Chronic and growing shortage of donor organs is a worldwide critical issue in organ transplantation, which has increased the use of donation after circulatory death (DCD) livers.1,2 Although utilization of DCD livers would substantially expand potential donor pools, it is very difficult to precisely estimate liver damage induced by DCD and subsequent hepatic function.
Nowadays, only limited transplant centers in some industrialized countries perform DCD liver transplant (LT), coupled with currently developed ex vivo machine perfusion techniques.3,4 Despite such innovative advancement, much concern still exists about the outcome after DCD-LT, in which the graft survival is approximately 10%–15% lower compared with the donation after brain death LT, because of higher rates of primary nonfunction and long-lasting biliary complications.5,6
Hepatic ischemia/reperfusion injury (IRI) is one of the main culprits for the poor outcome in DCD-LT. Attenuation or prevention of IRI remains an important clinical challenge to improve recipient outcome in DCD-LT. Warm ischemia in situ induces macrothrombosis and microthrombosis within hepatic vasculatures, leading to microcirculatory failure. In addition, subsequent cold storage (CS) significantly deteriorates vascular/sinusoidal endothelia, such as retraction, intercellular detachment, and apoptosis.7,8 Such ischemic damages during warm ischemic time (WIT) and subsequent CS are further exacerbated upon reperfusion by reactive oxygen species burst, Kupffer cell (KC) activation, upregulation of adhesion molecules, and subsequent leukocytes infiltration, finally leading to irreversible microcirculatory failure.
Human atrial natriuretic peptide (hANP) is a cardiovascular hormone, produced and secreted by heart atrium. hANP plays a major role in maintaining volume as well as pressure homeostasis in cardiovascular system, such as diuresis, natriuresis, and vasodilation.9 Several investigations have demonstrated its hepatocellular protection against hypoxia/reoxygenation.10,11 Furthermore, hANP preserves lung,12 heart,13 and kidney14 functions after warm ischemia. Most recently, perioperative hANP administration in patients undergoing resection of lung cancer significantly lowered the recurrence rate of hematogenous metastasis by suppressing inflammatory reactions in vascular endothelia.15 In addition, the interaction between hANP and hepatic endothelium was reported by a few investigations: hANP attenuates endothelin (ET)-1 upregulation from hepatic stellate cells (HSCs)16 and downregulates Na+/H+ and Ca+/H+ ions exchangers in hepatocytes, thus reducing cell swelling during hypoxia.10,17 ET-1 plays a major role in regulating sinusoidal blood flow by HSCs contraction.18
On the basis of this available evidence, we hypothesized that hANP supplementation into preservation solution might protect vascular endothelia, attenuate pro-inflammatory signals/cascades therein, and alleviate sinusoidal constriction, thereby improving damages and functions of DCD livers. This study was thus designed to evaluate the therapeutic potential of hANP on maintaining hepatic tissue integrity and function of cold-preserved DCD rat livers.
MATERIALS AND METHODS
Male Wistar rats (250–300 g; Japan SLC, Inc., Shizuoka, Japan) were used as liver donors. The rats were kept under specific pathogen-free conditions in a temperature- and humidity-controlled environment with a 12-hour light-dark cycle and had a free access to rat chow and water. All animal experiments were conducted under a protocol approved by the Animal Research Committee of Kyoto University, in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No 86–23, revised 1985).
Rat DCD Model
The rats were generally anesthetized with isoflurane (Escain; Mylan, Osaka, Japan) via a small animal anesthetizer (MK-A110; Muromachi Kikai, Tokyo, Japan). After midline laparotomy with bilateral subcostal extensions, the liver was carefully mobilized from all ligamentous attachments. After heparinization (300 IU/rat; Mochida Pharmaceutical, Tokyo, Japan), cardiac arrest was induced by phrenotomy and subsequent bilateral pneumothorax, followed by clamping the descending aorta. Donor animals remained untouched thereafter for 30 minutes. WIT in situ was counted from aorta clamping.19 Low body temperature was prevented by an isothermal heating pad and by covering the body, enabling to keep approximately 32.0 ± 0.3°C throughout WIT.
The rats were randomly assigned into 2 groups (n = 8 each), Group-hANP or -Control. First, splanchnic organs were flushed in situ from aorta with 50 mL of ice-cold University of Wisconsin (UW) solution (Viaspan; Astellas, Tokyo, Japan) with or without 2.5 μg/mL hANP (HANP; Daiichi-Sankyo Co., Ltd., Tokyo, Japan) supplementation (Group-hANP or -Control, respectively). During the initial washout, the superior and inferior vena cavae, respectively, were both incised to avoid hepatic outflow obstruction. The procedures hereafter were detailed elsewhere.20,21 In brief, the common bile duct was cannulated with a 24-gauge polyethylene tube (TERUMO, Tokyo, Japan). The hepatic artery was ligated, and portal vein (PV) was cannulated with a 14-gauge catheter (Argyle; COVIDIEN, Tokyo, Japan). The liver was perfused again with gravity from PV with 10 mL of cold UW with or without 2.5 μg/mL hANP supplementation. Superior vena cava was cannulated with a 14-gauge short stent for isolated perfusion. Livers were then excised, weighed (14.0 ± 0.2 g), and cold-stored at 4°C for 6 hours in the respective UW solutions.
Ex Vivo Oxygenated Warm Reperfusion
The isolated perfused rat liver (IPRL) system has been detailed elsewhere.20-22 In brief, the perfusion circuit was prerinsed and perfused with hemoglobin-free Krebs-Henseleit buffer (K3753, Sigma-Aldrich Inc., St. Louis), supplemented with 1 mg/L of hyaluronic acid (HA; CAS.9067-32-7, Wako Pure Chemical Industries, Japan). To account for slow rewarming during vascular reconstruction, all livers were transferred and immersed into saline at room temperature for 15 minutes just before reperfusion. Thereafter, the livers were positioned in a Plexiglas chamber and perfused via PV at a constant flow of 3 mL/g-liver/min at 37°C for 120 minutes using a roller pump. The perfusion fluid was 140 mL of modified Krebs-Henseleit buffer (pH = 7.4), saturated with Carbogen (95% O2 + 5% CO2). The perfusate PO2 was maintained above 500 mm Hg throughout reperfusion. Inflow and outflow perfusates were sampled at 0, 10, 30, 60, and 120 minutes of reperfusion. Bile production was monitored, and portal venous perfusion pressure (PVP) was continuously measured by a calibrated vertical column connected to the PV catheter.20,21
Aspartate aminotransferase and alanine aminotransferase release into hepatic effluent were both determined with a standard spectrophotometric method with an automated clinical analyzer (JCA-BM9030; JEOL, Ltd., Tokyo, Japan).
The 8-hydroxy-2-deoxyguanosine (8-OHdG) is a useful marker to estimate oxidative damage to liver tissues, mainly by hydroxyl radicals.23 Liver effluent was analyzed with a high-sensitive 8-OHdG enzyme-linked immunosorbent assay (8-OHdG check; Nikken seil, Sizuoka, Japan) according to the manufacturer’s protocol.
Hepatic Oxygen Consumption
Hepatic perfusate was sampled from both the portal inflow and the venous outflow, and oxygen contents were immediately determined using a blood gas analyzer (Rapid Point 405; Siemens, Tokyo, Japan). The oxygen consumption rate was calculated by the difference between the inflow and the outflow samples and provided as μL/g-liver/min according to the transhepatic flow and liver mass.20,21
Tissue Adenosine Triphosphate and Adenosine Concentrations
At the end of IPRL, liver tissues were snap-frozen and stored in liquid nitrogen until later analysis. Tissue ATP concentrations were determined by the luciferin-luciferase method with an ATP bioluminescence assay kit (Toyo B-Net Co., Ltd., Tokyo, Japan).21 Adenosine concentration in liver tissues was also measured with an enzyme-coupled fluorometric kit (Adenosine Assay Kit, Abcam, United Kingdom) according to the manufacturer’s instruction.24 The results were normalized to tissue protein concentrations, measured by bicinchoninic acid protein assay kit (Thermo Fisher Scientific K.K., Yokohama, Japan).
As a reference, normal, non-DCD livers, not subjected to warm ischemia but to 6-hour CS and subsequent 2-hour oxygenated reperfusion, were prepared in these assays to know the baseline values (Figures 2B and 4E, respectively), but not included in statistical analyses.
To estimate the remaining viability of sinusoidal endothelial cells (SECs) of DCD livers, HA clearance was calculated.25 Because SECs are the dominant site for uptaking and degrading HA,26 and HA production from other organs is completely excluded in the current isolated setting, HA clearance in IPRL precisely estimates the functional integrity of SECs. The HA concentration in perfusate samples was measured with enzyme-linked protein assay (Corgenix Inc., CO) according to the manufacturer’s protocol.
Quantitative Reverse-transcription Polymerase Chain Reaction
Total mRNA was extracted from liver tissue after 120-minute reperfusion using a NucleoSpin RNA Kit (MACHEREY-NAGEL GmbH & Co. Kg, Düren, Germany). Equal amounts of mRNA were adjusted with NanoDrop2000 (NanoDrop Technologies, Washington, DE). Complementary DNA was reverse-transcribed using an Omniscript RT kit (Qiagen, Tokyo, Japan). Quantitative reverse transcription-polymerase chain reaction was performed with the following amplification conditions: 50.0°C for 2 seconds and 95.0°C for 10 seconds during holding stage followed by 45 cycles of 95.0°C for 0.15 seconds and 60.0°C for 1.0 second. Polymerase chain reaction products were analyzed with the StepOnePlus Real-Time PCR System (Applied Biosystems, Life Technologies Japan, Tokyo, Japan). Target gene expressions were calculated relative to the housekeeping gene, β-actin. TaqMan probes and primers for endothelin-1 (ET-1: assay ID Rn9999901_m1), tumor necrosis factor-α (TNF-a ID Rn01410330_m1), endothelial nitric oxide synthase (eNOS: ID Rn00562055_m1), inducible nitric oxide synthase (iNOS: ID Rn00561646_m1), and β-actin (ID Rn00667869_m1) were obtained from TaqMan gene expression assays (Applied Biosystems, Tokyo, Japan).
Bile Production and Lactate Dehydrogenase Leakage Into Bile
Bile was collected throughout oxygenated perfusion and served as a functional parameter of DCD liver grafts. Lactate dehydrogenase (LDH) leakage into bile was also measured, given as a marker for biliary damages.21,27
At the end of reperfusion, tissue samples were obtained from the left lobe, stored, and fixed in 4% paraformaldehyde, paraffinized, and sectioned to 4 μm thickness. After hematoxylin-eosin staining, 2 independent pathologists assessed and graded hepatic tissue damage from 1 (excellent) to 4 (poor) in a blind fashion, according to the criteria reported elsewhere,28 as follows: (1) normal rectangular structure, (2) rounded hepatocytes with increased sinusoidal spaces, (3) apoptosis (vacuolization and nuclear pyknosis), and (4) necrosis. The scores were calculated from 10 independent observations (magnification × 400) and 8 livers per group.
At the end of reperfusion, additional 5 livers from the both groups were perfused through PV with 2% glutaraldehyde/4% paraformaldehyde in 0.1M phosphate buffer, pH = 7.4. After fixation, the samples were cut into 1-mm slices for scanning electron microscope analysis. After postfixation with 1% osmium tetroxide, tissue samples were dehydrated, substituted, freeze-dried with t-butyl-alcohol, ion sputter-coated, and then examined with S-4700 electron microscopy (Hitachi High-Technologies, Tokyo, Japan).
Silver impregnation staining for reticulin fibers was used to assess sinusoidal wall architecture, such as collapse/disconnection of the reticulin framework in ischemically damaged liver lobules.29,30 Ten randomly selected fields from 5 livers per each group were examined under an optical microscope. The area of the reticulin network was quantified using Image J (version 1.6.0_22; NIH).31 According to the instruction,32 the red channel was chosen on the software to emphasize reticulin fibers on the picture, thereby to binarize and quantify the reticulin fibers. The lower threshold was set as 55.
All statistical analyses were performed using Prism 6 (Graph Pad Software, Inc., San Diego, CA). All values are expressed as the mean ± SEM. Two-way repeated-measures ANOVA followed by Bonferroni post-test was used to assess time-dependent parameters including PVP, transaminase release, and O2 consumption. Statistical significance at each time point was annotated as follows: *P < 0.05; †P < 0.01; ‡P < 0.001.
The other comparisons between the 2 groups were performed by 2-sided Student t test or Mann-Whitney U test, as appropriate. P < 0.05 was considered significant.
As summarized in Figure 1, aspartate aminotransferase release was significantly lower in Group-hANP than that in Group-Control (159.3 ± 15.4 versus 241.3 ± 21.4 U/L at 2 hours, respectively, P < 0.001, Figure 1A). Alanine aminotransferase (134.2 ± 14.2 versus 223.3 ± 31.5 U/L, P < 0.001, Figure 1B) also exhibited significant reduction throughout reperfusion, indicating substantially less hepatocellular damage, achieved by hANP supplementation.
Tissue Oxygen Consumption, ATP Charge, and Oxidative Damages
Though oxygen consumption rate in Group-hANP was tended to be higher than in Group-Control, it could not reach statistical significance (Figure 2A). In contrast, ATP restoration was significantly higher in Group-hANP than in controls (P = 0.002, Figure 2B), indicating that hANP supplementation produced significantly better ATP charge with an equal amount of oxygen consumption, compared with untreated livers. The values in non-DCD livers are also presented in Figure 2B just for a reference.
Oxidative tissue damages were quantified by measuring 8-OHdG release into the perfusate. As shown in Figure 2C, hANP significantly lowered oxidative tissue damage than in Group-Control (0.54 ± 0.04 versus 0.73 ± 0.04 ng/mL, P < 0.05).
Taken all these together, hANP supplementation enabled efficient oxygen utilization to restore ATP charge, while in controls, a substantial amount of oxygen was consumed to generate oxidative stress.
Total Vascular Resistance and Sinusoidal Endothelial Function
During reperfusion, PVP, served as a parameter for total vascular resistance of DCD livers, showed time-dependent increase up to 22.5 ± 3.5 mm Hg. In contrast, PVP in hANP-treated livers maintained significantly lower level, retaining 12.2 ± 0.5 mm Hg at the end of reperfusion (P < 0.001, Figure 3A).
On the functional integrity of sinusoidal endothelia, HA clearance in Group-hANP was significantly higher than in Group-Control (2139.8 ± 257.9 versus 1381.2 ± 237.1 ng/g-liver/h, P < 0.05, Figure 3B).
Morphologically, scanning electron microscopy revealed that sinusoidal endothelial linings in DCD livers were severely deteriorated, and sinusoidal pores were fused and enlarged (Figure 3C), whereas hANP supplementation significantly attenuated such deleterious alterations in sinusoidal endothelia (Figure 3D).
Vasodilatory Effects by hANP
To examine the mechanisms underlying lowered PVP achieved by hANP, mRNA signals of eNOS, a well-known vasorelaxant, and of ET-1, a vasoconstrictor in hepatic sinusoids, were both determined by quantitative reverse transcription polymerase chain reaction. Of interest, hANP significantly upregulated eNOS from 0.36 ± 0.14 in the controls up to 0.86 ± 0.10 (P < 0.05, Figure 4A), while ET-1 was significantly downregulated to 35.1% of the controls (1.48 ± 0.25 versus 0.52 ± 0.05, P < 0.01, Figure 4B).
Tissue adenosine concentration in Group-hANP was significantly higher than that in Group-Control (P = 0.008). Of interest, the value in Group-hANP showed higher tendency even compared with normal, non-DCD livers (Figure 4E), indicating that hANP strongly upregulates tissue adenosine concentration.
Anti-inflammatory Effects by hANP
As shown in Figure 4C and D, mRNA expressions of tumor necrosis factor-α and iNOS were both significantly downregulated in Group-hANP than in Group-Control (1.26 ± 0.06 versus 1.79 ± 0.16, P < 0.05, and 0.66 ± 0.13 versus 1.41 ± 0.30, P < 0.05, respectively), directly reflecting strong anti-inflammatory effect by hANP.
Bile Production and Biliary Damage
Total remaining function of DCD livers was appraised by bile production during reperfusion. As displayed in Figure 5A, bile volume in Group-hANP was three-fold more than that in Group-Control (96.2 ± 18.2 versus 36.2 ± 15.2 μL/g-liver/2 h, P < 0.001).
Comprehensive biliary damage was estimated by LDH leakage into bile.21,27 In the controls, bile LDH was as very high as 1916.8 ± 331.9 IU/L, which was significantly improved by hANP to less than half (932.0 ± 194.3, P < 0.05, Figure 5B).
Microvilli formation in bile canaliculi was significantly decreased, and their height was also lowered in Group-Control (Figure 5C). In contrast, hANP significantly preserved the density and height of microvilli (Figure 5D).
Representative tissue sections of DCD livers after 6-hour CS and subsequent 2-hour oxygenated reperfusion are presented in Figure 6. As seen, hematoxylin/eosin staining revealed ballooning degeneration around pericentral zones, as well as scattering acidophilic bodies in the controls, representing necrosis and apoptosis of hepatocytes. Disturbed architecture of hepatic cords, sinusoidal dilatation, and focal necrosis were also found across hepatic lobules in untreated livers (Figure 6A and C); however, these damages were attenuated by hANP supplementation (Figure 6B and D). Thus, tissue damage scoring demonstrated significant improvement of hANP-treated livers compared with the controls (10.9 ± 0.8 versus 13.7 ± 0.7, P < 0.05, Figure 6E).
As shown in Figure 7A–F, silver staining of DCD liver tissues demonstrated that reticulin fibers were better maintained in Group-hANP compared with Group-Control. The relative ratio of reticulin fibers in Group-hANP was significantly higher than in Group-Control (4.8 ± 0.3 versus 3.3 ± 0.4, P < 0.05, Figure 7G), highlighting the substantial disruption of fibrous ultrastructure constituting sinusoidal architectures in DCD livers, as well as significant amelioration thereof achieved by hANP supplementation.
Cells, tissues, and organs communicate with each other to maintain or regulate surrounding circumstances for their better physiological functions. Various hormones, cytokines, and chemokines are involved in such endocrine homeostasis under not only physiological but also diverse pathological conditions. One of such warning signals from the heart is ANP, which exerts several biological action to reduce cardiac overload, including vasodilatation, diuresis, and natriuresis.33-35 Besides its remarkable cardioprotective properties, much attention has recently been paid to its protective action to vascular endothelia.36 Emerging evidence has shown that perioperative hANP administration just for 3 days attenuates inflammatory reactions in endothelial cells, such as E-selectin upregulation, and inhibits adhesion of circulating tumor cells to vascular endothelia in remote organs, thereby preventing hematogenous metastasis for years.15 In addition to its direct action to vascular endothelia, immunomodulatory effects of hANP were also reported, such as downregulation of KCs activation in hepatic IRI.37 Moreover, ANP was also proven to regulate p38-mitogen activated protein kinase activation in a porcine warm IRI model.38
These available evidence led us to hypothesize that hANP supplementation during preservation would possibly exert cytoprotective effects on DCD liver grafts, in which extremely large vascular beds in the liver are all severely predamaged by warm, as well as cold IRI.
As expected, we demonstrated for the first time that hANP supplementation could effectively attenuate IRI in DCD rat livers. The protection by hANP was associated with significant downregulation of ET-1 and iNOS, both of which led to increase in vascular resistance of the liver. Of endothelium-derived vasoconstricting factors, ET-1 plays a pivotal role in contracting sinusoidal microcirculation.39 In the present study, ET-1 mRNA was upregulated in DCD livers, which was significantly downregulated by hANP supplementation to one-third of the control value. Gorbig et al16 reported that ANP blocks the influx of intracellular calcium ion, thereby directly inhibiting ET-1–induced HSC contraction. In addition to such direct action to ET-1/HSC, hANP attenuated iNOS upregulation that further elicits ET-1 overexpression from HSCs, KCs, and SECs.40 Moreover, hANP supplementation also resulted in 2.4-fold increase in eNOS expression. Under physiological conditions, vasoactive nitric oxide (NO) is produced through eNOS in SECs and then diffuses into Disse space, promoting HSCs relaxation and subsequent sinusoidal dilatation. The bioavailability of eNOS remains crucial to maintain the balance between the vasodilatory and constrictive forces.41 As well demonstrated, tissue adenosine upregulation confers cytoprotection to ischemic tissue by inhibiting ET-1, as well as by increasing NO production. This, in turn, improved microcirculatory disturbance in DCD livers.42-44 In the present study, we demonstrated that hANP supplementation significantly increased tissue adenosine concentration in DCD livers, concomitant with significant downregulation of ET-1 and upregulation of eNOS. Surprisingly, the value in hANP-treated DCD livers showed higher tendency than in normal, non-DCD controls. Such adenosine enhancement by hANP contributed to reduce hepatic vascular resistance in DCD livers, as evidenced by lower PVP.
Furthermore, HA clearance and electron microscopy revealed that hANP significantly preserved functional and morphological viability of sinusoidal endothelia in DCD livers. Thus, hANP supplementation significantly lowered high vascular resistance of DCD livers down to the half in control livers. Moreover, silver staining of reticulin fibers also revealed better maintenance of ultrastructural architecture in sinusoidal framework by hANP. All these beneficial effects by hANP contributed with each other to protect sinusoidal wall structures comprising several components of SECs, HSCs and their multiple processes, nervous fibers, and extracellular matrixes, thereby ameliorating hepatic microcirculation in DCD livers.
Of interest, oxygen consumption rate in hANP-treated livers was similar to that in controls. Tissue ATP restoration after oxygenated reperfusion, however, exhibited two-fold improvement compared with controls. Such disparity between tissue oxygen uptake and resultant ATP charge may be attributed to the differences in simultaneous oxidative stress produced therein. In Group-Control, 8-OHdG, one of the most sensitive indices for oxidative cellular damages, was 33% higher than in hANP-treated livers, indicating that substantial amount of delivered oxygen was consumed to generate reactive oxygen species, not to restore tissue energy charge. In other words, hANP supplementation efficiently utilized delivered oxygen to restore ATP, thereby maintaining tissue integrity of DCD livers. We recently reported similar observations in macrosteatotic livers, in which subnormothermic machine perfusion preservation resulted in significantly higher ATP restoration with less oxygen uptake, whereas cold-stored fatty livers consumed more oxygen with significantly higher oxidative damage and less energy charge.21 These results may suggest a common pathology in predamaged ECD livers with less functional viability of hepatocellular mitochondria and sinusoidal microcirculation.
Of various disadvantages of DCD liver grafts, ischemic cholangiopathy is the leading cause of graft loss.45,46 Perioperative biliary hypoxia due to impaired microcirculation seems to be responsible for nonanastomotic strictures in intrahepatic bile ducts for a prolonged period.47 Of note, hANP supplementation significantly lowered LDH leakage into bile to less than half of that in untreated livers, directly reflecting significantly less biliary damage. This was followed by significantly more bile production, increasing up to four-fold higher bile secretion compared with the control. Early bile flow upon reperfusion is one of the most reliable parameters for graft injury, particularly for microcirculatory disturbance.48 Moreover, electron microscopy revealed that both density and height of microvilli in bile canaliculi were significantly better maintained by hANP. Although longer observation using a large animal model is required, these results suggest a therapeutic potential of hANP supplementation to ameliorate ischemic cholangiopathy of DCD-LT.
Since the first clinical application in 1995, hundreds of thousands of patients with congestive heart failure have received hANP administration. Because hANP is an endogenous hormone secreted by heart atrium, its unobtainable safety is very attractive for drug repositioning of this cardioprotective agent to different medical fields. Because the half-life of ANP in physiological blood circulation is approximately 2 minutes in human (4 minutes in rats),49 continuous intravenous administration is necessary to sustain its therapeutic effect in in vivo conditions. However, a previous study demonstrated its sustainable effect under low temperature, exerting renal protection during prolonged CS.50 Consistently, we also demonstrated significant protection of DCD liver grafts by hANP supplementation during CS. Such ex vivo treatments to isolated organs enable to avoid even relatively rare systemic adverse effects of this agent, such as hypotension.
As a limitation, this study adopted the ex vivo reperfusion model that has been widely used in various research fields including transplantation.20,21,27,51 This isolated setting enables us to avoid interferences from immunological alloreactions and technical dispersions in LT surgery in rodent, thereby allowing to assess organ preservation quality more precisely. However, validation of this novel preservation technique in an LT model is prerequisite before the next step toward its clinical translation.
In conclusion, hANP supplementation during preservation significantly protected DCD liver grafts from IRI by ameliorating predamaged hepatic microvasculature by warm and cold IRI. Given its simplicity and unobtainable safety, hANP supplementation may be a novel therapeutic option for DCD liver grafts, which is easily applicable worldwide, even though highly equipped MP devices are not available.
The authors are grateful to Dr Takuya Hiratsuka and his skillful team for helpful cooperation in histological assessments, Center for Anatomical, Pathological, and Forensic Medical Research, Graduate School of Medicine, Kyoto University.
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