Data Collection and Statistic Analysis. Data from the LDF and thc pulse oximeter were collected continuously on a laptop computer. The data were dculatcd.
,+nt
Effect of Ischemic Preconditioning on Hepatic Microcirculation and Function in a Rat Model of Ischemia Reperfusion Injury
'
Rabul S. Koti, * W e m a n Yang, Michael R. Dashwood, Brian R. Davidson, * and Alexander M. Seifalian' Ischemic premnditioning (IPC) may pmtcct the liver fmm ischemia reperfusion injury by nitric oxide formation. This d v has invatinated the dfcct of ischemic oreconditioning on hepatic microcirculation (HM), and ;he relationshiihetw& nitric oxide metabolism and HM in oreconditionine. Rats were allocated to 5 monos: 1. sham laparotomy; 2.45 minutes lobar ischemia followed by 2-hour reperfusion (IR); 3. IPC with 5 minutes ischemia and 10 minutes reperfusion before IR14.Garginine bcfore IR; and 5. GNAME + IPC before IR HM wu monitored by laser Doppler flowmeter. Liver transamin-, adenosine triphosphate, nitrites nitrates, and guanosine 3'5'-cyclic monophosphate (&MI') were m a sured. Nitric oxide synthase (NOS) distribution wu studied using nicotinamide adeninine dinuclwtide phosphate (NADPH) diaphorase histochemistry. At the end of reperhsion phase, in the IR group, flow in the HM recovered to 25.8% of baseline (P < .05 versus sham), whereas IPC improved HM to 49.5% of baseline (P< .O1 versus IR). With Gnrginine treatment, HM was 31.6% of baseline (NS versus IR), showing no attenuation of liver injury. In the preconditioned group treated with GNAME, HM declined to 10.2% of baseline, suggesting not only a blockade of the preconditioning - effect,but also an exacerbated liver injury. Hepatocellular injury was reduced bv IPC and Larpininc and was increased hv NO IPC also increased nit& + inhibition with GNAME nitrate (NOx) nod cGMP concentrations. NOS detected bv KADPH diaohorase staininn was associated with heoatoqIe.3 and wcular endothclium, and was induced by IPC. IPC induced NOS and attenuated HM impairment and heoatoccllular ininrv. Thae data strondv soppat a role for nitric oxid; in IPC. (Livrr ~ra;;;blf&2;8: 1182-1191.)
I
schema-reperfusion injury (IN) is a major cause of morb~dity .' and mortalicy in liver surgery and transplantation. Although the precise mechanism for I N remains to be defined,l key events in the pathophysiology of IRI include an amplified inflammatory response2 and failure of hepatic microcirculation (HM).3 The postischemic hepatic microcirculatory failure correlates with hepatocellular damage.4 Blood flow in the H M is modulated by vasoactive substances such as nitric oxide (NO) and endothelins (ET).ET evokes sinusoidal constriction by contraction of Ito cells.5 N O produces relaxation of hepatic stellate cells and opposes the vasoconstrictive effects of stellate cell acti~ation,~ and, as a result, may limit microcirculatory disturbances. Blocking ET receptors or ~rovidinga N O donor, protected HM, and reduced hepatic IRI in an experimental model.' Inhibition of N O synthase (NOS) in rats results in aggravated hepatic injury after the oxidative stress of endotoxaemia.sThese datasuggest that N O may influence liver injury either directly or through effects on blood flow. Ischemic preconditioning (IPC) is a potential therapeutic strategy, which may increase the tolerance of the liver to ischemic insults ofsurgery and preservation. It refers to the phenomenon of initial brief period of ischemia improving tolerance to subsequent sustained ischemia. An increase in ischemic tissue tolerance could decrease the morbidity and mortality associated with as well IRI. The existence of hepatic IPC in animal~9.'~ as humansll has been demonstrated but the mechanism Fmm thc * H ~ t i c H a r m o d ~ munit ; c uni-'igDPm~rof of the preconditioning effect in the liver is uncertain. Su'ge'y #ndLiwr Trmsphn~tionUnit, and the tDqamnmt ofClinBecause failure of the H M is key factor of I N , changes iral Biochmriroy, Roy1 F m nnd Univmi'y CoILgt Mrdicnl Schwl, uninm;ryc o b&,,don h n l F W H,,,~;YL don. u,,irrd~i,,~. in H M with IPC may give an important indication of the effect of IPC on IRI. An approach to study the role don. .. hacnrrdinpanarBn'rirhSoriqofGmmmohaAnnunlMm- of N O in heoatic IRI involves the use of antaeonisrs. ins March 18-21.ZWI. GLrgow, kitrdfingdom. /&mdingofthr hepatic IRI with NOS antagoan aggra;arion meeting inbrmm) publlhrd in: Gut 2Wl; 48 (Suppl I): A231 nists has been ~eported.~ IPC may therefore act through Addm nqwra AM SofiIia,,, Rql FFm local release of NO. U n i y m i ~~~Mdicd $ h L Un;-iv GUlpc Lmrdon R4rJ F m ~ r n p i d ' ~ o n d i Lmrdon C NW32QG. ~ n ~ L ; nTd& / ~ N : 44-25783& The present study has investigated the effects of IPC w ; F m : 44-20-7472-6444;E-mdil~A.S1~n@RF~~C~~~~ and N O and inhibitinn on H M and liver Co~~n'ght hsriationfm rhr 'f function in a rat model of lobar ischemia reperfusion,
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Li"" Disc',," 1527-6465/02IO812-WlU35.WIO doi:lo. 1 0 5 3 ~ l ~ . Z W Z . 3 ~ 6
Flow in the H M was continuously measured with laser Doppler flowmeter (LDF). LDF is a noninvasive tech-
nique allowing continuous evaluation of microvascular p e r h r ~ i o n . ' Its ~ use for measurement of the HM has been validated in many studies.'Z-14
Material and Methods Animal Preparation and Surgical Procedure The study was conducted under a project license granted by the home office in accordance with the Animals (Scientific Procedures) Act 1986. Male Sprague- Dawley rats, each weighing 250 to 300 g were used for the experiments. All animals werc kept in a temperature-controlled environment with a 12-hour lightdark cycle and allowed tap water and srandard rat chow pellets ad libitum. Animals werc anesthetized with urethane lmg/kg body weight intraperitoneally and prepared for aseptic surgery. Animals were allowed to breathe spontaneously through a concentric mask connected to an oxygen regulator during the procedure. The animal's body temperature was maintained at 37' to 39'Cwith a heating pad (Harvard apparatus Ltd, Kent, UK) and monitored with a rectal temperature probe. The arterial oxygen saturation (SaO,) and heart rate were continuosly monitored with a pulse oximcter (Ohmeda Biox 3740 pulse oximeter; Ohmeda, Louisville, CO). Polyethylene catheters (PE50, 0.38 mm inner diameter, Portex, Kent, UK) wcrc inserted into the right femoral artery and connected to a pressure transducer for monitoring of mean ancrial blood pressure (MABP), and in the right femoral vein for administeringnonnal saline (1 mUlOOg body weightlhr) to compensate for intraoperative blood loss. Laparommy was carried out through a midline incision. The ligamentous attachments from the liver to thediaphragm were severed, and the liver was exposed. Ischemia of the median and left lateral lobes of the liver was produced by clamping the corresponding vascular pedicle containing the p o d vein and hepatic artery branches with an atraumatic microvascular damp. The other hepatic lobes were not handled during the procedwc. This method produces ischemia to the left and median lobes of the liver (about 70% of the liver), leaving the blood supply to the right and caudate lobes uointcrrupted, and facilitata splanchnic decompression." At the end of the ischemia period, the vascular clamp was removed and the liver repetfused.
Measurement of Blood Flow in the HM HM was measured by a surface LDF (DRT4, Moor Instruments Limited, Axminster, UK)in flux units. The Doppler signal varies linearly with the product of the mtal number of moving red blood cells in the measured volume of a few cubic mm multiplied by the mean velocity of these red blood cells. The numeric product is termed petfusion units or blood cell flux units.16J' The LDF probe was placed on a h c d site on the left lateral lobe ofthe liver andwas held in place by a probe holder. LDF dam were collected continuously at sampling
rate of 2 Hz. LDF measurements at the relevant time points were collected as a mean of 1-minute dam.
Measurement of Hepatocellular Injury At the end of 2 hours of reperhion, a 2-mL blood sample was taken from the inferior vena cava. The measurements of liver enzymes alanine aminorransferase (ALT) and aspartate aminotransferase (AST) in plasma were performed by a standard spcctrophotomctric method with an automated clinical chemistry analyzer (Hitachi 747, Roche Diagnostics Ltd., Sussex, UK).
Measurement of Hepatic Tissue Adenosine Triphosphate (ATP) At the end of 2 houn of repetfusion, biopsies of ischemic loba ofliver were freae-damped in liquid nitrogen for determination. A T ' levels in ischemic liver tissue were assayed spcctrophotometrically (Unicam W 1 spectrophotometer).
Measurement of NO Production At the end of 2 hours of rcpcrfusion, a 2-mL blood sample was taken from the inferior vena cava. Plasma nitrite nitrate (NOx) was measured with a 280 Nitric Oxide Analyser (Sicven Instruments) by chemiluminescencemethod.'n
+
Measurement of Hepatic Guanosine 3'5' Cyclic Monophosphate (cGMP) cGMP accumulation in liver tissue was determinedl9 by using a commercial ELISA kit (Cayman Chemical, Ann Arbor, MI). Briefly, immediately at termination of the experiment, liver tissue was frozen in buffer, homogenized, and centrifuged, and the supernatant was collected for assay. Each sample supernatant was then individually acerylated by addition of 100 pLof4 M KOH and25 pLac~ticanhydride,vortexed for 15 seconds, and, finally,25 pL of 4 M KOH and vortex. Samples (50 pL) or standard solutions of cGMP (3 pmoll50 p L to 0.0234 pmoll50 pL), were incubated together with rabbit anti-cGMP (50 pL), and cGMP linked to acerylcholinesterase (50 pL), in prcwated platcs at room temperature for 18 hours. After the plates were washed, color development was initiated by the addition of Ellman's Reagent (200 pL) for 60 minuta. Absorbance was mcasured at 412 nm on a plate reader.
Nimtinamide Adeninine Dinuclcotide Phosphate (NADPH) Diaphonse Histochemical Stain Fresh liver tissue was frozen and stored at -70'C. Five-pm transverse sections wcrc cut with a cryostat at approximately -25°C and thaw-mounted onto polylysine-coated microscope slides. For staining, sections were allowed to equilibrate at room temperarurc (approx. 22°C) for 30 minutes and posth c d for 30 minutcs in 3% paraformaldehydein0.01M phosphate-buffered saline (PBS) buffer at 4°C. After rinsing in PBS anddryingin coldair, sections were incubated for 1 hour
1184
Koti rt al
rcularion (Hhr l ) in Flux UI,its, ar End o linures of Reperfusion (HiM-2), and at
1Cable 1. He1
of Warm Ischemia (HM-I), ar the En' urn of Reperfusion (HM-31
-
Group I (Sham) HM-I HM-2 HM-3 HR SaO2 MABP
154.3 f 150.2 f 155.0 235.0 f 98.4 f 55.0 f
*
15.7 21.4 14.9 11.9 1.1 2.2
Group 2 (IR)
Group 3 (IPC)
32.3 f 13.9. 42.4 + 16.9' 32.2 f 17.0229.6 + 7.75 97.7 f 0.65 51.0 + 1.45
30.0 2 12.9t 95.3 f 16.5t 78.7 f 17.8t 230.0 f6.2t 97.2 + 0.9t 53.0 + 1.Gt
*
Group 5 (LNAME + IPC)
Group 4 (Larginine + IR)
38.5 71.3 47.1 233.9 96.9 53.0
26.3 + 12.47 43.3 + 16.0$ 16.8 f 3.6s 230.0 ? 9.7t 97.4 + 1.Gt 50.0 2.4t
13.9t 19.8t 20.8t 7.7t + 1.4t 2.lt f
f ? 5
*
*
NOTE. Values arc mean SD of G animb in each group. Heart ntc (HR) (beanlmin),arterial oxygen saturation (Sa02) (%), man arterial blood pressure (MABP) (mmHg). Abbreviations: IR ischemia followed by reperfusion; IPC, ischcmia preconditioning;LNAME, I& nitro argininc methyl atcr. *P .05 us group 2) using unpaired r test. $P .05 m gmup I).
at 37°C with 1 mg/mL P-NADPH (cofactor) and 0.2 mg/mL nitroblue tetrazolium (chromagen) dissolved in PUS buffer (pH 7.G),containing 0.2% Triton X-100. The reaction was stopped by removing the incubation solution, blotting the sections and rinsing for 5 minutes in running tap water. The sections then were stained for 3 minutes with 1%eosin and prepared for microscopic examination. Controls were incubated with nitroblue tetrazolium and no NADPH. NADPH diaphorase-positive staining gives blue stain.
Experimental Protocoh The rats were randomly allocated to 5 study groups, 6 animals per gmup: Group 1: Sham-operated animals (controls). Thesc animals underwent an identical experimental pmtocol but without clamping of the hepatic blood vesscls. Group 2: Ischemia reperfusion (IR); ischemia was induced in the median and left lateral hepatic lobes for 45 minutes, followcd by a 2-hour period of reperfusion. Group 3: IPC + I R The median and left latcral lobes were preconditioned with 5 minutes of ischemia followed by 10 minutes of rcpcrfusion; this was followed by IR (group 2 procedure). Group 4: Larginine IR. Animals wcre treated with L-arginine (100 mglkg body wcight intravenously) 10 minutes before 1 R Gmup 5: Nu-Nitro-L-arginine methyl ester (LNAME) + IPC 1R: Animals were treated with LNAME (30 rng/kg body weight, intravenously) 10 minutes before group 3 procedure.
ischemia and at the end of30 and 180 minutes of reperfusion. The values are expressed as mean SD of 6 animals in each p u p . One-way analysis of variance (ANOVA) and Uonferroni adjustment for multiple comparisons were used unless otherwisestated where an unpaired Student t-test was used for statistic analysis between the groups. A P value of Im than .05 was considered sratistically significant. Thc relationship between H M changes, plasma transaminases, hepatic tissue ATP, and NOx was tested with Speatmans correlation coefficient.
+
Results Hemodynamic dataare shown in Table 1. I n all animals in the experimental groups the heart rate and arterial oxygen saturation did not change significantly throughout the experiment. I n the L-arginine treated group, a transient fall in blood pressure was observed immediately after L-arginine administration, but this was not statistically significant. I n the other groups, blood pressure did n o t change significantly throughout the experiment.
+
+
Data Collection and Statistic Analysis Data from the LDF and thc pulse oximeter were collected continuously on a laptop computer. The data were dculatcd as I-minute avenges at baseline at the end of 45 minutes of
Hepatic Microcirculation Figure 1 shows the changes in mean percentage (SD) of HM with respect to the preischemic baseline level (100%). Table 1 lists the changes in HM in absolute values. There was n o significant change in the HM at 1,2, o r 3 hours of recording in the sham-operated control group when compared with baseline (P = .5). There were significant differences between this group and groups 2 , 3 , 4 , and 5 (P< ,001) when HM changes a t
the end of45 minutes ischemia and at the end of30 and 180 minutess of reperfusion were compared (Fig. 1 and Table I). In IR group, at the end of 45 minutes of ischemia, the mean HM decreased to 20.7% of the preischemic level. After removal of the microsurgical clamp, the mean HM recovered to 31.1% duringfirst 30 minutes of reperfusion period and thereafter slowly declined to reach a value of 22.5% (P = .O versus bwline) at the end of 2 hours of reperfusion (Fig. 1). In the IPC group, during the preconditioning period, the mean HM rcu)vered rapidly to 97.3% on dedamping after 5 minutes of ischemia and remained steady during 10 minutes of reperfusion. After a subscquent 45 minutes of sustained ischemia, the mean HM dccreaxd to 18.1% and, on dcdunping, showed a rapid recovery to 58.5% during the i n i d 30 minutes of reperhion and th&r showed a slight decline without any puks to reach a final value of 49% (P = .005 wsus baseliie) during final15 minutes of recording (Fig. 1 and Table 1). There were signilicant differences between this group and group 2 and 4 (P < .05) at the end of 2 hours of reperfusion (Table 1). In the L-arginine-treated group, the mean HM decreased to 81.2 % (P= .01 versus baseline) immediately afcer L-arginineinjection. Subsequently, afcer 45
minutes of ischemia, the mean HM had decreased to 24.6 % (P < .05 versus baseliie). On declamping, the mean HM impmved to 46.8 % (P < .001 versus baseline; P < .05 versus IR) and then gradually declined to 31.6 % (P < .05 versus baseline) during the final 15 minutes of recording (Fig. 1). The d&nces were not significantwhen comparedwith the IRgroup at the end of 2 hours of reperfusion (P> .05) (Table 1). In the LNAME treated group, at the end of the preconditioning period, the mean HM recovexed to 89.3% (P < .05 versus baseline). After subsequent 45 minutes of sustained ischemia, the mean HM had d d to 15.8% (P < .05 versus &line). On dcdunping, the mean HM rccovucd to 26.3% and then dmeased pmgrasivcly to around 10.2% during the final 15 minutes of reperfusion (Fig. 1). There were significant &rences between this group and groups 2 and 3 at the end of 2 hours of reperfusion (P < .001) (Table 1).
IR (group 2) resulted in signilicant increase in plasma ALT and AST. Both IPC and L-arginine treatment reduced ALT and AST levels, whereas in the LNAME + IPC group (group 5), ALT and AST levels were increased signilicandy (Fig. 2).
1186
Koti et a1
DL-NAME
Figure 2. Plasma ALT and AST l m L (UIL). Vdua arc mean -C SD of 6 animals in each p u p . *P < .05 versus sham; **P< .05 versus IRI Student's t-tut.
-
ALT
AST
Hepatic T i u e ATP IR (group 2) resulted in significant decrease in hepatic tissue ATP as compared to Sham values. Both IPC and L-arginine treatment increased ATP levels as compared to 1R. ATP levels were not significantly different at the end of preconditioning period. Whereas, in LNAME + IPC group (group 5) ATP levels were significantly reduced as compared to IR (Fig. 3). NO Production and Hepatic cGMP IR (group 2) resulted in a significant decrease in NOx measured at the end of repehsion phase. Both IPC and L-arginine treatment increased NOx and cGMP, whereas in the LNAME + IPC group (group 5). NOx and cGMP levels were significantly reduced (Figs. 4 and 5).
Correlation of HcpatocclIulv Injury and NO Production W ~ t hHM At the end of 2 hours of reperfusion, there was a significant negative correlation berween plasma transaminases and HM and significant positive correlations between hepatic ATP and HM and between NOx and HM (Table 2). NADPH Diaphonue H i c h e m i c a l Stain NOS appraised with NADPH diaphorase staining was associated with hepatocytes and vascular endothelium in centrilobular wne. The distribution of NOS was similar in both IPC and L-arginine treated groups (groups 3 and 4). IR and IPC + L-NAME groups (groups 2 and 5) did not show positive staining for NOS (Fig. 6 ) .
..
.. sham
IR
IPC
L-Arg G N A M E
Figure 3. Hepatic t i m e ATP levels (pmoVg wet liver tissue). Values arc meanc! SD of 6 animals in w h p u p . *P < .05 v e m s sham+ **P< .05 versus IRI S~dent's :-test.
Sham
IR
L-arg
IPC
+
L-NAME
Figure 4. Changw in Plasma nitrite nit& (NOS)levels (ILM) Valocs arc mean f SD of 6 animals in each p u p . 'P < .05 versus sham+**P< .05 versua IRI SNdent's t-tat.
4
Sham
IR
IPC
L-arg
L-NAME
Figure 5. Hepatic cGMP lmls (pmoUmg). Vduu arc mean 2 SD of 6 animals in each p u p . *P< .05 versus sham, **P< -05 versus I& NS, not significant versus sham; Student's t-tut.
Discussion This study has investigated the effect of ischemic preconditioning on hepatic parenchymal perfusion and how it relates to N O metabolism. This study involved a rat model of lobar hepatic ischemia reperfusion. Blood flow to the left lateral and median lobes of the liver was temporarily interrupted while maintaining normal blood flow to the right and caudate lobes. This maintains splanchnic blood flow and prevents the systemic hemodynamic instability associated with mesenteric congestion and portal bacteraemia found with total inflow occlusion.'5 In this study, the systemic hemodynamic parameters, including MAPB, heart rate, body temperature, and oxygen saturation did not change significantly during the experiments excluding any systemic contributions to the liver injury. The experimental model of 45 minutes of ischemia with 2 hours reperfusion had no produre-related mortality and was developed to evaluate liver injury in the early phase (< 2 hours) of reperfusion after substantial but nonlethal ischemia. LDF is a reliable method for the continuous measurement of tissue blood flow16 and has been used The to monitor H M to assess the severity of application and reproducibility of LDF measurements for assessment of H M has been validated in both experimental animals'l' and human liver transplantation.14 LDF allows a continuous in vivo recording of the HM without directly affecting the HM.I4 IPC is a potential therapeutic strategy against hepatic IN,and a reccnt report by Clavien et all1 has shown efficacy in human liver surgery. Peralta et a12'-2s have proposed that endogenous production of N O may mediate IPC; in animal models, hepatic IRI was reduced with increase in endogenous NO. The protective role of endogenous N O in liver IRI is supported
indirectly by studies showing exacerbation of liver injury associated with failure of microcirculation in rats treated with nonselective NOS inhibitors.8 In this study, to evaluate the effect of N O on HM, additional groups had L-arginine (amino acid substrate for N O biosynthesis) and LNAME (nonspecific inhibitor of NOS) administered before I R Previously, improvement in microcirculation perfusion parameters has been shown by Zapletal et al,2* but the mechanism was not addressed. The results of this study indicate that the beneficial effect of preconditioning could be related to an improvement in microcirculation and implicates N O as a contributory mechanism. L-arginine treatment protected against IN similar to preconditioning, whereas LNAME administration aggravated microcirculatory impairment and cellular injury in preconditioned animals. LDF measurements calculated as a mean of l-minute data at each stage of experiment were expressed in arbitmy units. The preischemic baseline recordings of H M were expressed as a standard 100%in each individual experiment. LDF reflects the real blood flow in HM, which includes the collateral flow and backflow from hepatic veins in addition to portal vein flow and hepatic arterial flow. All groups subjected to 45 minutes of ischemia showed impairment of H M indicated by similar biphasic curve of initial partial recovery of blood flow followed by decline during 2 hours of reperfusion. This suggests that the failure of recovery of blood flow to baseline values coincide with the deleterious cascade of events associated with reperfusion injury. Several mechanisms contribute to the microcirculatory failure including, narrowing of the sinusoid lumens by endothelial cell swelling2' secondary to ischemia-induced ATP deficiency and the consequent failure of ion transport through the cell memhrane.26 A significant reduction of leukocyte velociry with subsequent stasis and intrasinusoidal plugging has been suggested as a hindrance for blood perfusion.27 Increased leukocyte adherence with increased perme-
Table 2. Correlation bewe ATP and luvx ( y )
HMn ALT HMvrAST HM nATP HM n N&
nd ALT, AST,
Carrelarian
P
Rcgrasion Anllysir
Coefficient
Value
y = -55.62~ + 7.1 y = -35.15~ 5.9
r = 0.95
e.001
