Laser speckle contrast imaging and Oxygen to See for assessing microcirculatory liver blood flow changes following different volumes of hepatectomy

    Laser speckle contrast imaging and Oxygen to See for assessing microcirculatory liver blood flow changes following different volumes of hepatectomy Chong Hui Li, Xin Lan Ge, Ke Pan, Peng Fei Wang, Yi Nan Su, Ai Qun Zhang PII: DOI: Reference:

S0026-2862(16)30152-2 doi: 10.1016/j.mvr.2016.11.004 YMVRE 3662

To appear in:

Microvascular Research

Received date: Revised date: Accepted date:

18 September 2016 4 November 2016 7 November 2016

Please cite this article as: Li, Chong Hui, Ge, Xin Lan, Pan, Ke, Wang, Peng Fei, Su, Yi Nan, Zhang, Ai Qun, Laser speckle contrast imaging and Oxygen to See for assessing microcirculatory liver blood flow changes following different volumes of hepatectomy, Microvascular Research (2016), doi: 10.1016/j.mvr.2016.11.004

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ACCEPTED MANUSCRIPT Laser speckle contrast imaging and Oxygen to See for assessing microcirculatory liver blood flow changes following different volumes of

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hepatectomy

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Chong Hui Li*, Xin Lan Ge, Ke Pan, Peng Fei Wang, Yi Nan Su, Ai Qun Zhang Institute of Hepatobiliary Surgery, Chinese PLA General Hospital, Chinese PLA Medical School, Beijing, China

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* Corresponding author: Institute of Hepatobiliary Surgery, Chinese PLA General Hospital, 28 Fuxing Road, Beijing 100853, China. E-mail address: Lich_ [email protected] (C.H.Li).

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Abstract

Objective: Portal hyperperfusion after extended hepatectomy or small-for-size liver transplantation may

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induce organ dysfunction and failure. This study was designed to monitor and characterize the hepatic

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microcirculatory perfusion following different volumes of hepatectomy in rats by using laser speckle

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contrast image (LSCI) and Oxygen to See (O2C), a spectrometric device. Methods: The microcirculatory liver blood flow of the rats that underwent 68%, 85% and 90% hepatectomy (68PH, 85PH and 90PH) was monitored with LSCI and O2C before and following the

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hepatectomy. The portal venous flow (PVF) and hepatic arterial flow (HAF) were measured with an ultrasonic flowmeter. Liver regeneration, liver injury, histologic evaluation and gene expression were also assessed at 12 h, 24 h, 3d and 7d post hepatectomy. Results: All the 68PH and 85PH rats survived, and 57% of the 90PH rats survived. After hepatectomy, both PVF and HAF decreased transiently, with the PVF of the 85PH and 90PH rats significantly lower than that of the 68PH rats. In contrast, the PVF and HAF per gram of liver weight were greatly increased after liver resection and were proportional to the volume of resected liver. Correspondingly, the microcirculatory liver blood flow of the 68PH, 85PH and 90PH rats, as assessed by both LSCI and O2C, were increased after hepatectomy, and the 90PH group was significantly higher than the 68PH and 85PH groups. The hyperperfusion continued for approximately 3 days and returned to baseline following the completion of liver regeneration. The liver venous oxygen saturation of the three groups decreased immediately after hepatectomy and returned to baseline from 24 h after hepatectomy. The 1

ACCEPTED MANUSCRIPT 90PH rats also showed delayed liver regeneration and the most severe liver injury, as reflected by increased serum ALT, AST and TBIL levels, hepatocellular vacuolization, and inflammatory and

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endothelial constriction gene expressions (TNF-, IL-1, MIP-1, ET-1 and TM-1).

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Conclusion: Hepatic microcirculation hyperperfusion resulting from major and extended liver

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resection could be assessed by LSCI and O2C methods. The 90PH in rats led to extraordinary sinusoidal hyperperfusion, severe endothelial injury and liver failure. Monitoring the changes of hepatic microcirculation perfusion following extended hepatectomy or small-for-size liver

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transplantation may help to analyze the extent of hyperperfusion.

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Keywords: Microcirculatory liver blood flow, Hepatectomy, Laser speckle contrast image, Oxygen to

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See (O2C)

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ACCEPTED MANUSCRIPT Introduction Extended hepatectomies and small-for-size liver transplantations result in portal hyperperfusion, which is a stimulus for liver regeneration but may simultaneously cause postoperative liver failure

strategy is currently available to improve the hepatic regeneration.

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(Garcea and Maddern, 2009; Gonzalez et al., 2010). However, no clinically established treatment

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The marked hemodynamic changes following hepatectomy or small-for-size liver transplantation may damage the hepatic microcirculation, which is an extended determinant for the development of liver failure (Sidler et al., 2008), but how the microcirculatory liver blood flow (microLBF) is related to

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the amount of liver resection is not known. Monitoring the changes of the hepatic microcirculatory perfusion following major or extended hepatectomy will help to further characterize the association of

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microLBF with liver failure.

Intravital fluorescence microscopy represents the standard method for the assessment of

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microcirculatory impairment in animal experiments (Vogten et al., 2003; Abshagen et al., 2006).

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However, intravital fluorescence microscopy is not suitable for the thick liver lobes in rats, pigs and humans. Currently, laser speckle contrast imaging (LSCI) (Basak et al., 2012) provides non-contact

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full-field imaging over wide areas with excellent spatial and temporal resolutions. LSCI technology is a simple, convenient and accurate method for the real-time monitoring of microLBF changes during ischemia and reperfusion (Sturesson et al., 2013; Li et al., 2014). The spectrometric device Oxygen to

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See (O2C) is a diagnostic device for the non-invasive determination of tissue perfusion, capillary-venous oxygen saturation and blood filling of microvessels at the same time. Pilot studies have proved that the O2C method allowed a reproducible intraoperative evaluation of the hepatic microcirculation and predicted organ function after kidney transplantation (Ladurner et al., 2009; Fechner et al., 2009). A 70% major hepatectomy is a typical and commonly used liver regeneration model, compared with 85% and 90% hepatectomies, which are both considered to be extended hepatectomies. In addition, all rats that underwent 85% hepatectomy survived, but 90% hepatectomy caused rats to die of liver failure (Emond et al., 1989; Zhang et al., 2015; Eipel et al., 2010). This study was designed to monitor and characterize the changes of hepatic hemodynamic and microcirculatory perfusion of rats that underwent 68%, 85% and 90% liver resections by using LSCI and O2C. The comparison between these models will be helpful in analyzing how the changes in increased hepatic microcirculation perfusion are related 3

ACCEPTED MANUSCRIPT to liver regeneration, injury and failure. The results may guide clinical liver resection, elucidate an unexplored aspect of pharmacological modulation of extended liver resection-induced liver failure, and

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motivate further experiments.

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ACCEPTED MANUSCRIPT Materials and methods Experimental animals Male Wistar rats (body weight 270–330 g) were purchased from the Animal Center of the

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Academy of Military Medical Science (Beijing, China). All experimental procedures were carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animal of

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the National Institutes of Health and were approved by the Committee on Ethics of Animal Experiments of the Chinese PLA General Hospital.

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Study design

We focused our experiment on the hemodynamic and microcirculatory changes of the remnant

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liver after different volumes of liver resection. Rat hepatectomies of 68%, 85% and 90% of total liver weight (68PH, 85PH and 90PH) were performed on 91 rats in this experiment. 28 rats were used in

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68PH group and 85PH group respectively, and 35 rats were used in 90PH group all together. Another 6

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rats were included as sham operation (Sham) group. The portal venous flow (PVF) and hepatic artery flow (HAF) were assessed using an ultrasonic flow meter. The microcirculatory liver blood flow

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(microLBF) was measured with LSCI and the spectrometric device O2C, respectively. Firstly, the measurements were performed at 5 min after laparotomy, which was taken as the baseline (Pre-PH), and at 10 min after the completion of the hepatectomy (10 min-Post-PH) for each rat. Then the

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measurements were performed at 12 h, 24 h, 3 d and 7 d after hepatectomy (n=6 for each time point of each group). However, for the time point of 7 d, at least 10 rats were used in each group for the determination of 7-day survival rate. When death occurred in 90PH group more rats were supplemented. From the time point of 12 h after hepatectomy, the rats were euthanized after all measurements in order to avoid the effects of tissue adhesion, repeat anesthesia and laparotomy. Then, blood and liver tissue were collected when the rats were sacrificed by euthanasia at the indicated time points after all measurements.

Surgical procedure After overnight fasting but with free access to water, the rats were anaesthetized with a continuous 1.5 vol % isoflurane/oxygen inhalation. As shown in Fig.1, 68PH was performed by the resection of the left lateral lobe (30 %) and left and right median lobes (38 %). For 85PH, the left lateral, median, 5

ACCEPTED MANUSCRIPT inferior right lobe (10 %) and anterior and posterior caudate lobes (8 %) were resected. For 90PH, all liver lobes except the caudate lobe (8 %) and caudate process (2-3 %) were resected. The resection was performed using the technique by Kubato with slight modifications [Kubota et al., 1997; Zhang et al.,

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2015]. Briefly, following a large midline incision, the liver was exposed and detached from the surrounding tissues. The portal vein and hepatic artery branches entering each liver lobe to be resected

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were ligated with 6-0 silk separately at the position of the liver parenchyma near the lobe pedicle. Then the pedicles of the lobes were ligated with 4-0 silk in two or three parts by piercing the liver parenchyma near the inferior vena cava. Finally, the ligated liver lobes were resected and weighted. The

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abdominal muscles and peritoneal sin were closed by continuous suture. Postoperatively, the animals were allowed to recover in warm boxes with free access to food and water ad libitum. To improve the

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survive rate of the 90PH rats, glucose that was dissolved in water to a final concentration of 20% was

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also administered to the rats immediately after surgery (Emond et al., 1989).

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Fig. 1. Graphical illustration of different volumes of rat hepatectomy. White lobes represent the resected liver lobes, and red lobes represent the reserved liver lobes after 68%, 85% and 90% partial hepatectomy (68PH, 85PH and 90PH). L, left lateral lobe; LM, left median lobe; RM, right median lobe; SR, superior right lobe; IR, inferior right lobe; AC, anterior caudate lobe; PC, posterior caudate lobe; CP, caudate process.

Ultrasonic Flow Measurement The hepatic artery and portal vein were separated from the surrounding tissue with microsurgical technique. An ultrasonic perivascular flow probe (0.5PSB; Transonic Systems, Ithaca, NY) was placed around the hepatic artery. The flow probe was connected to a flow meter (T206 Animal Research Flowmeter; Transonic Systems, Ithaca, NY). HAF was recorded for 1 minute at each observation time 6

ACCEPTED MANUSCRIPT point. Then, a 2.0PSB flow probe was positioned around the portal vein, and PVF was recorded accordingly.

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LSCI assessment of microLBF

MicroLBF was assessed using the PeriCam PSI System (Perimed AB, Sweden) according to the

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method of our previous study (Li et al., 2014) with modification. The distance between the scan head and the liver surface was approximately 15cm. The scanning area was 3.5×3.5 cm. The sampling frequency was 21 images per second, and the image acquisition rate was one frame per second in the

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normal resolution mode. The duration of recording was 15 seconds. Steady anesthesia with a continuous 1.5 vol % isoflurane/oxygen inhalation was kept during recording. The manufacturer's

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software PIMSoft was used to analyze the images and to quantify the perfusion in arbitrary laser speckle perfusion units (LSPU) as in Fig.2A . The size of region of interest (ROI) was 20-50 mm2. One

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or two ROIs were placed on the LSCI images of selected liver lobe. Artifact spots or areas where the

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flux value was not accurately measured were excluded from the ROIs.

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O2C assessment of liver microvascular perfusion Liver microvascular perfusion was also assessed using the spectrometric device O2C (LEA Medizintechnik Giessen, Germany). A specially designed probe (LFX-53) for rat liver was used with

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laser separation 1.2 mm and white separation 1.2 mm (Fig.2B). The flat probe with a diameter of 15 mm can be fixated to the liver surface by normal adhesive forces (Fig.2C).

The O2C can provide the

following four parameters of organ microcirculation: relative blood flow (Flow) [AU], blood flow velocity (Velo) [AU], venous oxygen saturation (SO2) [%], and relative tissue hemoglobin concentration (rHb) [AU]. Every point was recorded for 10 s according to a protocol suggestion, and six points were measured for each liver lobe.

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ACCEPTED MANUSCRIPT

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Fig.2. Assessments of liver microvascular blood perfusion of rat with LSCI and O2C. A: representative LSCI images and the setting of regin of interestin (ROI) of rat liver before partial hepatectomy (Pre-PH) and after 68%, 85% and 90% partial hepatectomy (Post-68PH, Post-85PH and

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Post-90PH). B: an O2C probe for rat liver use. C: the O2C probe fixated to the liver surface by normal adhesive forces.

L, left lateral lobe; LM, left median lobe; RM, right median lobe; SR, superior right

lobe; AC, anterior caudate lobe.

Liver regeneration The regenerating remnant liver lobes were weighted immediately after removal when the rats were sacrificed. The liver regeneration was calculated as the weight ratio of the regenerating liver to the whole liver before hepatectomy, which was estimated using the liver weight of the excised left and middle lobes divided by 0.68. The protein expression of Ki-67 was additionally assessed by immunohistochemistry as a marker to quantitatively rate hepatic regeneration. The fixed liver tissues were embedded, sectioned, and immunostained with a mouse anti-Ki-67 monoclonal antibody (BD 8

ACCEPTED MANUSCRIPT Pharmingen, USA). All immunostains were visualized by 3,3-diaminobenzidine staining and were then counter-stained with hematoxylin. The Ki-67 labeling index was determined as the percentage of Ki-67

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positive hepatocytes per total number of hepatocytes in 5 random visual fields (magnification × 400).

Blood biochemical analysis

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Levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin (TBIL) were measured using the Cobas 8000 serum analyzer (Roche Diagnostics, Manheim,

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Germany).

Histological assessments

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Tissue samples were harvested at the end of the experiments from remnant liver lobes, fixed in formalin (4% in phosphate-buffered saline) for 2 to 3 days at 4℃, and embedded in paraffin.

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Dehydrated, paraffin-embedded 5 µm sections were stained with hematoxylin-eosin and were analyzed

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under light microscopy. The hepatocytic necrosis, vacuolization, steatosis and liver sinusoids were

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observed in a blinded manner.

Quantitative reverse transcription real-time polymerase chain reaction (qRT-PCR) Total RNA was extracted from the frozen liver tissues using the RNASimple Total RNA Kit

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(Tiangen Biotech, Beijing, China), according to the manufacturer’s protocol. 4 g of RNA was reverse-transcribed with a RevertAid First Strand cDNA Synthesis Kit using Oligo-dT primers (Fermentas International Inc., Burlington, Canada). qPCR were performed with the SYBR Premix Ex Taq II (TakaRa Bio Inc, Dalian, China) on the StepOnePlus Real Time PCR System (Applied Biosystems CA, USA). The primers are shown in Table 1. The relative gene expressions were normalized to HPRT, calculated using the 2-Ct method as previously described (Livak and Schmittgen, 2001), and expressed as fold changes versus the Sham group. The analysis and quantification was done in a blinded manner by two individuals.

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Forward(5′- 3′)

Reverse(5′- 3′)

TNF-α

AAATGGGCTCCCTCTCATCAGTTC

TCTGCTTGGTGGTTTGCTACGAC

IL-1 β

CACCTCTCAAGCAGAGCACAG

GGGTTCCATGGTGAAGTCAAC

MIP-1

GCGCTCTGGAACGAAGTCT

GAATTTGCCGTCCATAGGAG

ET-1

CATCTGGGTCAACACTCCCG

TM-1

ACCAGTCGCCTCCACTTT

eNOS

TATTTGATGCTCGGGACTGC

HPRT

GCTGAAGATTTGGAAAAGGTG

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Genes

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Table 1 Primer sequences used for gene expression analysis by qRT–PCR

GGCATCTGTTCCCTTGGTCT TTCTCGCACGGCTTCTC AAGATTGCCTCGGTTTGTTG

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AATCCAGCAGGTCAGCAAAG

Statistical analysis

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All results are expressed as the mean  standard deviation. Comparisons of different groups of hepatectomy and Sham group were performed by one-way ANOVAs and post hoc tests of multiple

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comparisons after the normality and equal variance across groups and time points were tested using

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SPSS version 17.0 (SPSS Inc., Chicago, IL). Tukey test was used for equal variances, and Games-Howell test were performed if an equal variance test failed. Independent samples T-test was

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used for comparison of different time points post hepatectomy with pre-heptectomy. Values of P<0.05 were considered to be significant.

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Results

Liver regeneration after different volumes of liver resection All the rats that experienced 68PH and 85PH survived the observation period of 7 days. In contrast, the survival rate of the 90PH group was approximately 23%, which has been observed in our previous studies (Zhang et al., 2015; Ren et al., 2015), and the 7-day survival rate was improved to 57% (8/14) by the supplement of glucose in drinking water after hepatectomy. Most deaths of the 90PH rats occurred between 24 h and 72 h after the liver resection. Therefore, compared with the commonly used 68PH of the major hepatectomy model, 85PH was considered as a massive hepatectomy model without causing death while 90PH was a massive hepatectomy model that caused death because of liver failure.

The remnant liver grew constantly after the hepatectomy, returning to almost pre-operative weight at day 7 upon resection for the 68PH and 85PH groups (Fig. 3A). The remnant liver of the 90PH group increased significantly slower than did both the 68PH group and the 85PH group within the first 10

ACCEPTED MANUSCRIPT day post-hepatectomy. At 24 h after hepatectomy, the Ki-67 label index in the regenerating liver of 85PH group was significant higher than the other two groups, and the 90PH group had the lowest Ki-67 label index among the three liver resection groups (Fig. 3B, C). In the 90PH group, some rats displayed

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Liver regeneration

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considerably less Ki-67 positive hepatocytes than the others, as shown in Fig. 3C 90PH(1) and 90PH(2).

Fig. 3 Liver regeneration of rats after 68%, 85% and 90% partial hepatectomy (68PH, 85PH and 90PH) at 1, 3 and 7 days after hepatectomy. A: Ratio of the weight of regenerating liver to pre-hepatectomy whole liver. B: Ki-67 label index in the liver of Sham, 68PH, 85PH and 90PH rats. Values of six animals per group and time point are given in means±SD. ANOVA and post hoc comparison: && P<0.01 versus Sham, * P<0.05 and **P<0.01 versus 68PH, ## P<0.01 versus 85PH. C: Immunostains show Ki-67 positive hepatocytes in the liver of Sham, 68PH, 85PH and 90PH rats at 1 d after hepatectomy. 90PH (1) and (2) show the individual differences within the 90PH group. Hepatic hemodynamic changes after different volumes of liver resection The PVFs of the 68PH, 85PH and 90PH rats were measured with an ultrasonic flowmeter before and during liver regeneration. After hepatectomy, all PVFs decreased immediately, compared with the baseline (Fig. 4A), and the PVFs of the 90PH and 85PH rats decreased more than that of the 70PH rats. All the PVFs recovered to almost the level of pre-hepatectomy at 12 h and 24 h after hepatectomy. 11

ACCEPTED MANUSCRIPT However, when PVF was calculated as per gram of liver weight (Fig. 4B), partial hepatectomy resulted in several folds of increases of PVF, compared with pre-hepatectomy, and great differences were observed between the three groups. The value of PVF as ml/min/g liver tissue was positively correlated

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to the volume of resected liver, and this value remained high at 24 h after hepatectomy. The HAFs of the 68PH, 85PH and 90PH rats were also measured with an ultrasonic flowmeter

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before and after hepatectomy. Hepatectomy resulted in a significant decrease of HAFs in comparison with pre-hepatectomy (Fig. 4C, D), and no significant difference was found among the three groups (Fig. 4C). The same as PVF, the value of HAF as ml/min/g liver tissue was also positively correlated to

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the volume of resected liver, and a significant difference was observed between the three groups (Fig. 4D). Because the rat hepatic artery was very tenuous and the tissue adhesion occurred after operation, it

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was not possible to measure HAF during liver regeneration.

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Fig. 4. Portal venous blood flow (PVF) and hepatic artery blood flow (HAF) were measured with an ultrasonic flowmeter before and after 68%, 85% and 90% partial hepatectomy (68PH, 85PH and 90PH). A: PVF for the total liver; B: PVF per gram of liver weight; C: HAF for the total liver; D: HAF per gram of liver weight. Values of six animals per group and time point are given in means±SD. T-test 12

ACCEPTED MANUSCRIPT and ANOVA with post hoc comparison: & P < 0.05 and && P< 0.01 versus Pre-PH; * P < 0.05 and **P < 0.01 versus 68PH group; # P < 0.05 and ## P<0.01 versus 85PH group. Microcirculatory liver blood flow changes monitored by LSCI

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The microLBF of the rats with different volumes of liver resection was first assessed by the LSCI method (Fig. 5). All the microLBFs increased significantly after hepatectomy, compared with baseline,

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and the elevated microLBF continued for about three days and then decreased to baseline levels at day 7 after hepatectomy. There was a similar change of microLBF for the 68PH and 85PH groups following liver resection. However, the 90PH group showed significantly higher microLBF value than the 68PH

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and 85PH groups at 10 min and 3 days post hepatectomy, suggesting a higher extent of hyperperfusion in the 90PH group. In addition, the large standard deviation of the microLBF value of the 90PH group

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at 12 h and 24 h post hepatectomy suggested individual variations in the 90PH rats.

microLBF 68PH 85PH 90PH

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Fig. 5. Microcirculatory liver blood flow (microLBF) was measured with the LSCI method before and after 68%, 85% and 90% partial hepatectomy (68PH, 85PH and 90PH). Values of six animals per group and time point are given in means±SD. T-test and ANOVA and post hoc comparison: && P< 0.01 versus pre-hepatectomy; *P < 0.05 and **P < 0.01 versus 68PH group; #P < 0.05 and ## P<0.01 versus 85PH group. Hepatic microcirculation changes monitored by O2C method The spectrometric device O2C was applied to monitor the hepatic microcirculation perfusion flow (Flow), the blood flow velocity, the venous oxygen saturation (SO2) and the relative tissue hemoglobin concentration (rHb), as shown in Fig. 6. Accordingly, 68%, 85% and 90% hepatectomy resulted in significant increases of O2C Flow and Velo, and the increases continued for three days following 13

ACCEPTED MANUSCRIPT hepatectomy. The O2C Flow of the 90PH group was significant higher than the 68PH and 85PH groups. However, the Velo was comparable for the three groups (Fig. 6A, B). Despite the microvascular hyperperfusion post-hepatectomy, the hepatic tissue SO2 was significantly decreased (Fig. 6C) at 10

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much before or after the different volumes of hepatectomy (Fig. 6D).

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min-post-PH, and the decrease was comparable in the three groups. In addition, the rHb did not change

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Fig. 6 Hepatic microcirculation perfusion flow (Flow) (A), blood flow velocity (B), the venous oxygen saturation (SO2) (C) and relative tissue hemoglobin concentration (rHb) (D) were measured with the spectrometric device O2C before and after 68%, 85% and 90% partial hepatectomy (68PH, 85PH and 90PH). Values of six animals per group and time point were given in means±SD. T-test and ANOVA and post hoc comparison: & P<0.05 and && P<0.01 versus pre-hepatectomy; * P<0.05 and **P<0.01 versus the 68PH group; # P<0.05 and ## P<0.01 versus the 85PH group. Assessment of liver injury The serum levels of AST, ALT, and total bilirubin (TBIL) in the rats with different volumes of hepatectomy are shown in Fig. 7A. 85PH and 90PH induced more severe liver injury than did 68PH, which was reflected by significantly increased AST, ALT and TBIL levels at 12 h after hepatectomy. The levels of AST and TBIL were extraordinarily higher in the 90PH group than in the 85PH and 68PH groups at 24 h after hepatectomy. 14

ACCEPTED MANUSCRIPT Analysis of the H&E-stained remnant liver sections revealed that the histopathological differences between the 68PH, 85PH and 90PH rats became apparent mainly from 24 h post-hepatectomy, as shown in Fig.7B. Hepatocellular necrosis was not prominent in all groups. The obvious

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histopathological change was hepatocellular vacuolization in comparison with the Sham rats. In the 68PH group, irregular and large vacuoles resembling glycogen droplets were observed; in 85PH and

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90PH groups, small and round vacuoles resembling lipid droplets were observed. In addition, in the 90PH group, some rats with lower microLBF and more severe liver injury showed sinusoidal congestion. These results suggested that more severe liver injury and insufficiency of liver function

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might have induced liver failure and death in the 90PH group.

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Time after hepatectomy

Fig. 7. Serum levels of AST, ALT, and total bilirubin (TBIL) in the rats after 68%, 85% and 90% partial hepatectomy (68PH, 85PH and 90PH). Values are means±SD, n=6. ANOVA and post hoc comparison: & P<0.05 and && P<0.01 versus Sham group; * P<0.05 and **P<0.01 versus 68PH group; # P<0.05 15

ACCEPTED MANUSCRIPT and ## P<0.01 versus 85PH group. B: Representative H&E-stained remnant liver sections at 24 h after 68%, 85% and 90% partial hepatectomy (68PH, 85PH and 90PH). 90PH (1) and (2) show the individual differences within the 90PH group.

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Gene expression

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To further analyze the regulation of liver perfusion after major and extended hepatectomy, the

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mRNA expression of several genes involved in endothelial function and inflammatory response were detected in the remnant liver at 12 h and 24 h post-hepatectomy. Of interest, the gene expressions of endothelial regulatory gene endothelin-1 (ET-1), thrombomodulin-1 (TM-1) and endothelial nitric

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oxide synthase (eNOS), and inflammatory genes TNF-, IL-1 and MIP-1 were markedly up-regulated in the 90PH group and to a lesser extent in the 85PH group when compared to the Sham

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control and 68PH groups (Fig. 8). There were significant higher gene expressions in the 90PH group than in the 85PH group with respect to ET-1, TM-1, eNOS, IL-1 and MIP-1 at 12 h and/or 24 h post-hepatectomy. The expression of these genes in the 68PH group was only slightly increased,

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compared with the Sham group. In addition, the expression of up-regulated endothelial constriction

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genes ET-1 and TM-1 was extremely higher than the expression of the endothelial dilatation gene

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eNOS. These results demonstrated that the extent to which hyperperfusion correlated with endothelial regulatory gene expression.

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Fig. 8. Endothelial regulatory gene ET-1, TM-1 and eNOS, and inflammatory gene TNF-, IL-1 and MIP-1 expression in the rat livers with 68%, 85% and 90% partial hepatectomy (68PH, 85PH and 90PH) at 12 h and 24 h post-hepatectomy. Values are means±SD, n=6. ANOVA and post hoc comparison: & P<0.05 and && P<0.01 versus the Sham group; * P<0.05 and **P<0.01 versus the 68PH group; # P<0.05 and ## P<0.01 versus the 85PH group.

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ACCEPTED MANUSCRIPT Discussion In this study, the hepatic microcirculation following 68%, 85% and 90% hepatectomy in rats was assessed by the LSCI and the O2C techniques, respectively. Their possible application in monitoring

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the changes of hepatic microcirculation following major (68PH) and extended (85PH and 90PH) liver resection was analyzed. Both LSCI and O2C were superficial microvascular blood flow monitoring

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methods. The former is noncontact and suitable for the measurement of large areas, and the later can provide SO2 and rHb simultaneously in addition to microcirculatory blood flow and velocity. The results demonstrated that both LSCI and O2C were able to assess hepatectomy-induced increases of

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microcirculatory blood flow in the remnant liver. 90PH resulted in significantly higher microcirculatory blood flow than did 68PH and 85PH. This extraordinary hyperperfusion after 90PH was subsequently

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related to more severe liver injury, inhibited liver regeneration and liver failure.

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Following 68PH, 85PH and 90PH, the total blood inflow including PVF and HAF was decreased

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transiently because of the obvious reduction of liver volume and vascular bed. Extended (85% and 90%) hepatectomy exerted more effects on the PVF than did major (68%) hepatectomy. From 12 h after

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hepatectomy, the PVF of all groups returned to the level of pre-hepatectomy. Hepatectomy also resulted in a significant decrease of the HAF immediately after hepatectomy, and there was no significant difference among the decrease in the three groups. Unfortunately, because the rat hepatic artery was

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very tenuous and because the tissue adhesion occurred after operation, it was not possible to measure the HAF during liver regeneration. The decrease of the HAF possibly resulted from the reduced liver volume or the hepatic artery “buffer” response (HABR). However, when the PVF and the HAF were calculated as per gram of liver weight, the liver blood inflow increased several folds, and there was a great difference between the 68PH, 85PH and 90PH groups. Therefore, although partial hepatectomy only temporally reduced the total liver blood inflow, the liver blood inflow per gram of liver weight was increased and continued to be very high during the early time of liver regeneration. The hepatectomy-induced hyperperfusion has been proposed to regulate liver regeneration, including hepatocyte proliferation and death (Nobuoka et al., 2006; Abshagen et al., 2012).

It has been revealed with in vivo influence microscopy that the sinusoidal perfusion rate approached almost 100% and thus partly compensated for the marked reduction of the microvessel 18

ACCEPTED MANUSCRIPT beds after hepatectomy (Abshagen et al., 2006). In addition, Dold S et al. (Dold et al., 2015) found that the diameters of the liver sinusoids and the postsinusoidal venules were significantly increased after both 70% and 90% hepatectomies. Accordingly, our results also proved that the hepatic microvascular

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blood flow was increased after 68PH, 85PH and 90PH, in particular after 90PH. However, they concluded that the portal hyperperfusion after extended hepatectomy did not induce HABR because

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they observed that the portal hyperperfusion and the hepatic arterial blood flow, expressed as per 100 gram of liver tissue after 70% and 90% hepatectomy, were unchanged. The HABR was referred to as the diminished total hepatic arterial blood flow upon increased total portal blood inflow, not as the liver

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blood flow as per gram of liver tissue. We found both the total PVF and HAF decreased at 10 min after 68-90% hepatectomy. Therefore, if HABR had occurred, it could not have been determined. However,

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it was reported that splenectomy improves survival by increasing arterial blood supply in a rat model of

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reduced-size liver, suggesting HABR had occurred after extended hepatectomy (Eipel et al., 2010).

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However, the changes of microLBF after 68PH, 85PH and 90PH were not very consistently

associated with the changes of total liver blood inflow. All the microLBFs of the 68PH, 85PH and

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90PH groups increased immediately after liver resection. In particular, the increases of microLBF in 68PH and 85PH were similar, and the increase of microLBF in 90PH was significant higher than the increases of 68PH and 85PH, as demonstrated by both the LSCI and the O2C measurements. These

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results indicated that a greater extent of microcirculatory hyperperfusion was observed in the 90PH group. Considering the 100% survival rate for 85PH and 57% for 90PH, the microLBF of 90PH might have exceeded the tolerant limit of the liver microvasculature. The result might have been more severe liver injury and sinusoidal endothelial dysfunction, as demonstrated by the higher levels of ET-1, TM-1, IL-1 and MIP-1 gene expression in the 90PH group than in the 85PH and 70PH groups. Additionally, sinusoidal congestion, hepatocytic vacuolization and even liver failure were induced subsequently. Therefore, it was possible to detect the extended hepatectomy-induced extra hyperperfusion as in 90PH rats by LSCI or O2C, although the tolerant limit of liver microvascular still needs to be determined.

The most obvious change occurring after partial hepatectomy was an elevation in hyperperfusion-induced hemodynamic forces imposed on the liver cells. These changes occurred immediately upon resection. Liver regeneration depends on an adequate hepatic microcirculation in the 19

ACCEPTED MANUSCRIPT remnant liver (Vollmar and Menger, 2009). We found that the microcirculatory hyperperfusion continued for at least for three days following the 68PH, 85PH and 90PH hepatectomies. The hyperperfusion in the 68PH and 85PH groups was slightly relieved at day 3 after hepatectomy when

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over 60% of liver regeneration was completed. However, at day 3 after hepatectomy, the microLBF of the 90PH group was still significantly higher than the other two groups and the baseline level. These

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results demonstrated that the change of liver microLBF was closely related to the liver volume during liver regeneration, and meanwhile the total blood inflow to the liver was relatively stable. Sinusoidal hyperperfusion drives the liver regeneration continually. However, when the sinusoidal perfusion

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exceeds a limit after extended hepatectomy, such as 90PH, hyperperfusion-induced liver injury will inhibit the liver regeneration, which was demonstrated by the results of the liver regeneration

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assessment.

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The liver regeneration, assessed by liver weight and Ki-67 label index, matched with the liver

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microcirculation changes in the 68PH and 85PH groups. The 85PH group showed a greater growth rate and higher ki-67 label index than did the 68PH group. However, it was complicated for the 90PH group.

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We found a high level of individual variation in the 90PH group with respect to the microLBF, liver injury markers, ki-67 label index, histopathological changes, and gene expression at 24 h after hepatectomy. In our experiment, the 90PH model displayed a survival rate of 57%, and a large number

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of the rats died within the period of 24 h to 48 h post-hepatectomy. Therefore, the presumably dying rats at 24 h after hepatectomy might have shown higher levels of TBIL, lower levels of microLBF, inhibited liver regeneration, and significant upregulated endothelial and inflammatory gene expression. These results further demonstrate that hepatic hyperperfusion-induced endothelial dysfunction and liver injury are important factors for extended liver resection-induced liver failure.

In this study, the results of the hepatic microcirculatory blood flow (Flow) monitored by the spectrometric O2C device were consistent with the results by the LSCI method, except the arbitrary value of the LSCI was much higher than that of the O2C because of the influence of breath movement and “biological zero” (Li et al., 2014). The advantage of the O2C is that a specially designed small flat probe for liver was used and could be positioned stably on the liver surface, thus avoiding the influence of breath-induced liver movement and increasing the sensitivity of the monitoring. This advantage was 20

ACCEPTED MANUSCRIPT illustrated by the lower and more significant difference of liver microcirculation blood flow between the 85PH and 90PH groups, as measured with O2C than with LSCI. The disadvantage is that the O2C can only measure the flow of a single point (less than 1 mm3), and an average of the Flow of several

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measurement points at one liver lobe was needed. However, LSCI measurements were averaged over large surface areas, thereby reducing the spatial variability of cutaneous microcirculation (Tew et al.,

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2011; Millet et al., 2011).

Another advantage of the O2C is that it can measure SO2 simultaneously with Flow. Opposite to

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the arterial oxygen saturation, the capillary-venous oxygen saturation SO2 shows the balance between oxygen delivery and consumption. Therefore, the local oxygen measurement is an ideal parameter to

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determine the condition of local tissue hypoxia. In this study, despite the microvascular hyperperfusion, hepatic tissue SO2 was significantly decreased after 68%, 85% and 90% hepatectomies. Dold S et al

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(Dold et al., 2015) also reported that hepatic tissue pO2 was significantly decreased after 70% and 90%

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hepatectomies. They also found that the decreased SO2 was not due to a deterioration of microvascular perfusion but rather due to a relative hypermetabolism of the remnant liver after extended resection

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(Abshagen et al., 2006). The decreased liver tissue SO2 may also suggest the occurrence of HABR after partial hepatectomy.

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The expression of the vasoregulatory gene by the sinusoidal endothelial cells, the pericyte-like hepatic stellate cells and the Kupffer cells are mandatory in regulating vasotonus during the process of liver regeneration and therefore in maintaining intrahepatic shear stress as a trigger of hepatic proliferation. ET-1 and TM-1 are potent vasoconstrictors of microcirculation that are mainly produced by sinusoidal endothelial cells, HSC and Kupffer cells in the liver. The constricting action of the endothelin balances with the dilating action of NO, which is produced constitutively by eNOS in the liver (Greene et al., 2003). The gene expression levels of ET-1 and TM-1 were found to be significantly higher (a 6- to 10- fold difference for ET-1 and 35- fold difference for TM-1) in the 90PH group than in the 68PH and 85PH groups at 12 h and 24 h after liver resection in our study. Additionally, the difference of eNOS expression between them was much less (only 1- to 2- fold difference). These alterations resulted in an obvious imbalance between the levels of ET-1, TM-1 and eNOS-produced NO in the 90PH group. The imbalanced elevations in ET-1 and TM-1 expression would have caused 21

ACCEPTED MANUSCRIPT vasoconstriction and enhanced leukocyte- and platelet-endothelium interactions in the liver, subsequently aggravating inflammatory gene expression and liver injury. Abshagen K et al (Abshagen et al., 2008) reported that selective and long-lasting Kupffer cell elimination limited the

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resection-associated hyperperfusion, and Kupffer cell-depleted mice showed a reduction of hepatic ET-1, eNOS and HO-1 expression, demonstrating the contribution of microvascular regulatory gene to

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portal hyperperfusion and subsequent liver injury after liver resection.

Important limitations of the LSCI and O2C techniques are that the perfusion unit is not an

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arbitrary unit and that absolute values for single parameters are lacking. The measured values cannot be interpreted without clinical background or baseline values. The next aim is to determine regular values

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for hepatic microcirculation in a homogenous patient cohort. The pilot studies have proved that the O2C method allowed a reproducible intraoperative evaluation of the hepatic microcirculation and

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predicted organ function after kidney transplantation, provides a starting point for establishing these

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values (Ladurner et al., 2009; Fechner et al., 2009).

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In conclusion, with this study we were able to demonstrate that hepatic microcirculation hyperperfusion resulting from major and extended liver resection could be assessed by the LSCI and O2C methods. Compared with nonlethal 70PH and 85PH in rats, extended hepatectomy with the risk of

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liver failure, such as with 90PH, resulted in an extraordinary high hepatic microcirculatory blood flow. This change in blood flow, which could be monitored by LSCI and O2C, can induce significantly increased expressions of vasoconstrictor factors such as ET-1 and TM-1. Although this is an animal study, these methods seem reproducible and reliable.

Acknowledgments This work was supported by the Natural Science Foundation of China (grant number 81271738) and the Natural Science Foundation of Beijing (grant number 7153173). References 1. Abshagen, K., Eipel, C., Menger, M.D., Vollmar, B., 2006. Comprehensive analysis of the regenerating mouse liver: an in vivo fluorescence microscopic and immunohistological study. J. Surg. Res. 134, 354-62. 22

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9. 10. 11.

12. 13.

14.

15. 16.

17.

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Abshagen, K., Eipel, C., Kalff, J.C., Menger, M.D., Vollmar, B.. 2008. Kupffer cells are mandatory for adequate liver regeneration by mediating hyperperfusion via modulation of vasoactive proteins. Microcirculation 15, 37–47. Abshagen, K., Eipel, C., Vollmar, B., 2012. A critical appraisal of the hemodynamic signal driving liver regeneration. Langenbecks Arch. Surg. 397, 579–590. Basak, K., Manjunatha, M., Dutta, P.K., 2012. Review of laser speckle-based analysis in medical imaging. Med. Biol. Eng. Comput. 50, 547–558. Dold, S., Richter, S., Kollmar, O., von Heesen, M., Scheuer, C., Laschke, M.W., Vollmar, B., Schilling, M.K., Menger, M.D., 2015. Portal hyperperfusion after extended hepatectomy does not induce a hepatic arterial buffer response (HABR) but impairs mitochondrial redox state and hepatocellular oxygenation. PLoS One 10, e0141877. Eipel, C., Abshagen, K., Ritter, J., Cantré, D., Menger, MD., Vollmar, B., 2010. Splenectomy improves survival by increasing arterial blood supply in a rat model of reduced-size liver. Transpl. Int. 23, 998-1007. Emond, J., Capron-Laudereau, M., Meriggi, F., Bernuau, J., Reynes, M., Houssin, D., 1989. Extent of hepatectomy in the rat. Evaluation of basal conditions and effect of therapy. Eur. Surg. Res. 21, 251-9. Fechner, G., von Pezold, J., Luzar, O., Hauser, S., Tolba, R.H., Müller, S.C., 2009. Modified spectrometry (O2C device) of intraoperative microperfusion predicts organ function after kidney transplantation: a pilot study. Transplant. Proc. 41, 3575-9. Garcea, G., Maddern, G.J., 2009. Liver failure after major hepatic resection. J. Hepatobiliary Pancreat. Surg. 16, 145-55. Gonzalez, H.D., Liu, Z.W., Cashman, S.,Fusai, G.K., 2010. Small for size syndrome following living donor and split liver transplantation. World J. Gastrointest. Surg. 2, 389－394. Greene, A.K., Wiener, S., Puder, M., Yoshida, A., Shi, B., Perez-Atayde, A.R., Efstathiou, J.A., Holmgren, L., Adamis, AP., Rupnick, M., Folkman, J., O'Reilly, M.S., 2003. Endothelial-directed hepatic regeneration after partial hepatectomy. Ann. Surg. 237, 530–535. Kubota, T., Takabe, K., Yang, M., 1997. Minimum sizes for remnant and transplanted livers in rats. J. Hepatobiliary Pancreat. Surg. 4, 398–403. Ladurner, R., Feilitzsch, M., Steurer, W., Coerper, S., Königsrainer, A., Beckert, S., 2009. The impact of a micro-lightguide spectrophotometer on the intraoperative assessment of hepatic microcirculation: a pilot study. Microvasc. Res. 77, 387-8. Li, C.H., Wang, H.D., Hu, J.J., Ge, X.L., Pan, K., Zhang, A.Q., Dong, J.H., 2014. The monitoring of microvascular liver blood flow changes during ischemia and reperfusion using laser speckle contrast imaging. Microvasc. Res. 94,28-35 Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25, 402–408. Millet, C., Roustit, M., Blaise, S., Cracowski, J.L., 2011. Comparison between laser speckle contrast imaging and laser Doppler imaging to assess skin blood flow in humans. Microvasc. Res. 82,147–151. Nobuoka, T., Mizuguchi, T., Oshima, H., Shibata, T., Kimura, Y., Mitaka, T., Katsuramaki, T., Hirata, K., 2006. Portal blood flow regulates volume recovery of the rat liver after partial hepatectomy: molecular evaluation. Eur. Surg. Res. 38, 522–532. Ren, W., Wang, X., Zhang, A., Li, C., Chen, G., Ge, X., Pan, K., Dong, J.H., 2015. Selective

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2.

23

ACCEPTED MANUSCRIPT

T

IP

SC R

NU

24.

MA

23.

D

22.

TE

21.

CE P

20.

AC

19.

bowel decontamination improves the survival of 90% hepatectomy in rats. J. Surg. Res. 195,454-64. Sidler, D., Studer, P., Küpper, S., Gloor, B., Candinas, D., Haier, J., Inderbitzin, D., 2008. Granulocyte colony-stimulating factor increases hepatic sinusoidal perfusion during liver regeneration in mice. J. Invest. Surg. 21, 57-64. Sturesson, C., Milstein, D.M., Post, I.C., Maas, A.M., van Gulik, T.M., 2013. Laser speckle contrast imaging for assessment of liver microcirculation. Microvasc. Res. 87, 34–40. Tew, G.A., Klonizakis, M., Crank, H., Briers, J.D., Hodges, G.J., 2011. Comparison of laser speckle contrast imaging with laser Doppler for assessing microvascular function. Microvasc. Res. 82,326–332. Vogten, J.M., Smakman, N., Voest, E.E., Borel Rinkes, I.H., 2003 Intravital analysis of microcirculation in the regenerating mouse liver. J. Surg. Res. 113, 264-9. Vollmar, B., Menger, M.D., 2009. The hepatic microcirculation: mechanistic contributions and therapeutic targets in liver injury and repair. Physiol. Rev. 89, 1269-339. Zhang, D.X., Li, C.H., Zhang, A.Q., Jiang, S., Lai, Y.H., Ge, X.L., Pan, K., Dong, J.H., 2015. mTOR-dependent duppression of remnant liver regeneration in liver failure after massive liver resection in rats. Dig. Dis. Sci. 60, 2718-29.

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Highlights

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1. First use of LSCI and O2C for assessing hepatectomy-induced changes of liver microLBF

2. Both LSCI and O2C could be used to monitor the excessive increase of microLBF

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after hepatectomy

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3. Liver venous oxygen saturation decreased transiently after 68%, 85% and 90% hepatectomy

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4. Extended hepatectomy also led to severe liver injury, delayed liver regeneration and

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liver failure

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