1. Why did you decide to do this research project and what prior work led up to this latest paper?

We have seen many articles dealing with liver steatosis without a consideration of qualitative diagnosis of macrosteatosis (MaS) and microsteatosis (MiS). For example, large number of previous studies have predominantly focused on MaS without a consideration for MiS with a simple comment like "because microsteatosis seems to have limited impact according to our clinical experiences" or "as in many transplant centers" without supporting references [1-5]. Indeed, the exact clinical importance of MiS has been an issue of controversy for last 15 years in the field of liver transplantation since Selzner M. and Clavien PA. concluded "until more accurate data are available, MaS and MiS should probably be considered as a comparable risk for liver surgery" in 2001 [6]. The same group and others have subsequently reported conflicting results regarding the importance of MiS. Many of them have inspired the authors to initiate our clinical research project on the issues of each steatosis in the living donor liver transplantation (LDLT).

Following are the examples of studies which motivated us to our papers. First, we would like to mention an article by McCormack, L. et al. published in 2007 [7]. This interesting article involving the patients who underwent major hepatectomy for malignant or benign liver diseases has recommend researchers to distinguish pure MaS, pure MiS, and mixed steatosis, and a possibility of the interaction between MaS and MiS. Second, two recent large studies performed by Spitzer, A. L. et al. and Dutkowski, P. et al., respectively [8,9], have demonstrated the insignificance of MiS in deceased donor liver transplantation (not in LDLT) using multivariate regression analyses. The two studies led us to embark a study in LDLT because the data from deceased donors may not be extrapolated into living donors mainly due to small-for-size graft and donor safety issues of LDLT.

In addition, a study of patients who underwent deceased donor liver transplantation suggesting the feasibility of severe steatosis grafts further prompted us to perform studies to distinguish respective importance of MaS and MiS in LDLT. Finally, we would like to mention an animal study in mice by Selzner, N. et al. published in 2006 comparing MaS-dominant livers and MiS-dominant livers [10]. Although this study failed to generate pure MiS or pure MaS model, the study clearly showed that the impact of MaS is greater than MiS, being in agreement with our papers.

2. How did the study series proceed.

The above-mentioned and other articles led us to our paper titled "The effect of pure microsteatosis on transplant outcome after living donor liver transplantation: A matched case-control study" [11]. We designed this study to determine pure effects of MiS with the exception of confounding effects of MaS and demonstrated that MiS alone does not impair the post-transplant donor and recipient outcomes. Afterward, since the majority of donor organs with steatosis are likely to have a mixed pattern of both MaS and MiS, our group designed a more recent study titled "Comparison of the tolerance of hepatic ischemia reperfusion injury in living donors: Macrosteatosis versus microsteatosis" to show differences in clinical importance of MaS and MiS and to validate the prevailing paradigm which suggests limited risks related to MiS [12]. This study is simple and clear but very interesting because there have been no studies to show the clinical impact of MaS and MiS simultaneously in one study. Also, we did not use multivariate analysis enrolling MaS and MiS in a multivariate model together as independent covariables because we believed that multicollinearity between MaS and MiS could significantly confound the results even if model fitness test like the Hosmer-Lemeshow test, I called it a generous test, says "it is appropriate (P > 0.05)". We gave deep thought on this matter and reached the decision to generate a figure comparing the tolerance of hepatic ischemia reperfusion injury between MaS and MiS. To depict the tolerance of hepatic ischemia reperfusion injury, we have chosen a surrogate parameter once used in a study of patients undergoing hepatectomy for liver diseases, however, never been used in liver transplantation [13]: the coefficient of the regression line between the cumulative intermittent hepatic inflow occlusion (IHIO) time, easily speaking the liver ischemia time, and the peak postoperative transaminase concentrations.

After clarifying the different response of MaS and MiS to hepatic ischemia, we felt that the remaining path is clear. The only thing we have was to confirm the importance of MiS greater than the degree which was studied in the 'Pure MiS study'. We made up mind to use propensity score-based matching again as in the 'Pure MiS study' to control MaS degree between low MiS group and high MiS group. The thing was that I have used many-to-one matching this time instead of one-to-one matching which was used in the 'Pure MiS study' as an effort to secure more study population and maximize statistical power. The exposed subjects who had ≥ 30% MiS (high MiS) were rare (~60 in > 10-year LDLT experience in our hospital) and the small exposed group was a major limitation of the study. After confirming study design, everything went straightforward and the results of the study has demonstrated that > 30% MiS has limited impact on early liver graft regeneration and post-transplant donor/recipient outcomes. Our results could also identify that there is no interaction between MaS and MiS because we only included MiS grafts mixed with MaS (please remind that we only include pure MiS in our previous study). From the clinical standpoint, our results should help expand the living donor pool by including living donors with < 60% MiS, which had been resulting in reluctance to proceed LDLT.

3. How do you plan to take this work forward? What are the implications for future research?

Surely, the fore-mentioned studies have their own limitations. I would simply suggest four issues which should be better studies in future research. First, the methodology to evaluate liver steatosis depends on pathologists' experience, and thus, is too subjective for now. More objective techniques need to report more consistent results. As of now, both the inter-rater variability and intra-rater variability are severe [14]. Second, our studies cannot conclude the impact of more severe degree MiS (for example > 60% MiS) in LDLT. Although it is very rare, we often encounter living donors who have > 60% MiS in some cases. Third, we should focus on how to attenuate ischemia reperfusion injury, which is a major mechanism decreasing graft regenerative capacity, of macrosteatotic livers. The strategies may include ischemic preconditioning, thermal management, and improvement of preservation solutions [15].

Finally, as an anesthesiologist, I would like to open up the new filed which has never been elucidated. That is, I would explore that how various anesthetic measurements performed during LDLT can affect graft injury, graft regeneration, and transplant outcomes. Intraoperative postreperfusion period was very critical for the success of LDLT because numerous signals for triggering liver regeneration, including growth hormones and cytokines, are initiated immediately after graft reperfusion [16]. The anesthetic measurements may include ischemic preconditioning, pharmacologic preconditioning, hemodynamic management, vasoactive drug use, and transfusion [17,18]. I feel the lack of knowledge in the field of basic science and translational research, and seek kind advices and corroborations which would boost my future research related to liver graft regeneration, steatosis in LDLT, preconditioning/postconditioning to ameliorate graft injury, and transfusion medicine in LDLT.

References

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[2] Park JB, Joh JW, Kim SJ, Kwon CH, Chun JM, Kim JM, et al. Effect of intermittent hepatic inflow occlusion with the Pringle maneuver during donor hepatectomy in adult living donor liver transplantation with right hemiliver grafts: a prospective, randomized controlled study. Liver Transpl 2012;18:129-137.

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[4] Briceno J, Ciria R, Pleguezuelo M, de la Mata M, Muntane J, Naranjo A, et al. Impact of donor graft steatosis on overall outcome and viral recurrence after liver transplantation for hepatitis C virus cirrhosis. Liver Transpl 2009;15:37-48.

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[10] Selzner N, Selzner M, Jochum W, Amann-Vesti B, Graf R, Clavien PA. Mouse livers with macrosteatosis are more susceptible to normothermic ischemic injury than those with microsteatosis. J Hepatol 2006;44:694-701.

[11] Han S, Ko JS, Kwon G, Park C, Lee S, Kim J, et al. The effect of pure microsteatosis on transplant outcome after living donor liver transplantation: A matched case-control study. Liver Transpl 2014.

[12] Han S, Kim G, Lee S, Kwon CD, Gwak M, Lee S, et al. Comparison of the tolerance of hepatic ischemia reperfusion injury in living donors: Macrosteatosis versus microsteatosis. Liver Transpl 2014.

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[15] Casillas-Ramirez A, Mosbah IB, Ramalho F, Rosello-Catafau J, Peralta C. Past and future approaches to ischemia-reperfusion lesion associated with liver transplantation. Life Sci 2006;79:1881-1894.

[16] Michalopoulos GK. Liver Regeneration. Science 1997;276:60-66.

[17] Beck-Schimmer B, Breitenstein S, Urech S, De Conno E, Wittlinger M, Puhan M, et al. A randomized controlled trial on pharmacological preconditioning in liver surgery using a volatile anesthetic. Ann Surg 2008;248:909-918.

[18] Beck-Schimmer B, Breitenstein S, Bonvini JM, Lesurtel M, Ganter M, Weber A, et al. Protection of pharmacological postconditioning in liver surgery: results of a prospective randomized controlled trial. Ann Surg 2012;256:837-844; discission 844-835.