Liver, 1989: 9, 6-13 Key words: energy metabolism; liver ischemia; prosta- glandin E l ; reticuloendothelial function.

Protective effect of prostaglandin E 1 (PGE 1) on energy metabolism and reticuloendothelial function in the ischemically damaged canine liver

YUJl UEDA, KENlCHl MATSUO, TAKASHI KAMEI, KOHJl

KAYASHIMA AND KOHKl KONOMI

First Department of Surgery, Kyushu University, Faculty of Medicine, Fukuoka, Japan

ABSTRACT - The protective effects of PGEl on ischemia-related liver damage were evaluated in dogs. Ninety minutes warm hepatic ischemia was induced by the total clamping of hepatic inflow vasculatures with portal bypassing. The survival rate improved up to 62.5% when PGEl was administered intravenously prior to ischemia, while no dog survived for longer than 1 week in the nontreated group. Hepatic ATP content was restored up to 80% of preischemic level 2 h after reflow in the PGEl pretreated group, compared to 55% recovery in the nontreated group. Complete normalization of hepatic energy charge and rapid decrease of lactate were also seen in the PGEl group. The clearance rate of intravascular lipid emulsion remained fairly normal in the PGE 1 group, thereby suggesting well-preserved hepatic reticuloendothe- lial functions. The serum activities of P-glucuronidase, GOT and GPT were suppressed in the PGE 1 -pretreated group, thereby implying a well-protected hepatic integrity. The histology revealed well-preserved hepatic architecture. The remarkable cytoprotective effect of PGEl on hepatic ischemia shown in this study indicates that PGEl warrants further study for protection of ischemically compromised hepatic allografts.

Accepted for publication 25 May 1988

The search has continued for an improved means to protect hepatic allografts from ischemic injury ( I , 2). Current investigations have demonstrated the potent cytoprotective effects of PGEl on vari- ous types of organ damage (3-6). There is, how- ever, little information concerning the effect of this agent on ischemia-related liver damage. Al- though a clear interpretation of the pathogenesis of ischemic liver failure remains to be elucidated, there are several possible explanations. Hepato- cytes seem to be most susceptible to anoxic con- ditions (7). It has been emphasized that the im- pairment of mitochondria1 energy metabolism and the consequent ATP depletion play a most critical role in the course of functional and struc- tural deteriorations which develop in the ischemic

liver (8-10). Leaked lysosomal enzymes have been considered to be one of the causative factors lead- ing to irreversible liver damage and subsequent systemic organ failure (1 1-13). Postischemic or- gan hypoperfusion designated as “no reflow phen- omenon” appeared to be caused by destruction of microvascular beds, microsludging and vascular spasm, and would contribute to organ damage (8, 12, 14). The hepatic reticuloendothelial phago- cytic function has also been considered to play a key role in host-defense mechanisms against ischemia-related liver failure (1 5).

It seems to be of paramount importance to evaluate PGEl as a potential agent against ische- mic insult and to elucidate its role in protection, since PGEl has numerous biological activities in-

EFFECT OF PGEl ON LIVER ISCHEMIC DAMAGE 7

cluding vasodilation, anti-platelet aggregation and lysosomal membrane stabilization.

Material and methods Ischemic model Fifty-nine adult mongrel dogs weighing 12-16 kg were used. After a 24-h fast, they were anesthetized with ketamine hydrochloride (5 mg/kg) and pentobarbital so- dium (25 mg/kg). An endotracheal tube was inserted and the animals were artificially ventilated with equal volumes of nitrous oxide and oxygen. The direct arterial pressure and esophageal temperature were monitored continuously. After laparotomy, the liver hilus was dis- sected free, followed by resection of all hepatic ligaments and ligation of phrenic vessels. Following splenectomy, the Anthron Bypass Tube (VTT-51160; 5 mm (OD) x 4 mm (ID) x 600 mm (L): Toray Medical Co., Ltd., Japan) which is heparin-coated on the inner surface, was placed between the splenic and left external jugular vein for portal bypass. Hepatic ischemia was induced by clam- ping of the portal vein, common hepatic and gastroduo- denal artery.

After completion of 90 min of ischemia, the liver was declamped and systemic acidosis was corrected by sodium bicarbonate injection. About 80 ml/kg of lac- tated Ringer’s solution, which contains 5% dextrose, and 2 g of Ampicillin was infused throughout the oper- ation.

Experimental design Six dogs were used to measure both the Indocyanine Green (ICG) retention rate and the portal pressure in the anhepatic phase. Seven dogs were used in the normal and six in the sham-operated group in the phagocytic function study. The other 40 dogs were separated into 6 groups (Table 1). Four groups were prepared for acute experiment. In Group 1, as nontreated control, lactated Ringer’s solution was infused before clamping. In Groups 2,3 and 4, PGEl (On0 Pharm. Co., Ltd., Osaka) in doses of 0.1, 0.5 and 1 .O pg/kg/min, respectively, was administered intravenously with an infusion pump for 30 min before clamping. Groups 5 and 6 were prepared for chronic experiment to estimate survival rates. In- fusion and antibiotic therapy were continued for 3 days after operation. Animals living for 2 weeks were regard- ed as long survivors and were killed for histology studies.

Cellular content of adenine nucleotides and lactate About 1 g of liver tissue was taken before, at 30 and 90 min of ischemia and also I and 2 h following reflow. The sample was crushed within a few seconds with aluminum tongs precooled in liquid N2, and was then powdered.

Table I Experimental design for pre-ischemic treatment in dogs

Group (n) Pre-treatment Dose

1 (6) Solvent* 2 (6) PGEI** 0.1 pg/kg/min x 30 min 3 (6) PGEl 0.5 pg/kg/min x 30 min 4 (6) PGEl 1 .O pg/kg/min x 30 min

5 (8) Solvent 6 (8) PGEl 0.5 pg/kg/min x 30 min

-

-

* 200 ml of lactated Ringer’s solution. **Prostaglandin El.

Adenine nucleotides were extracted twice with 8.5% and 6.5% perchloric acid, respectively. The supernatants were neutralized with 3 mol KK03 and recentrifuged at 25 000 g for 10 min and stored at -80°C. The adenosine 5’-triphosphate (ATP), adenosine 5’-diphosphate (ADP) and adenosine 5’-monophosphate (AMP) levels were as- sayed enzymatically (16). Lactate was assayed using a Boehringer-Mannheim kit.

Phagocytic activity of the reticuloendothelial system Two hours after reflow, lipid emulsion (Intralipid 10%; Vitrum Stockholm, Sweden) in a dose of 1 .O ml(100 mg)/ kg was injected through a foreleg vein. The turbidities of the serum at 2, 4, 6, 8 and 10 min following injection were measured spectrophotometrically. Phagocytic ac- tivity was estimated by the determination of the intravas- cular clearance half-time (T1/2) of lipid emulsion (17).

Serum chemistry Blood samples were taken from Groups 5 and 6 in the pre- and postischemic periods and on the following postoperative days. Serum glutamic oxaloacetic trans- aminase (sGOT) and glutamic pyruvic transaminase (sGPT) were assayed using Nissui’s assay kit (Nissui Co., Ltd., Tokyo). For the assay of serum P-glucuronidase activity, para-nitrophenyl P-D-glucuronid (Boehringer- Mannheim) was used for the substrate according to Kato et al. (18).

Histology Liver tissues were taken 2 h after reflow and on the 14th postoperative day. The specimens were fixed in 10% formalin and stained with hematoxylin and eosin for light microscopical examination.

Statistical study All values of experimental data were presented as meanf standard deviation (mean & SD) and were ana-

8 UEDA ETAL.

lysed by Student’s t-test for paired and unpaired vari- ables. A P-value less than 0.05 was considered to be significant.

Results Evaluation of the ischemic model The liver quickly shrank and turned dark brown after clamping of the vasculatures. The ICG reten- tion rate was 94.58 f4.36%, thereby implying the completion of hepatic ischemia. The systemic hemodynamics was well maintained during is- chemia under portal bypassing with the anti- thrombogenic Anthron Bypass Tube. There was a transient increase in portal pressure from 9.75f0.50 mmHg of the preischemic value to 28.00k 11.60 mmHg immediately after portal clamping, and thereafter it gradually decreased to 16.75k3.86 mmHg. There was no apparent congestion in the splanchnic vascular beds. The esophageal temperature remained between 32°C and 33°C during ischemia, in each group.

Survival time PGEl pretreatment led to a marked improvement in the survival of dogs with warm hepatic ischemia (Table 2). Five of eight dogs (62.5%) given 0.5 pg/kg/min of PGEl survived for at least 2 weeks. The mean survival time in Group 6 was 10.75+ 4.98 days, or more, while no dogs lived longer than 7 days in the nontreated group (P<O.OOl). The livers in Group 6 dogs looked fairly normal, micro- and macroscopically, when the dogs were killed 2 weeks after operation. ,The only change was a slight atrophy with decoloration. In con- trast, engorged livers with massive hemorrhage were found in the nontreated dogs at autopsy.

Table 2 Survival of dogs after 90 min of warm hepatic ischemia

I ?8 - 2.0 E -

0 2 .- s $

g 1.0 -

k

c

3 a

\ I

4

0 0.5 1.5 2.5 3.5 Time (hours)

Fig. I . Changes in hepatic ATP content in the course of hepatic ischemia and reperfusion. Comparison between PGEl pretreated groups and nontreated group. Each symbol represents mean value of each group (Group 1; + 2, -A- 3, -0- 4, -0-). *P<O.O5, **P<0.005; as compared with Group 1 .

Adenine nucleotides, energy charge and lactate As shown in Fig. 1 and Table 3, the preischemic ATP level in the liver of dogs pretreated with PGEl was not significantly changed compared to the nontreated group, while the livers in Groups 3 and 4 were bright red during PGEl administra- tion, indicating an improved circulation and oxy- genation. At the end of ischemia, both the levels of ATP and the energy charge decreased to less than 13% and 35% of the preischemic values, respectively, in each group. After declamping, in Groups 3 and 4, there was a rapid reversion to a normal color.

The ATP levels were restored significantly up to about 80% and 74% of preischemic value, compared to 55% recovery in Group 1 (P<O.OOl,

Mean survival Survival rate Group (n) Survival time (hour, day) time (days) (14th day)

5 (8) 6 h 12h 12h 16h 2 4 h 2 d 2 d 7 d 1.74k2.23 0 Yo 6 (8) 24h 6 d 9 d 14d 1 4 d - 1 4 d 14d 14d 10.75 +4.98* 62.5%

*P<O.OOl; as compared with Group 5.

EFFECT OF PGEl ON LIVER ISCHEMIC DAMAGE 9

Table 3 Adenine nucleotides content and energy charge in hepatic tissue

(wmol/g wet weight)

Group (n) Time (h) ATP

1 (6) 0 1.5 3.5

2 (6) 0 1.5 3.5

1.5 3.5

3 (6) 0

4 (6) 0 1.5 3.5

2.20 k 0.28 0.24k0.09 1.20 * 0.20

2.14kO. 11 0.22k0.09 1.12 k 0.49

2.21 f 0.13 0.29k0.08 1.77+0.17*

2.14k0.13 0.23 k0.02 1.56 k 0.36**

ADP AMP TAN? EC§ 0.57+0.07 0.27+0.08 2.98k0.33 0.84k0.02 0.52k0.09 1.21k0.29 1.97k0.45 0.25+0.02 0.37k0.05 0.19k0.08 1.76k0.25 0.78k0.05

0.56+0.09 0.44kO.15 0.36+0.05

0.53 k0.13 0.58+0.15 0.40 k 0.04

0.54k0.06 0.37 k0.07 0.39k0.07

0.22k0.06 1.12*0.11 0.19k0.09

0.14k0.03 1.06k0.21 0.12kO.02

0.19k0.05 1.08+0.14 0.17 k0.04

2.93 + O . 16 1.78 k0.26 1.76f0.46

2.88 k0.24 1.92k0.30 2.30kO. 16*

2.88 k0.20 1.69 k 0.16 2.15k0.49

0.83 k 0.03 0.28 k0.06 0.77+0.l1

0.86+0.02 0.30+0.05 0.85 kO.Ol**

0.84k0.02 0.25k0.03 0.83 k0.02

t TAN; total adenine nucleotides (ATP + ADP+ AMP). EC; energy charge (ATP+O.5 ADP/TAN). All values are represented as mean f SD. *P<O.005, **P<O.O2; as compared with Group 1.

0.05), concomitant with complete normalization of the energy charge measured 2 h after reflow. Total adenine nucleotides in Groups 3 and 4 were also increased and reached 80% and 75% of preis- chemic value 2 h after reflow, respectively. On the other hand, resynthesis of ATP and total adenine nucleotides in Groups 1 and 2 were poor despite re-establishment of hepatic circulation. These re- sults indicate that the precursors of adenine nucle- otides in these groups might be further degraded and washed out of liver following reflow, much more so than those rephosphorylated to AMP, ADP and ATP. Fig. 2 shows the changes in lac- tates which accumulated in the liver to about 5 times preischemic level, in each group, at the end of ischemia. The levels declined more rapidly in Groups 3 and 4 after reflow, compared to the nontreated group (P < 0.01). These results indicate that the dogs pretreated with PGEl had a more satisfactory hepatic microcirculation as well as resynthesis of high energy phosphate bonds, de- spite serious ischemic insult.

Reticuloendothelial function Reticuloendothelial phagocytic activities were as- sessed at 2 h after reflow (Fig. 3). The clearance half-time of intravascular lipid emulsion was 16.56k2.56 min in normal and 15.08+4.08 min

in sham-operated dogs. The clearance half-time in the nontreated Group 1 was significantly pro- longed to about 1.9 times over that of the sham- operated group (P < 0.02), while those in Groups 3 and 4 were little affected (17.56k6.80 and 17.54k 4.53 min) and were significantly shorter than that in Group 1 (P<O.O5). These results suggest that PGEl pretreatment leads to a fairly intact phagocytic activity of Kupffer cells.

we-ischemia end of 1 hour reflow 2 hours reflow ischemia

Fig. 2. Changes in hepatic lactate content in the course of hepatic ischemia and reperfusion. Each bar represents mean valuekSD in each group. Group number. * P < 0.05, ** P < 0.005; as compared with Group 1.

10 UEDAETAL.

Serum enzyme levels As shown in Table 4, the postischemic elevation of serum P-glucuronidase activity was significantly suppressed in the PGEl group (Group 6), as com- pared to the nontreated group (Group 5) (P < 0.01). On the first postoperative day, these levels had already returned to within the normal range in both groups. The serum GOT and GPT values in Group 6 were also significantly lower than those of Group 5 (P<O.O5). These values, however, reverted to almost normal levels within 1 or 2 weeks after surgery.

Histological findings As presented in Fig. 4, the marked degeneration of hepatocytes and derangement of sinusoidal

5c

h

s 4c E s t- hi .- E 3c

v

c

'c KJ .s a, 2 20 !!

0

(d a, -

10

0

Fig. 3. T

\lormalt Shamtt 1 2 3 4 Group

reticuloendothelial phagocytic activity repre- ~ ~.

sented by intravascular clearance half-time of lipid emul- sion. Each bar represents mean value f SD with n num- ber in parenthesis. t: In the normal group, lipid emulsion was injected after induction of anesthesia. tt: In the sham-operated group, lipid emulsion was injected after splenectomy and skeletonization of the liver. * P < 0.02, ** P < 0.005; as compared with Group I .

architecture with congestion occurred 2 h after reflow in the nontreated dogs, while the appear- ance of hepatocytes in the PGEI-pretreated dogs was fairly well maintained despite prolonged is- chemia.

Discussion We obtained evidence that the administration of PGEl prior to induction of 90 min of hepatic ischemia improved the survival rate in dogs re- markably. Ninety minutes hepatic ischemia is gen- erally thought to lead to irreversible damage (7, 10). The extremely augmented survival rate ob- tained in this experiment showed the excellent protective effect of PGEl against hepatic ischemic insult.

ATP depletion and increase of plasma mem- brane permeability occurring with ischemia would compromise hepatocellular Ca2+ metabolism as well as Na+-K+ active transport. The increased Caz+ in the cytosol subsequently inactivates the mitochondrial phosphorylative function, in ad- dition to increasing the activities of membrane- bound phospholipase, and then finally disrupts cellular structures and functions (9). It was sug- gested by Sikujara that increased levels of hepato- cellular ATP and cyclic nucleotides might play a key role in accelerating the efflux of Caz+ during and after ischemia, and thereby regulating intra- cellular CaZ+ homeostasis (2). It has been reported that PGEl could increase cyclic AMP levels in liver slices, perfused liver and isolated hepatocyte by stimulating PGE-sensitive adenylate cyclase activity in the rat (19, 20). Smigel also showed that there were high affinity binding receptors for PGEl in rat liver plasma membranes (21). In our in vivo experiment, however, significant differ- ences in hepatic tissue cyclic AMP concentrations were nil between nontreated and PGEl-pretreated dogs (unpublished data). Nevertheless, a marked recovery of mitochondrial synthetic activity was evident in the PGEl-pretreated dogs. Thus, PGEl may exert a cytoprotective action on the ischemic liver not only through its action on the adenylate cyclase-cyclic-AMP system in hepatic cells but also through other mechanisms. The ameliorated ATP resynthesis in the postischemic liver showed a well-preserved and enhanced mitochondrial

EFFECT OF PGEI ON LIVER ISCHEMIC DAMAGE 11

Table 4 Serum enzyme levels before and after 90 min of warm henatic ischemia

Group (n) Time (h) ~-glucuronidase (R.U.)? GOT (K.U.) GPT (K.U.)@

5 (8) 0 23.6f5.9 42.0f 8.2 38.4k 12.4 3.0 87.0f23.1 845 f 721 658 f401 4.5 91.5k21.2 4860 2650 5200 & 3880

6 (8) 0 28.4+ 8.5 39.2+ 11.0 40.8 f 10.4 3.0 49.5+ 16.6** 199+87* 185+69 4.5 42.3+ 18.8** 698 460* 598 f 480

?The activity was expressed as a relative unit (R.U.) which shows the amount of released p-nitrophenol (pg) per hour of incubation time per ml of serum sample. SKarmen Unit. *P<O.O5, **P<O.Ol; as compared with Group 5.

phosphorylative function indispensable to main- tain various intracellular metabolic activities such as protein synthesis or active iron transport (7, 10).

The leakage of hydrolytic enzymes from lyso- somes and their activation by cellular acidosis in the ischemic liver seem to be critical contributing factors for the irreversible changes of the cell (8, 11, 12). Even after 30 min of hepatic ischemia in the dog, lysosomal enzymes could be detected in the systemic circulation (22). It was demonstrated that the life-supporting effects of PGEl on hemor- rhagic and endotoxin-shocked dogs might be due in part to stabilizing effects on lysosomal mem- brane (3, 4, 23). Raflo and co-workers also showed in an in vitro study that PGEl could directly inhibit the release of cathepsin D from

isolated hepatic and pancreatic lysosomes (24). The marked suppression of serum P-glucuronid- ase activity recognized in this experiment suggests the stabilizing effect of PGEl on lysosomal mem- brane in ischemic liver.

It was demonstrated by Di Luzio and col- leagues that reticuloendothelial phagocytic dys- function might be contributory to fatal outcome in shock caused by hepatic ischemia in baboons, and that in all animals undergoing 90 min of hepatic ischemia systemic endotoxemia occurred (15). Endotoxin, if not cleared from the blood stream by Kupffer cells, might cause hepatic cell injury and death, not only by aggravating the hemodynamic circulation but also by a direct un- coupling effect on the mitochondria and sub- sequent inhibition of ATP production (25,26). On

Fig. 4. Light photomicrographs of liver specimens from dogs subjected to 90 min of warm hepatic ischemia. (a) Liver biopsy taken from Group 3 two hours after reflow. Slight sinusoidal widening is evident. Note the well- preserved hepatocellular architecture (H & E x 200). (b) Liver biopsy taken from Group 1 two hours after reflow. Hepatocellular degeneration with cytoplasmic vacuolation and sinusoidal congestion are evident (H & E x 200).

12 UEDAETAL.

the other hand, the decreased ATP levels in the liver might in part contribute to the impaired phagocytic activity in Kupffer cells (27). Although we did not check the endotoxin level in this experi- ment, the fairly normal clearance of lipid emulsion in PGEl-pretreated dogs, in accord with the marked suppression of serum 0-glucuronidase ac- tivity, suggested the well-preserved phagocytic ac- tivity in reticuloendothelial systems. The lysoso- ma1 enzymes accumulating in the systemic circu- lation, if not cleared by Kupffer cells, would cause deleterious effects on systemic organs (1 3). The lysosomal stabilizing effects of PGEl might con- tribute to protecting Kupffer cells in the ischemic liver, as these cells contain numerous lysosomes (1 1). However, it must be taken into account that other factors such as hepatic blood flow or serum opsonin activity can also modify such functions (25).

We found that PGEl protected the kidney from prolonged warm ischemic damage and we sug- gested that marked improvement of ischemic renal cortical blood flow might in part be attributed to the vasodilating and anticoagulating actions of PGEl (5). The cellular swelling and destruction, vascular spasm and microsludging which follow ischemic insult would disturb sinusoidal microcir- culation (14, 28). The subsequent hypoperfusion in the liver would produce persistent tissue hypox- ia and impair aerobic energy production in the mitochondria, even after reperfusion (7, 8). In the present study, we observed that the liver quickly recovered a normal color concomitant with rapid wash-out of hepatic tissue lactate in the PGE1- pretreated dogs, thereby suggesting well-preserved hepatic mcirocirculation by its vasodilating and anticoagulating actions, in addition to stabiliza- tion of cellular membranes.

In case of clinical transplantation, liver grafts can be preserved safely for only 8 h or less (29). An improved method of liver preservation is urgently needed. The remarkable cytoprotective effects of PGEl on hepatic ischemia evidenced in the pres- ent study indicate that PGEl may be applicable for the protection of ischemic liver grafts.

Acknowledgements We thank Dr. H. Nakao, Nagoya University Faculty of Medicine, for pertinent advice, Ono Pharmaceutical Co.,

Osaka for the gift of prostaglandins, K. Hagio and M. Fukiyama for technical assistance and M. Ohara for comments on the manuscript.

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Address: Kohki Konomi, M.D. First Department of Surgery Kyushu University Faculty of Medicine 3- 1 -1 Maidashi, Higashi-ku Fukuoka 812, Japan