ANALYTICALBIOCHEMISTRY 2 0 6 , 131-136 (1992)
Changes in the Redox Distribution of Rat Liver by Ischemia Toshiyuki Kitai, Akira Tanaka, Atsuo Tokuka, Kazue Ozawa, Shingo Iwata,* and Britton Chance* Second Department of Surgery, Faculty o[ Medicine, Kyoto University, 54 Kawaracho Shogoin, Sakyoku, Kyoto, 606 Japan; and *The Johnson Research Foundation, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
R e c e i v e d M a r c h 11, 1992
The changes in the redox distribution of the ischemic r a t l i v e r w e r e s t u d i e d u s i n g t h e r e d o x s c a n n e r , w h i c h is a time-sharing, tissue fluorescence and reflectance spectroscopy with fine spatial resolution, with the results p r e s e n t e d a s a h i s t o g r a m a n d a g r a y - s c a l e d p i c t u r e . Hep a t i c i n f l o w w a s c l a m p e d f o r 1 0 rain ( g r o u p 1), 2 0 m i n ( g r o u p 2), a n d 3 0 m i n ( g r o u p 3). T h e r a t i o o f o x i d i z e d f l a v o p r o t e i n to r e d u c e d p y r i d i n e n u c l e o t i d e f l u o r e s cence (FP/PN), representing the intracellular redox state, and hemoglobin reflectance were measured prior to c l a m p i n g a n d at s u b s e q u e n t t i m e s f o l l o w i n g d e c l a m p ing in each group. The mean and the range width of FP/PN histogram decreased with the prolongation of i s c h e m i c t i m e . I n g r o u p 1, F P / P N h i s t o g r a m s h o w e d a r e m a r k a b l y w i d e r a n g e at 2 rain a f t e r d e c l a m p i n g b u t w a s a l m o s t c o m p l e t e l y r e c o v e r e d to p r e i s c h e m i c l e v e l s at 1 0 m i n a f t e r d e c l a m p i n g . I n c o n t r a s t , i n g r o u p s 2 a n d 3, t h e m e a n o f F P / P N h i s t o g r a m w a s o n l y p a r t i a l l y rec o v e r e d at 1 0 m i n a f t e r d e c l a m p i n g a n d t h e r a n g e w i d t h c o n t i n u e d to i n c r e a s e . T h e c o r r e l a t i o n b e t w e e n F P / P N fluorescence and hemoglobin reflectance pictures sugg e s t e d t h a t t h e l o c a l s t a g n a t i o n o f b l o o d f l o w c a u s e d tissue hypoxia, preventing the recovery of the redox state p r o b a b l y i n t h e c e n t r i l o b u l a r a r e a . © 1992 Academic Press, Inc.
A liver acinus forms a microcirculatory u n i t of the liver p a r e n c h y m a . Since the hepatic blood flow is unif o r m l y directed along the sinusoids from the t e r m i n a l p o r t a l venule to the t e r m i n a l hepatic venule, gradients of oxygen, substrate, h o r m o n e s , and various metabolites p r o d u c e d by cellular activity along the capillaries are more p r o m i n e n t in the liver t h a n in o t h e r organs (1,2). As R a p p a p o r t a n d K a t z p r o p o s e d in their zonation of hepatic p a r e n c h y m a (3,4), cell e n v i r o n m e n t s are quite different b e t w e e n the p e r i p o r t a l area a n d the centrilobular area of an acinus, a n d h e p a t o c y t e s are also 0003-2697/92 $5.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
morphologically and functionally organized in the f r a m e w o r k of the microcirculatory unit. It is well k n o w n t h a t various liver damage occurs h e t e r o g e n e o u s l y in the liver acinus (3,6). C e n t r a l necrosis often seen in the hypoxic liver indicates t h a t the c e n t r i l o b u l a r area is far more susceptible to hypoxic stress t h a n the p e r i p o r t a l area (5,6). Although hepatic ischemia is one of the surgical models m o s t f r e q u e n t l y studied f r o m the aspect of energy m e t a b o l i s m (7-10), the changes at the level of the liver acinus r e m a i n to be clarified. Intracellular redox state of pyridine nucleotide ( N A D + / N A D H , N A D P + / N A D P H ) has been used as an indicator of intracellular o x y g e n a t i o n of the tissue {6,7). Quistorff and C h a n c e have developed a scanning fluorim e t e r t h a t measures the oxidized f o r m of flavoprotein (FP) 1 a n d the reduced form of pyridine nucleotide (PN) (11,12). T h e fluorescence signal of F P originates m o s t l y from the m i t o c h o n d r i a l c o m p a r t m e n t . T h e b a c k g r o u n d signal of PN, which is not associated with the redox transition, is relatively small in the liver a n d the P N signal f r o m the m i t o c h o n d r i a is severalfold e n h a n c e d at low t e m p e r a t u r e . T h e r e f o r e , the ratio of F P / P N represents the intracellular, p r e d o m i n a n t l y mitochondrial, redox state (13). T h e redox s c a n n e r has some advantageous functions: (i) the spatial resolution is a b o u t 100 #m, which is fine enough for m e a s u r e m e n t at the level of the liver acinus. (ii) F P / P N is insensitive to changes in m i t o c h o n d r i a l c o n c e n t r a t i o n , i n t e r f e r i n g pigments, and red blood cells in the tissue (14). T h i s is an i m p o r t a n t quality which m a k e s it possible to m e a s u r e the redox state of tissue c o n t a i n i n g blood. (iii) Since the measurem e n t is carried out with a f r e e z e - t r a p p e d sample at subzero t e m p e r a t u r e , F P / P N should give a still profile of the redox state at the time of sampling. A previous s t u d y with hemoglobin-free p e r f u s e d rat liver showed the peri1Abbreviations used: FN, flavoprotein; PN, pyridine nucleotide; ALAT, alanine aminotransferase; LDH, lactate dehydrogenase. 131
132
KITAI ET AL. TABLE 1
Mean
SD
Min
Max
Range
Control Before clamp (n = 7)
0.84 ± 0.02
0.07 ± 0.01
0.57 ± 0.06
1.10 ± 0.04
0.53 ___0.07
Group 1 After clamp (n = 4) 2 rain after declamp (n = 7) 10 min after declamp (n = 5)
0.67 - 0.04* 0.74 ± 0.05*** 0.73 _ 0.06
0.07 ± 0.01*'t 0.13 _ 0.02***'t 0.07 _ 0.01t
0.44 - 0.08t 0.33 ± 0.05**'t 0.48 ± 0.04*'t
1.03 ± 0.08** 1.34 _ 0.14"** 1.02 _ 0.10
0.59 ± 0.14 1.00 ± 0.16"** 0.53 _ 0.08
Group 2 After clamp (n = 4) 2 min after declamp (n = 6) 10 min after declamp (n = 5)
0.61 - 0.02*'tt'§ 0.58 ± 0.03***'tt'§ 0.63 - 0.08tt
0.06 -+ 0.00" 0.08 ± 0.01"** 0.08 ± 0.00
0.46 - 0.035 0.32 ± 0.01"*.:~ 0.32 - 0.08"$
0.85 ± 0.03**'t 0.80 _ 0.06***'t 0.94 ± 0.13t
0.40 - 0.06 0.48 _+0.05*** 0.62 ± 0.08
Group 3 After clamp (n = 5) 2 min after declamp (n = 4) 10 min after declamp (n = 4)
0.50 -+ 0.04*'tt'§ 0.55 ± 0.02***'tt'§§ 0.56 ± 0.04~t'§
0.05 -+ 0.01*'t 0.07 ± 0.01***'t 0.11 ± 0.03t
0.34 _+0.04**'tt 0.33 - 0.01*'tt 0.22 ± 0.08*'tt
0.70 - 0.06**'tt'§ 0.81 ± 0.12***'tt 0.97 ± 0.05tt
0.36 - 0.05t 0.48 ± 0.11***'t 0.75 ± 0.11t
Note. Mean, SD, rain, max, and range correspond to the mean value, the standard deviation, the minimal value, the maximal value, and the range width of the FP/PN histogram. * Significantly different between groups 1, 2, and 3 by analysis of variance. P < 0.05. ** P < 0.01. *** P < 0.001. t Significantly different in time course changes of each group by analysis of variance. P < 0.05. :~P < 0.01. t t P < 0.001. § Significantly different from the control value by two-sample t test. P < 0.05. §§ P < 0.01.
portal to c e n t r i l o b u l a r redox gradient, which changes a c c o r d i n g t o v a r i o u s m e t a b o l i c p e r t u r b a t i o n s (11). L i v e r a n o x i a decreased b o t h the average a n d the g r a d i e n t of the redox state in an acinus. However, the changes in t h e in situ l i v e r m u s t b e d i f f e r e n t f r o m t h o s e i n t h e b l o o d - f r e e p e r f u s e d liver, s i n c e t h e o x y g e n - c a r r y i n g cap a c i t y o f w h o l e b l o o d is m u c h g r e a t e r t h a n t h a t of h e m o globin-free perfusate, and the changes in the systemic hemodynamics and the blood viscosity should profoundly influence the hepatic microcirculation. I n t h i s s t u d y , we i n v e s t i g a t e d t h e c h a n g e s i n t h e r e d o x d i s t r i b u t i o n of the rat liver after c l a m p i n g a n d declamping the portal vein and the hepatic artery, using the r e d o x s c a n n e r . I n a d d i t i o n , t h e effects of t h e h e p a t i c microcirculation on the redox distribution were simultan e o u s l y assessed by reflectance of hemoglobin.
MATERIALS AND METHODS
Animalpreparation. M a l e r a t s w e i g h i n g f r o m 200 t o 300 g w e r e u s e d . E a c h r a t w a s a n e s t h e s i z e d b y i n t r a p e r i t o n e a l i n j e c t i o n of s o d i u m p e n t o b a r b i t a l (5 m g / 1 0 0 g). T h e r a t s w e r e d i v i d e d i n t o t h r e e g r o u p s ; g r o u p 1, 10 m i n o f h e p a t i c i s c h e m i a ( n = 16); g r o u p 2, 20 r a i n o f h e p a t i c i s c h e m i a ( n = 15); g r o u p 3, 30 m i n of h e p a t i c i s c h e m i a ( n = 13). H e p a t i c i s c h e m i a w a s p r o d u c e d b y c r o s s - c l a m p ing the hepatoduodenal ligament including the hepatic
artery, the portal vein, a n d the biliary duct. One h u n d r e d u n i t s of h e p a r i n w a s i n t r a v e n o u s l y i n j e c t e d b e fore c l a m p i n g t o p r e v e n t i n t r a h e p a t i c c o a g u l a t i o n . I n each group, hepatic tissue was freeze-trapped by alumin u m tongs which were sufficiently precooled in liquid n i t r o g e n . S a m p l e s w e r e o b t a i n e d (i) b e f o r e c l a m p i n g ( c o n t r o l ) , (ii) a f t e r c l a m p i n g , (iii) a t 2 m i n , a n d (iv) a t 10 min after declamping.
Measurements of tissue fluorescence and reflectance. O p t i c a l m e a s u r e m e n t of t h e h e p a t i c t i s s u e w a s d o n e foll o w i n g t h e m e t h o d d e s c r i b e d b y Q u i s t o r f f et al. (11,12), e x c e p t for h e m o g l o b i n r e f l e c t a n c e . T h e i n s t r u m e n t u s e d is a t i m e - s h a r i n g , t w o - d i m e n s i o n a l t i s s u e f l u o r e s c e n c e and reflectance spectroscopy, coupled with a minicomp u t e r for d a t a a n a l y s i s . A f r o z e n s a m p l e w a s i m m e r s e d in liquid n i t r o g e n b a t h in order to hold the redox t r a n s i t i o n , a n d a flat s u r f a c e for s c a n n i n g was m a d e b y t h e l o w - t e m p e r a t u r e milling system. S c a n n i n g was perf o r m e d b e t w e e n 50 a n d 500 # m f r o m t h e s u r f a c e , s i n c e i t h a s b e e n r e p o r t e d t h a t t h e r e d o x s t a t e was h e l d c o n s t a n t at such depths. T h e light source was a 100-W s h o r t m e r c u r y arc c o m b i n e d w i t h a 1 0 - m W h e l i u m c a d m i u m l a s e r a t 442 n m . T h e w a v e l e n g t h s o f e x c i t a t i o n a n d e m i s s i o n filters e m p l o y e d for m e a s u r e m e n t o f P N were 355 a n d 450 n m , r e s p e c t i v e l y . T h o s e for m e a s u r e m e n t o f F P w e r e 460 a n d 520 n m . I n a d d i t i o n , t h e reflect a n c e s i g n a l s a t t h e w a v e l e n g t h s of 540 a n d 560 n m ,
ANALYSIS OF REDOX H E T E R O G E N E I T Y IN ISCHEMIC RAT LIVER
control (before clamping)
200t
(
0.86, mean,0.620-1.13 rain - max}I
100 5O 00,2 0,4 06 08 1 12 14
group 1
group 3
group 2 after clamping 0,63, 0 48 - 0,82 I
%:2 0', 06 06
I
I
i
i
I
112 14 02 0~4 06 08 1 12 1.4 0,2 04 06 0.8 1 12 1.4
133
I n order to assess the association b e t w e e n the c h a n g e s in the redox state a n d the liver cell damage, s e r u m alanine a m i n o t r a n s f e r a s e ( A L A T ) a n d lactic deh y d r o g e n a s e ( L D H ) were m e a s u r e d at 2 h after restoration of hepatic blood flow in rats in s h a m operation, group 1 a n d group 3 (n = 4, in each group). A n i m a l p r e p a r a t i o n s were the same as t h o s e described above. All animals received h u m a n e care a n d the e x p e r i m e n tal protocol was in c o m p l i a n c e with the i n s t i t u t i o n ' s guidelines. All values were p r e s e n t e d as m e a n _ SE. Statistical analysis of the c h a n g e s in the t h r e e groups was done using analysis of variance. In cases in which statistical difference was s h o w n by analysis of variance, a twosample t test was additionally p e r f o r m e d . A P value less t h a n 0.05 was c o n s i d e r e d significant.
2 minutes after declampin, 1~ 2 0 50 1 .975.000 056 ~ 0- 1.63 0
00.20,4060.9
1 121,4020.4
' '
-
i
0,53, 0.32 - 0.69
0.60.8 1 121.40.20.40.60'8
1 1'2 '14
10 minutes after declamping
°°loo00o I 0 80
0{~'.2 0'4 0'6 0.8 1 1'2 1 ' 4 0 2 0 4 0 6 0 8
1 12 14 02 04 0.6 08 1 1.2 t4
FIG. 1. Typical changes in FP/PN histogram in each group. The x axis shows the FP/PN value and the y axis shows the number of points in the scan in which a particular FP/PN value was recorded. In group 1, the histogram showed a remarkably wide range at 2 min after declamping and almost recovered at 10 min after declamping. In groups 2 and 3, the mean of histogram did not recover to the control levels at 10 rain after declamping and the range width continued to increase.
which are the a b s o r p t i o n p e a k s of oxy- a n d d e o x y h e m o globin, respectively, were s i m u l t a n e o u s l y m e a s u r e d a n d the s u m of the two was u s e d as an indicator of hemoglobin c o n c e n t r a t i o n . A q u a r t z microlight guide with a dia m e t e r of 90 #m, c o n t a i n i n g the excitation a n d emission light, a u t o m a t i c a l l y s c a n n e d the flat surface of the tissue. T h e distance b e t w e e n the tissue surface a n d the light guide end was a b o u t 50 #m, a n d the s e p a r a t i o n of m e a s u r i n g points was 100 #m. In one m e a s u r e m e n t , d a t a of 30 × 30 p o i n t s in the area of 3 × 3 m m 2 were acquired. Since the size o f one hepatic acinus is of the order of 0.5 to 1 m m in diameter, one acinus should be covered by 25 to 100 m e a s u r i n g points. Optical d a t a r e a d o u t from the p h o t o m u l t i p l i e r was stored in the c o m p u t e r a n d p r e s e n t e d as a f r e q u e n c y - d i s t r i b u t e d hist o g r a m or a t w o - d i m e n s i o n a l gray-scaled picture.
RESULTS T h e results of F P / P N h i s t o g r a m are listed in T a b l e 1. Typical c h a n g e s in F P / P N h i s t o g r a m in each group are shown in Fig. 1. As the ischemic time increased, b o t h the m e a n a n d the SD of F P / P N h i s t o g r a m decreased. I n group 1, F P / P N h i s t o g r a m showed a r e m a r k a b l y wide range at 2 min after declamping. Small p o p u l a t i o n s of h e p a t o c y t e s showed F P / P N values higher t h a n t h o s e before c l a m p i n g (overshoot of F P / P N ) . B o t h the m e a n a n d the SD were a l m o s t c o m p l e t e l y recovered to preischemic levels at 10 min after declamping. In c o n t r a s t , in groups 2 a n d 3, the m e a n of F P / P N h i s t o g r a m did n o t recover to the c o n t r o l levels. T h e SD a n d range width c o n t i n u e d to increase until 10 min after declamping, a t e n d e n c y t h a t was more p r o n o u n c e d in group 3. While the m a x i m a l value of F P / P N h i s t o g r a m recovered to some extent, the m i n i m a l value r e m a i n e d at low levels in group 2 a n d decreased f u r t h e r in group 3. Figures 2A, 2B, a n d 2C show a set of gray-scaled pictures o f F P / P N a n d hemoglobin. Figures 2A, 2B, a n d 2C r e p r e s e n t n o r m a l liver, liver 2 min after 10-min ischemia, a n d liver 10 min after 20-min ischemia, respectively. T h e d a r k areas in each picture depict a low F P / P N value in F P / P N pictures a n d a high h e m o g l o b i n c o n c e n t r a t i o n in h e m o g l o b i n pictures. A l t h o u g h t h e acinus image was p o o r l y defined in Fig. 2A, it was m o r e clearly s h o w n in Figs. 2B a n d 2C, where t h e encircled areas p r o b a b l y c o r r e s p o n d to liver acini. T h e s e pictures would indicate t h a t the areas of m o r e reduced redox state generally c o r r e s p o n d to the areas of higher h e m o globin c o n c e n t r a t i o n . Figures 2D a n d 2E show the pixel correlation between F P / P N values a n d h e m o g l o b i n reflectance signals in Figs. 2B a n d 2C, respectively (P < 0.001). Figure 3 shows the s e r u m A L A T a n d L D H levels at 2 h after r e s t o r a t i o n of hepatic b l o o d flow in rats in s h a m operation, group 1, a n d group 3. T h e r e is no significant difference between s h a m o p e r a t i o n in group 1, w h e r e a s
134
KITAI
A
FP/PN
E T AL.
B
HEMOGLOBIN (540 nm + 560 nm)
--
FP/PN
HEMOGLOBIN (540 nm + 560 nm)
1 mm
C
1 mm
D
FP/PN
1,200 0
1,150
8 1,100
g
O0
0
o o~
o
o°
~o
o/~--"
Oo qb 0000 % o~oo o ~ g ~o ° ° g~_
o
oo Oo
c 1,050 ~o 1000
~ o ~ oO
°
~oo
950 900 60
11
I
I
I
I
0
80
90
1O0
1 10
E
1,200
1,150
o°
8
o o °
1,100
oo o O0 0
g 1,050
0 0
1000
•.=
1 mm
120
FP/PN
HEMOGLOBIN (540 nm + 560 nm)
950 45
o
O8o.~
o ~ ~" oO0 o 9 ~ / o o°(9° j oO 0 0
O0 0%
0 0 0
OGgO 0
5'0 5'5 6'0 5'5 7'0 7'5 8'0 FP/PN
85
ANALYSIS OF REDOX HETEROGENEITY IN ISCHEMIC RAT LIVER A
8
t
p
Changes in the Redox Distribution of Rat Liver by Ischemia Toshiyuki Kitai, Akira Tanaka, Atsuo Tokuka, Kazue Ozawa, Shingo Iwata,* and Britton Chance* Second Department of Surgery, Faculty o[ Medicine, Kyoto University, 54 Kawaracho Shogoin, Sakyoku, Kyoto, 606 Japan; and *The Johnson Research Foundation, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
R e c e i v e d M a r c h 11, 1992
The changes in the redox distribution of the ischemic r a t l i v e r w e r e s t u d i e d u s i n g t h e r e d o x s c a n n e r , w h i c h is a time-sharing, tissue fluorescence and reflectance spectroscopy with fine spatial resolution, with the results p r e s e n t e d a s a h i s t o g r a m a n d a g r a y - s c a l e d p i c t u r e . Hep a t i c i n f l o w w a s c l a m p e d f o r 1 0 rain ( g r o u p 1), 2 0 m i n ( g r o u p 2), a n d 3 0 m i n ( g r o u p 3). T h e r a t i o o f o x i d i z e d f l a v o p r o t e i n to r e d u c e d p y r i d i n e n u c l e o t i d e f l u o r e s cence (FP/PN), representing the intracellular redox state, and hemoglobin reflectance were measured prior to c l a m p i n g a n d at s u b s e q u e n t t i m e s f o l l o w i n g d e c l a m p ing in each group. The mean and the range width of FP/PN histogram decreased with the prolongation of i s c h e m i c t i m e . I n g r o u p 1, F P / P N h i s t o g r a m s h o w e d a r e m a r k a b l y w i d e r a n g e at 2 rain a f t e r d e c l a m p i n g b u t w a s a l m o s t c o m p l e t e l y r e c o v e r e d to p r e i s c h e m i c l e v e l s at 1 0 m i n a f t e r d e c l a m p i n g . I n c o n t r a s t , i n g r o u p s 2 a n d 3, t h e m e a n o f F P / P N h i s t o g r a m w a s o n l y p a r t i a l l y rec o v e r e d at 1 0 m i n a f t e r d e c l a m p i n g a n d t h e r a n g e w i d t h c o n t i n u e d to i n c r e a s e . T h e c o r r e l a t i o n b e t w e e n F P / P N fluorescence and hemoglobin reflectance pictures sugg e s t e d t h a t t h e l o c a l s t a g n a t i o n o f b l o o d f l o w c a u s e d tissue hypoxia, preventing the recovery of the redox state p r o b a b l y i n t h e c e n t r i l o b u l a r a r e a . © 1992 Academic Press, Inc.
A liver acinus forms a microcirculatory u n i t of the liver p a r e n c h y m a . Since the hepatic blood flow is unif o r m l y directed along the sinusoids from the t e r m i n a l p o r t a l venule to the t e r m i n a l hepatic venule, gradients of oxygen, substrate, h o r m o n e s , and various metabolites p r o d u c e d by cellular activity along the capillaries are more p r o m i n e n t in the liver t h a n in o t h e r organs (1,2). As R a p p a p o r t a n d K a t z p r o p o s e d in their zonation of hepatic p a r e n c h y m a (3,4), cell e n v i r o n m e n t s are quite different b e t w e e n the p e r i p o r t a l area a n d the centrilobular area of an acinus, a n d h e p a t o c y t e s are also 0003-2697/92 $5.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
morphologically and functionally organized in the f r a m e w o r k of the microcirculatory unit. It is well k n o w n t h a t various liver damage occurs h e t e r o g e n e o u s l y in the liver acinus (3,6). C e n t r a l necrosis often seen in the hypoxic liver indicates t h a t the c e n t r i l o b u l a r area is far more susceptible to hypoxic stress t h a n the p e r i p o r t a l area (5,6). Although hepatic ischemia is one of the surgical models m o s t f r e q u e n t l y studied f r o m the aspect of energy m e t a b o l i s m (7-10), the changes at the level of the liver acinus r e m a i n to be clarified. Intracellular redox state of pyridine nucleotide ( N A D + / N A D H , N A D P + / N A D P H ) has been used as an indicator of intracellular o x y g e n a t i o n of the tissue {6,7). Quistorff and C h a n c e have developed a scanning fluorim e t e r t h a t measures the oxidized f o r m of flavoprotein (FP) 1 a n d the reduced form of pyridine nucleotide (PN) (11,12). T h e fluorescence signal of F P originates m o s t l y from the m i t o c h o n d r i a l c o m p a r t m e n t . T h e b a c k g r o u n d signal of PN, which is not associated with the redox transition, is relatively small in the liver a n d the P N signal f r o m the m i t o c h o n d r i a is severalfold e n h a n c e d at low t e m p e r a t u r e . T h e r e f o r e , the ratio of F P / P N represents the intracellular, p r e d o m i n a n t l y mitochondrial, redox state (13). T h e redox s c a n n e r has some advantageous functions: (i) the spatial resolution is a b o u t 100 #m, which is fine enough for m e a s u r e m e n t at the level of the liver acinus. (ii) F P / P N is insensitive to changes in m i t o c h o n d r i a l c o n c e n t r a t i o n , i n t e r f e r i n g pigments, and red blood cells in the tissue (14). T h i s is an i m p o r t a n t quality which m a k e s it possible to m e a s u r e the redox state of tissue c o n t a i n i n g blood. (iii) Since the measurem e n t is carried out with a f r e e z e - t r a p p e d sample at subzero t e m p e r a t u r e , F P / P N should give a still profile of the redox state at the time of sampling. A previous s t u d y with hemoglobin-free p e r f u s e d rat liver showed the peri1Abbreviations used: FN, flavoprotein; PN, pyridine nucleotide; ALAT, alanine aminotransferase; LDH, lactate dehydrogenase. 131
132
KITAI ET AL. TABLE 1
Mean
SD
Min
Max
Range
Control Before clamp (n = 7)
0.84 ± 0.02
0.07 ± 0.01
0.57 ± 0.06
1.10 ± 0.04
0.53 ___0.07
Group 1 After clamp (n = 4) 2 rain after declamp (n = 7) 10 min after declamp (n = 5)
0.67 - 0.04* 0.74 ± 0.05*** 0.73 _ 0.06
0.07 ± 0.01*'t 0.13 _ 0.02***'t 0.07 _ 0.01t
0.44 - 0.08t 0.33 ± 0.05**'t 0.48 ± 0.04*'t
1.03 ± 0.08** 1.34 _ 0.14"** 1.02 _ 0.10
0.59 ± 0.14 1.00 ± 0.16"** 0.53 _ 0.08
Group 2 After clamp (n = 4) 2 min after declamp (n = 6) 10 min after declamp (n = 5)
0.61 - 0.02*'tt'§ 0.58 ± 0.03***'tt'§ 0.63 - 0.08tt
0.06 -+ 0.00" 0.08 ± 0.01"** 0.08 ± 0.00
0.46 - 0.035 0.32 ± 0.01"*.:~ 0.32 - 0.08"$
0.85 ± 0.03**'t 0.80 _ 0.06***'t 0.94 ± 0.13t
0.40 - 0.06 0.48 _+0.05*** 0.62 ± 0.08
Group 3 After clamp (n = 5) 2 min after declamp (n = 4) 10 min after declamp (n = 4)
0.50 -+ 0.04*'tt'§ 0.55 ± 0.02***'tt'§§ 0.56 ± 0.04~t'§
0.05 -+ 0.01*'t 0.07 ± 0.01***'t 0.11 ± 0.03t
0.34 _+0.04**'tt 0.33 - 0.01*'tt 0.22 ± 0.08*'tt
0.70 - 0.06**'tt'§ 0.81 ± 0.12***'tt 0.97 ± 0.05tt
0.36 - 0.05t 0.48 ± 0.11***'t 0.75 ± 0.11t
Note. Mean, SD, rain, max, and range correspond to the mean value, the standard deviation, the minimal value, the maximal value, and the range width of the FP/PN histogram. * Significantly different between groups 1, 2, and 3 by analysis of variance. P < 0.05. ** P < 0.01. *** P < 0.001. t Significantly different in time course changes of each group by analysis of variance. P < 0.05. :~P < 0.01. t t P < 0.001. § Significantly different from the control value by two-sample t test. P < 0.05. §§ P < 0.01.
portal to c e n t r i l o b u l a r redox gradient, which changes a c c o r d i n g t o v a r i o u s m e t a b o l i c p e r t u r b a t i o n s (11). L i v e r a n o x i a decreased b o t h the average a n d the g r a d i e n t of the redox state in an acinus. However, the changes in t h e in situ l i v e r m u s t b e d i f f e r e n t f r o m t h o s e i n t h e b l o o d - f r e e p e r f u s e d liver, s i n c e t h e o x y g e n - c a r r y i n g cap a c i t y o f w h o l e b l o o d is m u c h g r e a t e r t h a n t h a t of h e m o globin-free perfusate, and the changes in the systemic hemodynamics and the blood viscosity should profoundly influence the hepatic microcirculation. I n t h i s s t u d y , we i n v e s t i g a t e d t h e c h a n g e s i n t h e r e d o x d i s t r i b u t i o n of the rat liver after c l a m p i n g a n d declamping the portal vein and the hepatic artery, using the r e d o x s c a n n e r . I n a d d i t i o n , t h e effects of t h e h e p a t i c microcirculation on the redox distribution were simultan e o u s l y assessed by reflectance of hemoglobin.
MATERIALS AND METHODS
Animalpreparation. M a l e r a t s w e i g h i n g f r o m 200 t o 300 g w e r e u s e d . E a c h r a t w a s a n e s t h e s i z e d b y i n t r a p e r i t o n e a l i n j e c t i o n of s o d i u m p e n t o b a r b i t a l (5 m g / 1 0 0 g). T h e r a t s w e r e d i v i d e d i n t o t h r e e g r o u p s ; g r o u p 1, 10 m i n o f h e p a t i c i s c h e m i a ( n = 16); g r o u p 2, 20 r a i n o f h e p a t i c i s c h e m i a ( n = 15); g r o u p 3, 30 m i n of h e p a t i c i s c h e m i a ( n = 13). H e p a t i c i s c h e m i a w a s p r o d u c e d b y c r o s s - c l a m p ing the hepatoduodenal ligament including the hepatic
artery, the portal vein, a n d the biliary duct. One h u n d r e d u n i t s of h e p a r i n w a s i n t r a v e n o u s l y i n j e c t e d b e fore c l a m p i n g t o p r e v e n t i n t r a h e p a t i c c o a g u l a t i o n . I n each group, hepatic tissue was freeze-trapped by alumin u m tongs which were sufficiently precooled in liquid n i t r o g e n . S a m p l e s w e r e o b t a i n e d (i) b e f o r e c l a m p i n g ( c o n t r o l ) , (ii) a f t e r c l a m p i n g , (iii) a t 2 m i n , a n d (iv) a t 10 min after declamping.
Measurements of tissue fluorescence and reflectance. O p t i c a l m e a s u r e m e n t of t h e h e p a t i c t i s s u e w a s d o n e foll o w i n g t h e m e t h o d d e s c r i b e d b y Q u i s t o r f f et al. (11,12), e x c e p t for h e m o g l o b i n r e f l e c t a n c e . T h e i n s t r u m e n t u s e d is a t i m e - s h a r i n g , t w o - d i m e n s i o n a l t i s s u e f l u o r e s c e n c e and reflectance spectroscopy, coupled with a minicomp u t e r for d a t a a n a l y s i s . A f r o z e n s a m p l e w a s i m m e r s e d in liquid n i t r o g e n b a t h in order to hold the redox t r a n s i t i o n , a n d a flat s u r f a c e for s c a n n i n g was m a d e b y t h e l o w - t e m p e r a t u r e milling system. S c a n n i n g was perf o r m e d b e t w e e n 50 a n d 500 # m f r o m t h e s u r f a c e , s i n c e i t h a s b e e n r e p o r t e d t h a t t h e r e d o x s t a t e was h e l d c o n s t a n t at such depths. T h e light source was a 100-W s h o r t m e r c u r y arc c o m b i n e d w i t h a 1 0 - m W h e l i u m c a d m i u m l a s e r a t 442 n m . T h e w a v e l e n g t h s o f e x c i t a t i o n a n d e m i s s i o n filters e m p l o y e d for m e a s u r e m e n t o f P N were 355 a n d 450 n m , r e s p e c t i v e l y . T h o s e for m e a s u r e m e n t o f F P w e r e 460 a n d 520 n m . I n a d d i t i o n , t h e reflect a n c e s i g n a l s a t t h e w a v e l e n g t h s of 540 a n d 560 n m ,
ANALYSIS OF REDOX H E T E R O G E N E I T Y IN ISCHEMIC RAT LIVER
control (before clamping)
200t
(
0.86, mean,0.620-1.13 rain - max}I
100 5O 00,2 0,4 06 08 1 12 14
group 1
group 3
group 2 after clamping 0,63, 0 48 - 0,82 I
%:2 0', 06 06
I
I
i
i
I
112 14 02 0~4 06 08 1 12 1.4 0,2 04 06 0.8 1 12 1.4
133
I n order to assess the association b e t w e e n the c h a n g e s in the redox state a n d the liver cell damage, s e r u m alanine a m i n o t r a n s f e r a s e ( A L A T ) a n d lactic deh y d r o g e n a s e ( L D H ) were m e a s u r e d at 2 h after restoration of hepatic blood flow in rats in s h a m operation, group 1 a n d group 3 (n = 4, in each group). A n i m a l p r e p a r a t i o n s were the same as t h o s e described above. All animals received h u m a n e care a n d the e x p e r i m e n tal protocol was in c o m p l i a n c e with the i n s t i t u t i o n ' s guidelines. All values were p r e s e n t e d as m e a n _ SE. Statistical analysis of the c h a n g e s in the t h r e e groups was done using analysis of variance. In cases in which statistical difference was s h o w n by analysis of variance, a twosample t test was additionally p e r f o r m e d . A P value less t h a n 0.05 was c o n s i d e r e d significant.
2 minutes after declampin, 1~ 2 0 50 1 .975.000 056 ~ 0- 1.63 0
00.20,4060.9
1 121,4020.4
' '
-
i
0,53, 0.32 - 0.69
0.60.8 1 121.40.20.40.60'8
1 1'2 '14
10 minutes after declamping
°°loo00o I 0 80
0{~'.2 0'4 0'6 0.8 1 1'2 1 ' 4 0 2 0 4 0 6 0 8
1 12 14 02 04 0.6 08 1 1.2 t4
FIG. 1. Typical changes in FP/PN histogram in each group. The x axis shows the FP/PN value and the y axis shows the number of points in the scan in which a particular FP/PN value was recorded. In group 1, the histogram showed a remarkably wide range at 2 min after declamping and almost recovered at 10 min after declamping. In groups 2 and 3, the mean of histogram did not recover to the control levels at 10 rain after declamping and the range width continued to increase.
which are the a b s o r p t i o n p e a k s of oxy- a n d d e o x y h e m o globin, respectively, were s i m u l t a n e o u s l y m e a s u r e d a n d the s u m of the two was u s e d as an indicator of hemoglobin c o n c e n t r a t i o n . A q u a r t z microlight guide with a dia m e t e r of 90 #m, c o n t a i n i n g the excitation a n d emission light, a u t o m a t i c a l l y s c a n n e d the flat surface of the tissue. T h e distance b e t w e e n the tissue surface a n d the light guide end was a b o u t 50 #m, a n d the s e p a r a t i o n of m e a s u r i n g points was 100 #m. In one m e a s u r e m e n t , d a t a of 30 × 30 p o i n t s in the area of 3 × 3 m m 2 were acquired. Since the size o f one hepatic acinus is of the order of 0.5 to 1 m m in diameter, one acinus should be covered by 25 to 100 m e a s u r i n g points. Optical d a t a r e a d o u t from the p h o t o m u l t i p l i e r was stored in the c o m p u t e r a n d p r e s e n t e d as a f r e q u e n c y - d i s t r i b u t e d hist o g r a m or a t w o - d i m e n s i o n a l gray-scaled picture.
RESULTS T h e results of F P / P N h i s t o g r a m are listed in T a b l e 1. Typical c h a n g e s in F P / P N h i s t o g r a m in each group are shown in Fig. 1. As the ischemic time increased, b o t h the m e a n a n d the SD of F P / P N h i s t o g r a m decreased. I n group 1, F P / P N h i s t o g r a m showed a r e m a r k a b l y wide range at 2 min after declamping. Small p o p u l a t i o n s of h e p a t o c y t e s showed F P / P N values higher t h a n t h o s e before c l a m p i n g (overshoot of F P / P N ) . B o t h the m e a n a n d the SD were a l m o s t c o m p l e t e l y recovered to preischemic levels at 10 min after declamping. In c o n t r a s t , in groups 2 a n d 3, the m e a n of F P / P N h i s t o g r a m did n o t recover to the c o n t r o l levels. T h e SD a n d range width c o n t i n u e d to increase until 10 min after declamping, a t e n d e n c y t h a t was more p r o n o u n c e d in group 3. While the m a x i m a l value of F P / P N h i s t o g r a m recovered to some extent, the m i n i m a l value r e m a i n e d at low levels in group 2 a n d decreased f u r t h e r in group 3. Figures 2A, 2B, a n d 2C show a set of gray-scaled pictures o f F P / P N a n d hemoglobin. Figures 2A, 2B, a n d 2C r e p r e s e n t n o r m a l liver, liver 2 min after 10-min ischemia, a n d liver 10 min after 20-min ischemia, respectively. T h e d a r k areas in each picture depict a low F P / P N value in F P / P N pictures a n d a high h e m o g l o b i n c o n c e n t r a t i o n in h e m o g l o b i n pictures. A l t h o u g h t h e acinus image was p o o r l y defined in Fig. 2A, it was m o r e clearly s h o w n in Figs. 2B a n d 2C, where t h e encircled areas p r o b a b l y c o r r e s p o n d to liver acini. T h e s e pictures would indicate t h a t the areas of m o r e reduced redox state generally c o r r e s p o n d to the areas of higher h e m o globin c o n c e n t r a t i o n . Figures 2D a n d 2E show the pixel correlation between F P / P N values a n d h e m o g l o b i n reflectance signals in Figs. 2B a n d 2C, respectively (P < 0.001). Figure 3 shows the s e r u m A L A T a n d L D H levels at 2 h after r e s t o r a t i o n of hepatic b l o o d flow in rats in s h a m operation, group 1, a n d group 3. T h e r e is no significant difference between s h a m o p e r a t i o n in group 1, w h e r e a s
134
KITAI
A
FP/PN
E T AL.
B
HEMOGLOBIN (540 nm + 560 nm)
--
FP/PN
HEMOGLOBIN (540 nm + 560 nm)
1 mm
C
1 mm
D
FP/PN
1,200 0
1,150
8 1,100
g
O0
0
o o~
o
o°
~o
o/~--"
Oo qb 0000 % o~oo o ~ g ~o ° ° g~_
o
oo Oo
c 1,050 ~o 1000
~ o ~ oO
°
~oo
950 900 60
11
I
I
I
I
0
80
90
1O0
1 10
E
1,200
1,150
o°
8
o o °
1,100
oo o O0 0
g 1,050
0 0
1000
•.=
1 mm
120
FP/PN
HEMOGLOBIN (540 nm + 560 nm)
950 45
o
O8o.~
o ~ ~" oO0 o 9 ~ / o o°(9° j oO 0 0
O0 0%
0 0 0
OGgO 0
5'0 5'5 6'0 5'5 7'0 7'5 8'0 FP/PN
85
ANALYSIS OF REDOX HETEROGENEITY IN ISCHEMIC RAT LIVER A
8
t
p
