non-IP group were considered to be in poor clinical condition and 6 animals died spontaneously during the first 24 h after reperfusion (Fig. 4).
In this study, we demonstrated that IP temporarily improves cellular high energy metabolism of obese rat liver grafts undergoing 4 hours of cold ischemia. At 8 hours of cold ischemia, the beneficial effect on high energy metabolism was absent in livers from obese donors. Recipients of organs from obese donors exposed to IP had a significantly more frequent survival at 24 hours when compared with recipients of non-IP organs after 4 hours of cold ischemia.
The increased susceptibility of livers from obese Zucker rats to IR injury has been recently documented (16,17). Organs with fatty changes as a result of obesity are frequently not used for transplantation because they have been associated with more frequent poor initial function and primary nonfunction (18,19).
Experimental studies of warm ischemia have demonstrated that I/R injury of fatty livers can be effectively ameliorated by using IP (11,20). The influence of ATP status on hepatic viability during cold preservation has been emphasized (2). IP preserves high energy nucleotides in a warm ischemia model (21). IP also has a protective effect when using fatty livers by preserving ATP content (22). However, often only the ATP concentrations have been documented. Documented absolute ATP levels, as reported in most studies, provide an incomplete picture of energy metabolism. Most of the energy-requiring processes in the cell use the high energy phosphate bonds of ATP to provide energy. Active ion transport, thermogenesis, and the biosynthesis of macromolecules are all energy-requiring processes in which ATP is hydrolyzed to ADP and inorganic phosphate. In addition, the degradation of ATP is not a “one-way” reaction. The ATP can be regenerated from ADP and inorganic phosphate in the presence of oxygen (23). Therefore, energy balance (the ratio of ATP/ADP) provides a more accurate reflection of high energy metabolism than ATP levels alone. In contrast to conventional biochemical analysis, where usually only a single metabolite is quantified, MRS provides quantitative information on the entire metabolome (e.g., on all metabolites in the concentration range above 10 μM). This allows for the assessment of metabolic pathways and their changes, which otherwise may be neglected.
We have compared the energy balance at different time points during cold ischemia in livers from obese and lean rats. At baseline (T0) the energy balance was lower in livers from obese rats when compared with lean rats. This indicates an already compromised hepatic energy metabolism in the obese rat. The smaller NAD concentration in this group suggests a compromised oxidative phosphorylation. To compensate for an impaired mitochondrial energy production, anaerobic glycolysis, as indicated by increased lactate concentrations, was significantly higher in livers from obese rats when compared with lean controls at baseline. These metabolic changes in lactate and NAD, in combination with statistically unchanged concentrations of ADP, indicate that livers from obese rats may experience impaired ATP production instead of an increased ATP degradation. In addition to these specific differences in hepatic energy metabolism between livers from obese and lean rats, an increase in the concentrations of triglycerides and total fatty acids was seen in livers from obese rats. The metabolic differences are of importance because histological examination of the livers did not reveal significant steatosis (Fig. 1). This may imply that standard hematoxylin & eosin histology may not be sufficient for the evaluation of potential donor livers from obese donors. As expected, the IP exposed lobes of both lean and obese rats experienced a worsened energy balance at baseline as a result of the depletion of ATP during the IP phase.
Our results confirm the previously published results that IP has a beneficial effect on lean liver metabolism (21). Moreover, we demonstrate that IP significantly improves cellular bioenergetics in obese livers during the first hours of cold ischemia. However, in livers from obese rats, this metabolic protection lasted for only the first hours of cold ischemia, whereas in lean rats the protective effect of IP lasted throughout the study period, at least to 8 hours. Even though hepatic lactate levels were highly increased in livers from obese rats (both IP and non-IP) at time points T6 and T8 when compared with livers from lean rats, the increased activity of anaerobic glycolysis failed to compensate for the declining energy balance. In contrast, livers from lean rats with IP demonstrated a significantly increased energy balance and decreased lactate production when compared with non-IP lean livers at the time point T8. No significant changes in NAD were seen between IP and non-IP groups at 4 hours, implying that IP prevents ATP degradation but does not stimulate ATP production.
This study demonstrated that IP is able to slow the reduction of energy balance in both lean and obese rats. However, ischemic preconditioning does not prevent the depletion of energy balance and within our comparatively short cold ischemia time, we demonstrated a significant depletion in all groups but the IP lean group.
This finding questions the concept that ATP preservation is solely responsible for IP induced protection. Despite the observed significant decreases in energy balance in lean rats at 8 hours of cold ischemia, lean rats have excellent survival after extended cold ischemia times and transplantation (24,25).
According to our original hypothesis—that a preserved energy balance is essential for IP-induced protection—we chose 4 hours of cold ischemia for our transplantation model. Our original end-point was 7-day animal survival. However, in our model the mortality was frequent and the survival rate after 24 hours was already significantly less in recipients of nonpreconditioned livers from obese rats after 4 hours of cold ischemia. We, therefore, shortened the observation period to 24 hours, as has been done previously in studies with frequent mortality (17,26). Nevertheless, such a shortened observation period fails to demonstrate the prolonged protective effects of IP and is a potential limitation of the present study. Several studies have shown that transplantation of livers from obese Zucker rats after 4 hours of cold ischemia resulted in a 14-day survival of 40% (27,28). Treatment of livers from obese Zucker rats with p-selectin inhibitors and heme oxygenase-1 gene transfer has been shown to significantly improve survival in recipients in several studies (27,28). The increased survival rates could have been the result of different experimental protocols or better organ protection with p-selectin and heme oxygenase-1 gene transfer.
In summary, we were able to demonstrate that IP results in distinctly different metabolic profiles in obese and lean rats during 8 hours of cold ischemia. Furthermore, IP resulted in a significant survival benefit at 24 hours in recipients of livers from obese donors after 4 hours of cold ischemia. A clear IP survival benefit using livers from obese Zucker rats has yet to be demonstrated.
We thank Linda Ferell, MD, Department of Pathology, University of California San Francisco for her time and expertise in evaluating the histological slides.
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