Shanghai,China

Introduction

The incidence of neurological complications (NCs)following liver transplantation (LT) ranges from 13% to 47%,especially within the first 2 weeks after surgery.The NCs include disturbance of consciousness,cerebrovascular disorders,infections,seizures and central pontine myelinolysis,[1]which contribute to a prolonged intensive care unit and/or in-hospital stay.Despite the remarkable improvement of surgical technique,the availability of immunosuppressive agents with low neurotoxicity and the development of perioperative management during the past decade,the incidence of NCs has not shown a significant decline.

Along with the improvements in understanding of the pathological effects of ischemic reperfusion,the important role of the mitochondrial ATP-sensitive potassium channel (mitoKATP) is becoming increasingly evident.Studies of mitoKATPin ischemic preconditioning of the heart have been reported as a causative factor.However,Bajgar et al[2]suggested that mitoKATPmay also play a key role in brain protection.His report also indicated that brain mitochondria contain six to seven times more mitoKATPper milligram of mitochondrial proteins than those in the liver or heart.Recently,many studies have demonstrated that mitoKATP,as a trigger or effector of brain ischemic preconditioning,may have a cerebral protective effect both in vivo and in vitro.[3,4]

Nicorandil,a clinically useful drug for the treatment of ischemic heart disease,has also been shown to reduce brain injury in animal models of brain ischemia in recent studies,and activation of mitoKATPunderlies this effect.[5,6]Clinical research has also indicated that nicorandil premedication has a cerebral protective effect in patients undergoing coronary artery bypass grafting with cardiopulmonary bypass.[7]We investigated in the present study whether nicorandil has a cerebral protective effect against ischemic reperfusion in patients undergoing LT.

Methods

Patients

Fifty patients,scheduled for LT,were selected from the Shanghai First People's Hospital,China from June 2008 to November 2009.The patients were randomly divided into a nicorandil group (group N) (n=25) in which patients received 10 mg nicorandil through a nasogastric tube 30 minutes before induction of anesthesia and a control group (group C) which received 10 mL normal saline (n=25).

Patients were not enrolled if they had hepatic encephalopathy or a low Mini-Mental State Examination(MMSE) score (below 24) preoperatively.Patients diagnosed with concurrent diseases such as renal insufficiency,diabetes mellitus,and neuroendocrine cell carcinoma were also excluded.Patients were excluded if severe reperfusion syndrome (mean arterial pressure below 60 mmHg sustained over 15 minutes) or renal insufficiency occurred perioperatively.

Anesthesia protocols

Midazolam 50 μg/kg,fentanyl 10-15 μg/kg,propofol 1-1.5 mg/kg,and pipecuronium 0.15 mg/kg were used for anesthesia induction.PETCO2was maintained within 32-35 mmHg during the operation.Anesthesia was maintained with 0.7-1.3 MAC iso flurane.Fentanyl and pipecuronium were administered according to clinical judgment.Fluid balance was adjusted with normal saline,succinylated gelatin and albumin.Autoreinfusion was used when applicable.Red blood cells were infused when hemoglobin was below 70 g/L.To maintain an intraoperative mean arterial pressure above 60 mmHg during reperfusion,vasoactive drugs such as phenylephrine or dopamine were used.Acid-base and electrolyte levels were balanced according to blood gas analysis during the operation.

Neuropsychological assessment

The MMSE was performed by an observer unaware of the grouping before anesthesia,and 3 and 7 days after the surgery in the intensive care unit.

Serum neruron-specific enolase (NSE) and S100β protein (S100β) analysis

Peripheral blood samples for NSE and S100β were obtained just before induction of anesthesia (T1),and at 12 (T2) and 36 hours (T3) after the surgery.Clotted blood was centrifuged within 10 minutes (4000 r/min)at 4 ℃.The serum was stored at -80 ℃ for subsequent analysis.ELISA kits were used at room temperature to determine serum NSE (CanAg Co.,Sweden) and S100β(Alpco Co.,USA) concentrations in 96-well plates.The results were calibrated by hemoglobin values:

Calibrated value=analytic result*100/hemoglobin value (g/L).[7]

Ethical approval

The study was carried out according to the principles of the Helsinki Declaration.It was approved on January 5,2008 (code number QY040101-2-04) by the Ethics Committee of Shanghai First People's Hospital and written informed consent was obtained from each participant.

Statistical analysis

The data were analyzed using the SPSS 17.0 statistical software package.Descriptive variables are expressed as mean±SD or median and range.Differences between the groups were assessed by Student's t test or the Mann-Whitney U test.Difference within the groups were assessed by ANOVA with Dunnett's post-test.A P value less than 0.05 was considered statistically significant.

Results

General information

During surgery,5 patients of each group were eliminated due to low blood pressure (mean arterial pressure below 60 mmHg sustained over 15 minutes) or renal insufficiency.Therefore,20 patients were left in each group.Among the 40 patients,25 suffered from liver cancer and 15 from liver cirrhosis.

There were no significant differences in age,gender,body weight,preoperative liver function (total serum bilirubin,Child-Pugh classification),operation time,anhepatic phase time,blood loss and infusion (including auto-reinfusion),and fluid infusion between the groups C and N (Table 1).Two patients in group N suffered from massive blood loss (22 000 mL and 20 000 mL).Because of good perioperative management including fluid infusion and the use of vasoactive drugs,no severe reperfusion syndrome occurred,thus the 2 patients were not eliminated.

Classic non-bypass orthotopic LTs were performed in all patients.FK506 and mycophenolic acid were selected as the postoperative immunosuppressive agents.

We investigated the mean arterial pressure,heart rate,central venous pressure,blood gases and hemoglobin of the 40 patients before the operation,at 10,30,and 60 minutes of the anhepatic phase,at 10 and 30 minutes of the new hepatic phase,and at the end of the operation.There were no significant differences in these data between the two groups (Tables 2,3).

MMSE scores

The MMSE scores at days 3 and 7 were lower than those at day 0 in group C (P＜0.05).However,there was no difference at days 3 and 7 compared with day 0 in group N.

There was no significant difference in MMSE scoresbetween the two groups at day 0.However,the MMSE scores at days 3 and 7 in group N were higher than those in group C (P＜0.05; Fig.1).

Table 1.Clinical characteristics of the study population

Serum NSE and S100β concentrations

Serum NSE concentrations at T2 and T3 were higher than the baseline (at T1) in both groups (P＜0.05),except for NSE at T3 in group N.Serum S100β concentration after the surgery was higher than the baseline (at T1) in both groups (P＜0.05).

There was no significant difference in serum NSE and S100β concentrations between the two groups at T1.However,the concentrations at T2 and T3 in group N were lower than those in group C (P＜0.05; Fig.2).

Fig.1.Comparison of MMSE scores of the two groups.Data were presented as mean±SEM.Day 0:before the surgery; day 3:3 days after the surgery; day 7:7 days after the surgery.*:P＜0.05,compared with day 0; # :P＜0.05,compared with group C.

Table 2.Perioperative hemodynamic change (each group n=20,mean±SD)

Table 3.Perioperative blood gas analysis and hemoglobin change (each group n=20,mean±SD)

Fig.2.Serum NSE and S100β concentrations in the two groups.Data were presented as mean±SEM.T1:before induction of anesthesia; T2:12 hours after the surgery; T3:36 hours after the surgery.＊:P＜0.05,compared with T1; #:P＜0.05,compared with group C.

Discussion

There are two principal findings in the present study.First,compared to day 0,the MMSE scores of patients in the control group at days 3 and 7 showed a significant decrease.However,the levels of NSE and S100β in blood increased significantly 12 and 36 hours after the surgery.These results indicate that neurological injury occurred in the early period of post LT.Second,nicorandil improved the postoperative MMSE scores and attenuated the increase of serum NSE and S100β,indicating its cerebral protective effect.

In order to obtain accurate data and to reduce misinterpretation,a corresponding strategy was adopted in our study.At first we strictly selected the participants.Patients with alcoholic or metabolic liver diseases,or preoperative renal insufficiency were not enrolled.[8]Those with hepatic encephalopathy or MMSE scores below 24 were also excluded because they may have increase NCs post-LT.[9,10]Patients were excluded if their reperfusion syndrome (mean arterial pressure below 60 mmHg sustained over 15 minutes) or renal insufficiency occurred perioperatively.Secondly this study was a prospective randomized,double blind,placebo-controlled trial.There were no significant differences in age,gender,body weight,preoperative liver function (serum total bilirubin,Child-Pugh classification),operation time,anhepatic phase time,blood loss and infusion,mean arterial pressure,heart rate,central venous pressure,blood gas and hemoglobin analysis between the two groups.

We used the MMSE as a neuropsychological index to assess mental status.The MMSE is a brief structured test of cognitive function.The total MMSE score is widely accepted as an indicator of the severity of cognitive impairment.[11]It is highly sensitive (87%),and highly specific (82%) in the detection of dementia.[12]The test has been widely used in clinical practice and in research.[11]

NSE and S100β proteins are neurobiochemical markers of brain damage.[13]NSE is an isoform of the glycolytic enzyme,enolase,and is found in neurons and neuroendocrine cells.Neurological injury is associated with neuronal dysfunction and vascular redistribution of brain NSE resulting in a measurable increase in serum NSE.[14]S100β protein is a dimeric acidic calciumbinding intracellular and extracellular protein found in astrocytes and Schwann cells.S100β serum levels indicate neurological injury and correlate with the severity of damage.[15,16]It has potential as a new,noninvasive indicator of brain damage.[15]

NCs are common after LT and become a major cause of high mortality in recipients.The etiology of NCs remains obscure,and is most likely multifactorial.[17]Poor clinical condition,hemodynamic instability,renal insufficiency and the toxic effects of immunosuppressive drugs such as cyclosporine and FK506 may cause a wide spectrum of NCs.[18]Data from other series indicate that alcoholic and metabolic liver diseases,pre-LT mechanical ventilation,model for end-stage liver disease (MELD)score ＞15,and non-elective LT may be independent predictive factors for mental status changes after LT.[8]

Neurons contain two distinct KATPchannels:the classic one in the cell membrane (surface KATPchannel)and the other in the mitochondrial inner membrane(mitoKATPchannel).[2]Nicorandil is a selective mitoKATPchannel opener.Pooled dose-response data confirms that nicorandil concentration as low as 10 μg/L activates mitoKATPchannels.[19]Single-dose premedication with nicorandil can last 8 hours and may fully cover the period of low brain perfusion and reperfusion during surgery.Activation of mitoKATPmay inhibit oxidative stress-induced apoptosis in neurons.[20]This may also attenuate mitochondrial calcium overload which inhibits mitochondrial permeability transition pore opening during brain ischemia and reperfusion.[21]Further research indicates that opening mitoKATPchannels expressed in neurons protects primary mesencephalic neurons against MPP+-induced cytotoxicity by inhibiting reactive oxygen species overproduction and subsequently ameliorating mitochondrial function.[22]Recent studies have shown that activation of mitoKATPchannels improves the function of astrocytic gap junctions,indicating that the effect of astrocytic mitoKATPchannels on neurotoxin-induced neurodysfunction might be,in part,through regulation of the gap junction-coupled spatial buffering in the hippocampus.[23]However,the mechanism of mitoKATPactivation in neuronal protection remains obscure.

In summary,nicorandil,given orally as premedication before LT,improves postoperative MMSE score.It also attenuates the increase of NSE and S100β in blood,indicating a cerebral protective effect.However,these observations only cover the early period after LT.Further investigations of the long-term neuronal protective effect of nicorandil are required.

Acknowledgement:We appreciate helpful discussion with Prof.Ji-Jian Zhen who helped with the writing of this article.

Contributors:WZP proposed the study and wrote the first draft.XYF collected and analyzed the data.All authors contributed to the design and interpretation of the study and to further drafts.WZP is the guarantor.

Funding:This study was supported by a grant from the Shanghai Clinical Medical Center of organ transplantation (QY040101-2-04).

Ethical approval:The study on human participants was approved by the Ethics Committee of the Hospital and adhered to international ethical standards.

Competing interest:No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

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