Functional recovery after liver resection - Chapter 5 Hepatobiliary function assessed by Tc-99m mebrofenin cholescintigraphy in the evaluation of severity of steatosis in a rat model

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Functional recovery after liver resection

Veteläinen, R.L.

Publication date

2006

Link to publication

Citation for published version (APA):

Veteläinen, R. L. (2006). Functional recovery after liver resection.

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Reetaa L. Vetelainen

Roeloff J. Bennink

Koraa de Bruin

Arlènee van Vliet

Thomass M. van Gulik

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-INTRODUCTION N

Thee liver has a unique regeneration capacity after various types of liver damage (7). Therefore,, the importance of liver regeneration as the basis of treatment for many liver diseasess is growing (7). Basic research aims at a better understanding of the regulatory mechanismss governing liver regeneration eventually leading to new therapeutic strategies forr encouraging liver regeneration and avoiding fibrosis (7). In vivo experiments however, aree important because there have been found to be significant differences in physiological responsess of hepatocytes in culture (2). Therefore, multiple animal models have been describedd {1,3,4)- The ultimate choice of animal species depends on the scientific problem.

However,, mice and rats are frequently used because they are easy to manage and present minimall logistical problems (7).

Noninvasivee evaluation of global and regional liver function in animal models remains a challenge.. A real time test of liver function should give a measure of current hepatocellular capacity,, rather than reflect past damage (5). Standard tests for liver enzymes and bilirubinn plasma levels are not appropriate for describing actual liver function before and afterr surgery {6,7) or chronic liver conditions (8). Therefore, quantitative liver function testss have been evaluated (5). These tests however, show global liver function without informationn on regional function distribution.

Recently,, hepatobiliary scintigraphy (HBS) has been shown to be able to assess total

andd regional liver function, both for uptake and excretion (9). Technetium-99m (99mTc)

mebrofeninn HBS has been used extensively in larger animals for the measurement of hepatocellularr function. HBS has been performed successfully in rats suggesting a role forr scintigraphic liver function assessment {8,10). However, these experiments were performedd on routine gamma cameras without calculation of a hepatic clearance rate. Thee aim of this study was to validate liver function assessment with dedicated pinhole HBSS in rats. To illustrate an application of this technique, liver function was assessed in 2 surgicall models of liver regeneration.

MATERIALSS AND METHODS

Studyy Design

Alll animal experiments were performed with approval of the Animal Research Ethics Committeee of the University of Amsterdam and following its guidelines. Male specific-pathogen-freee Wistar rats (250-300 g) were purchased from Harlan (Zeist, The Netherlands).. The animals were acclimatized minimal 7 days to laboratory conditions at

constantt C with a 12h light and dark cycle. The animals were fed a standard rodent

choww and water ad libitum.

CharacterizationCharacterization and Normal Values. 12 rats were anaesthetized and injected intravenously withh 99mTc-labeled (2,4,6 trimethyl-3-bromo) iminodiacetic acid (99mTc-mebrofenin). Upon

injection,, a dynamic acquisition was started. The hepatic mebrofenin uptake rate was calculatedd twice on separate days by the same observer to establish a normal range and thee reproducibility of processing. Furthermore, a hepatic time-activity curve was made

forr determination of the time at which maximal hepatic activity occurred (Tmax), as welll as the time required for peak activity to decrease by 50% (TV2). After scintigraphy,

bloodd was collected by heart puncture, centrifuged (10 min, 3,000 RPM, ) and plasma

sampless were immediately analyzed using standard laboratory methods. The degree of hepatocellularr injury was assessed by serum levels of AST and ALT. For evaluation of hepatocellularr synthesis function, plasma levels of albumin and prothrombin time (PT) weree used. Livers were removed and weighed.

Reproducibility.Reproducibility. 3 groups of 3 rats were anaesthetized and scanned on 3 separate days to assesss the reproducibility of scintigraphic liver function testing. The hepatic mebrofenin

uptakee rate was calculated as well as the Tmax and TVi.

Intervention// Surgical Procedure

Eighteenn rats were randomized into 2 experimental groups. Surgery was performed under inhalationn anesthesia by a mixture of 02/N20 (1:1 V/V, 2 L/min) and isoflurane (1-2 %

Florene,, Abbott laboratories Ltd, Queensborough, UK} and pain medication buprenorphine (Temgesicc i.v. 0,033 mg/ 0,1 kg).

Too induce liver regeneration by hypertrophy, 2 commonly used surgical models were used. Inn the first group (n = 9), 70 % hepatectomy was performed to assess liver function duringg liver hypertrophy (11). In the second group (n = 9), a simplified and standardized methodd of portal vein embolization was used for evaluation of both the liver atrophy andd hypertrophy. A ligation of the portal vein to the median and left lateral liver lobes wass performed, comprising the portal blood perfusion of 70% of total liver mass (12). In bothh groups, 3 rats were anaesthetized and scanned 1, 3 and 7 days after surgery. After imagining,, blood was collected by heart puncture. The degree of hepatocellular injury wass assessed by serum levels of ALT. For evaluation of hepatocellular synthesis function, plasmaa levels of albumin were used. Livers were removed and weighed. The hepatic mebrofeninn uptake rate was calculated as well as the Tmax and TVa. Data was compared too measured liver weight.

Cameraa Design

Forr imaging of 99rnTc-mebrofenin uptake in the rat liver, a gamma camera (Philips ARC

3000,, Eindhoven, The Netherlands) situated in a dedicated animal care facility was equippedd with a pinhole collimator fitted with a 3 mm tungsten insert. The pinhole collimatorr is facing up. On the detector, a mechanical support was mounted in which the animall is fixed in a perspex cylinder positioned exactly above the pinhole collimator (Fig. 1).. The mechanical support was designed such that the midline of the cylinder is exactly in thee middle of the pinhole. The position of the animal is adjustable in the axial dimension. Furthermore,, the distance from the cylinder to the pinhole aperture is adjustable. Therefore,, this gantry permits optimal pinhole scintigraphy of anterior projections of rats standardizingg magnification and orientation. The gamma camera is interfaced to a NUD (Nuclearr Diagnostics, Stockholm, Sweden) Hermes acquisition station.

Scintigraphyy and Interpretation

Thee animals were sedated with ketamine/xyiazine i.m. (40 mg/kg and 2 mg/kg, respectively).. Once sedated, the rats were injected intravenously with a bolus of 40 MBq

99rT

Tc-mebrofeninn (Bridatec; GE-Amersham Health) in 0.3 mL saline in a tail vein. The animalss were scanned upon injection of the radiopharmaceutical in anterior position with thee liver and the mediastinum in the field-of-view. Dynamic images were obtained for 30 minn (10 min at 5 sec per frame and 20 min at 60 sec per frame) at the 140 KeV 99mTc-peak withh a 20% window in a 128x128 matrix. Data was processed on a Hermes workstation (Nuclearr Diagnostics). The liver uptake was calculated based on a technique described byy Ekman et al. {13) The algorithm was adapted for rat hepatobiliary scintigraphy based onn the faster hepatic extraction of mebrofenin in rats (8). Regions of interest (ROI) were drawnn around the liver, the heart and large vessels within the mediastinum (serving as bloodd pool) and around the total FOV (indicative of total activity). The fiver ROI was drawn automaticallyy on a threshold-based algorithm using 20% of the maximum liver value on aa summed image of the first 2.5 min of the acquisition as cut-off. Three different time-activityy curves were generated based on the liver, blood pool and total FOV. Liver uptake wass calculated in %/min, based on these three parameters, Calculations of hepatic 99rT Tc-mebrofeninn uptake were performed using scanned radioactivity values acquired between 300 and 120 sec postinjection, to make sure that calculations were made during a phase off homogenous distribution of the agent in the blood pool and before the rapid phase of hepaticc excretion. Furthermore, a second liver ROI was drawn excluding large bile ducts andd superimposing bowel loops. This ROI was used to create a hepatic time activity curve forr calculation of the time at which maximal hepatic activity occurred (Tmax), as well as thee time required for peak activity to decrease by 50% (TV2).

Statisticall Analysis

Commerciall computer packages were used for the analysis of the data (GraphPad Prism, SPSS).. Values are given as mean SEM. The relation between the liver uptake of 99rT1 Tc-mebrofeninn and other parameters was tested using the standard Pearson correlation coefficientt r. All statistical tests were 2-tailed and differences were evaluated at the 5% levell of significance.

RESULTS S

Dedicatedd animal pinhole dynamic hepatobiliary scintigraphy was very feasible in rats. Underr anesthesia, intravenous injection is possible with the animal positioned before thee pinhole collimator (Fig. 1). Dynamic scintigraphy shows a rapid hepatic uptake of the radiopharmaceutical,, with excretion into the bowel (Fig. 2A). After partial hepatectomy, thee hepatic uptake is decreased (Fig. 2B). Liver uptake can be quantified and expressed ass percentage uptake per min using ROI's encompassing the liver and the heart as blood pooll correction (Fig. 3). Determination of Tmax and TY2 based on the kinetics of the entire

(orr remaining) liver volume was sometimes difficult due to over projection of bowel loops HBSS caused no mortality or excessive morbidity except for the anesthesia

FIGUREE 1. A rat is placed in a perspex cylinder and is being injected intravenously with 99mTc-mebrofenm in aa tail vein at the start of a dynamic acquisition (A) The rat is positioned with the upper abdomen before the aperturee of the pinhole collimator The transaxial position is fixed and centered in the midline of the cylinder Thee axial position can be adjusted manually for optimal positioning of the target organ (B).

FIGUREE 3 HBS in a rat (A) Summed image from 300 - 120 sec after intravenous injection of 40 MBq 99mTc-mebrofeninn A ROI is drawn around the entire liverr A second ROI is drawn in the mediastinum FIGUREE 2. Dynamic pinhole HBS in a rat after

intravenouss injection with 40 MBq 99mTc-mebrofemn Imagess are reframed to 30 sec/frame (A) Normal HBSS in a rat showing fast and homogenous liver uptakee with visible excretion into the bowel starting fromm 3 mm post injection (B) Abnormal HBS in a ratt after partial resection HBS shows delayed and inhornogeneouss liver uptake with visible excretion intoo the bowel starting from 6 mm post injection

Characterizationn and Normal Values

Thee animal characteristics and results are illustrated in Table 1 The mean body weight was

3500 7 g The mean liver weight was 10 92 0 44 g The mean scintigraphic liver uptake

ratee was 7729 1 29 %/min Calculation of the liver uptake percentage per mm was highly

reproducible,, with an excellent correlation (r= 0 95. P <0 001) between both calculations Thee variation of liver uptake was low in rats assessed 3 times within 1 week (Fig 4) All measuredd values stayed within the normal range There was no influence of previous anesthesiaa on behavior or functional results

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Tablee 1. Characterization and normal values 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 0 11 1 12 2 Mean n SEM M SD D 335 5 342 2 336 6 400 0 335 5 342 2 336 6 400 0 335 5 342 2 336 6 336 6 350 0 7 7 24 4 100 89 1029 9 9 6 0 0 13.96 6 10.99 9 10.11 1 9.56 6 133 89 11.00 0 11.01 1 9 7 8 8 99 90 10.92 2 0.44 4 11 51 79.57 7 711 84 80.17 7 855 18 72.59 9 79.01 1 70.21 1 755 77 80.25 5 8178 8 74.58 8 76.55 5 77.29 9 1.29 9 4.47 7 79.14 4 70.09 9 79.57 7 8 4 7 9 9 744 11 81.15 5 72.16 6 777 04 78.96 6 811 98 74.75 5 79.14 4 777 74 1.23 3 4.27 7 22 00 22 83 22 33 22 00 11 92 2 0 0 0 2.33 3 22 50 2.42 2 2.33 3 22 08 22 08 22 24 0.08 8 0.27 7 66 50 66 67 6 0 0 0 55 08 6.50 0 6.17 7 6.83 3 55 83 6.25 5 55 92 675 5 55 83 66 19 0.14 4 0.50 0 86 6 62 2 78 8 88 8 96 6 78 8 52 2 59 9 67 7 54 4 47 7 59 9 69 9 5 5 16 6 56 6 68 8 64 4 92 2 88 8 56 6 56 6 68 8 64 4 65 5 65 5 56 6 67 7 4 4 12 2 30 0 36 6 34 4 32 2 34 4 36 6 34 4 34 4 32 2 32 2 34 4 32 2 33 3 1 1 2 2 174 4 17.8 8 177 5 164 4 165 5 171 1 17.3 3 174 4 165 5 161 1 1 6 4 4 166 5 169 9 0 2 2 0.6 6

Interventionn / Surgical Procedure

Liverr weight was decreased 1 and 3 days after resection w i t h recuperation within the normall range 7 days after surgery (Fig 5A) Liver weight was increased 1 and 3 days afterr ligation with normalization 7 days after surgery (Fig 5A). After surgery, there was a significantlyy (P < 0 01) decreased liver uptake rate, w i t h an inferior uptake rate (P < 0 05) afterr partial resection as compared t o portal ligation (Fig 5B) There is regeneration of liverr function 3 and 7 days after surgery, which was significantly higher (P < 0 01) after resectionn as compared to ligation (Fig 5B). After surgery, there is a significant (P<0.01) increasee of serum ALT This increase is significantly higher (P < 0 01) after ligation as comparedd to resection (Fig 6A) Seven days afterr surgery, serum ALT normalizes in both groups.. After resection, the albumin level in thee plasma decreases, w i t h normalization withinn 1 week After portal ligation, the albuminn level remains w i t h i n the normal rangee (Fig 6B) There is a strong positive associationn (r = 0 85 P < 0 001) between liverr weight and the functional uptake rate afterr resection (data not shown) There

FIGUREE 4 Liver uptake rate measured with HB5 in 3

ratss on 3 different occasions The variation HBS was w a s a

9 0 o d a s s o c i a t i o n b e t w e e n liver loww and all measured values were within the 95% CI

(horizontall dashed lines) based on the normal values (Tablee 1) Ral 1 .. Ral? Rat 3 D;iy y

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FIGUREE 5. (A) Liver weight of rats (mean 1 SD) att baseline and after resection ( A ) or ligation Theree is a significant reduction (P < 0 01) in liver weightt 1 and 3 days after resection with recuperation withinn the normal range 7 days after surgery The horizontall dashed line indicates the 9 5 % CI based onn the normal values (Table 1) (B) Scintigraphic liver uptakee rate of rats (mean 1 SD) at baseline and after resectionn ( A ) or ligation ) There is a significant reductionn (P < 0 01) in uptake rate 1 and 3 days after resectionn with recuperation 7 days after surgery. The horizontall dashed line indicates the 95% CI based on thee normal values (Table 1)

FIGUREE 6. (A) Serum ALT of rats (mean 1 SD) att baseline and after resection ( A ) or ligation Theree is a significant increase (P < 0.01) in serum ALTT 1 and 3 days after resection with normalization withinn the normal range 7 days after surgery The horizontall dashed line indicates the 9 5 % CI based on thee normal values (Table 1) (B) Serum albumin of rats (meann 1SD) at baseline and after resection ( A ) or ligationn ( D There is a significant decrease (P < 0 01) off serum albumin 1 and 3 days after resection with recuperationn 7 days after surgery Serum albumin afterr ligation remains within the normal range The horizontall dashed line indicates the 9 5 % CI based on thee normal values (Table 1)

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DISCUSSION N

HBSS has been described as a valid alternative for the indocyanine green (ICG) clearance testt in the preoperative assessment of liver f u n c t i o n (6) Iminodiacetic acid (IDA) enters t h ee hepatocyte via a carrier-mediated non-sodium-dependent organic anion path, by a mechanismm very similar t o that of bilirubin or ICG (74) Both ICG and IDA-analogues aree excreted in the bile by hepatocytes via t h e ATP-dependent export p u m p multidrug-resistancee associated protein 1 and 2 (MRP1.2) (75) Furthermore, preoperative assessment

off remnant liver function can be performed with HB5 (9). Moreover, liver function regenerationn can be monitored in vivo with HBS and correlates better with quantitative liverr function testing than liver volume regeneration (9). Therefore, quantitative HBS for researchh purpose was evaluated in rats where repeated measurement of liver function is neededd for an accurate evaluation of chronic liver disease or regeneration after an acute eventt otherwise requiring sacrificing a number of animals per measurement time point.

HBSS using 99mTc-labeled IDA-analogues has been performed in rats {8,10,16,17). However,

liverr function was determined by time activity curve derived parameters {8,16), invasive samplingg (70) or uptake scintigraphy without correction for blood pool activity (17). The liverr uptake function measured by lodida clearance rate was described by Ekman et al. (73).. This technique was adapted and validated for 99mTc-mebrofenin (6). For use in rats, thee dynamic acquisition has been adapted to the faster metabolism to make sure that calculationss were made during a phase of homogenous distribution of the agent in the bloodd pool and before the rapid phase of hepatic excretion. Calculation of the liver uptake ratee is performed between 30 and 120secpostinjection, which is before the average Tmax. Calculationn of the liver uptake rate from a dynamic acquisition was highly reproducible. Furthermore,, there was little inter-subject variability under standardized conditions within time.. Moreover, we have demonstrated that HB5 can be performed multiple times in an animall creating the possibility for repetitive measurement within an experiment increasing statisticall power and possibly reducing the number of animals needed for experiments.

Besidess the hepatic uptake rate, Tmax and T1/z were calculated. Unlike Tmax, which can

bee calculated easily for the entire liver, TVa was sometimes difficult to determine due to over-projectionn of bowel loops. This precludes characterization of the excretory phase off the entire liver or evaluation of regional excretion obscured by bowel uptake. Finally, Tmaxx could be influenced by intrahepatic cholestasis, which can occur initially without repercussionn on the hepatic uptake function (74).

Studiess have been performed to estimate the hepatic functional reserve by making use of

99m

Tc-DTPA-gatactosyll human serum albumin (99mTc-GSA) liver scintigraphy in rats (18,19).

99m

To-GSAA is a liver scintigraphy agent that binds to the asialoglycoprotein receptor on hepatocytess (20). It was found that the total counts and counts per unit hepatic volume

basedd on 99mTc-GSA in the entire liver significantly decreased in patients with more

extensivee liver disease (21). The disadvantage of 99mTc-GSA is that it does not provide any informationn on hepatic excretory function.

Improvementt of scintigraphic resolution, enabling imaging of small laboratory animals, cann be achieved by using a pinhole collimator, both for planar or SPECT (22,23). Pinhole collimatorss are routinely available in most nuclear medicine facilities. Moreover, dedicated animall gamma cameras have become commercially available. To obtain magnified images withh high spatial resolution in small-animal studies, system sensitivity is sacrificed and moree radioactivity or longer acquisition time is needed. In rats, increasing the amount of radioactivityy administered relative to body weight (compared with humans at a factor of 40)) results in statistically useful pinhole images with a 7-fold magnification.

Pilott experiments assessing the possibilities of HBS in rats using interventional models show aa good correlation between liver weight and function after resection. After ligation, liver functionn decreases and liver weight increases, probably due to edema. Serological liver

functionn parameters show diverging results in function of the intervention with higher ALT valuess after ligation, which can be explained by increased cell damage and lower serum albuminn after resection, which can be explained by a decreased functional volume. HBS showss a consistent pattern of decreased liver function, both after resection and ligation. Furthermore,, HBS can be repeated to assess functional recovery within 1 animal, enabling aa longitudinal study design.

CONCLUSION N

Dedicatedd animal pinhole hepatobiliary scintigraphy using 9 9 m_{Tc-mebrofenin offers a}

uniquee combination of functional liver uptake and excretion assessment w i t h the ability to determinee the liver function reserve before and after an intervention. HBS is a repeatable noninvasivee imaging tool providing visual and quantitative information and enabling serial measurementt within an animal potentially improving statistical power and reducing the numberr of animals needed for research.

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