Cholestasis in the male rat is associated with naloxone-reversible antinociception

Journal of Hepatology 1994; 20:85-90 Prhtted in Denmark All rights reserved Munksgaard. Copenhagen

Copyright © Jotlrnalof Hepatolog), 1994 Journal of Hepatology ISSN 0168-8278

Cholestasis in the male rat is associated with naloxone-reversible antinociception N o r a Valeria Bergasa, D a v i d W. Ailing, J o h n Vergalla a n d E. A n t h o n y Jones Liver Diseases Section, Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, and Division of Intramural Research, National hTstitute of Allergy and blfectious Diseases; National Institutes of Health, Bethesda, Maryland, USA

(Received 4 August 1992)

Clinical observations have suggested that cholestasis is associated with increased neurotransmission mediated by the opioid system in the central nervous system. As opiate agonists (e.g. morphine) mediate analgesia, increased opioidergic tone in cholestasis should be associated with a decreased response to pain. To test this hypothesis, the response of rats with acute cholestasis to a nociceptive stimulus was measured by the use of the tail-flick test, an extensively validated assay for measuring opiate-induced antinociception. Five and 7 days after bile-duct resection, the mean tail-flick latency was longer than before surgery (p<0.05), whereas the corresponding means for unoperated and sham-resected controls were not significantly different from their respective baseline values. The increase in the mean tail-flick latency in the bileduct resection group was reversed by (-)-naloxone (1 mg/kg subcutaneously), but not by its enantiomer (+)-naloxone (10 mg/kg subcutaneously) (p<0.001). The stereoselective reversal of antinociception in cholestasis by naloxone indicates that this phenomenon is opioid-receptor mediated. In contrast, prolongation of the mean T F L found in the rat model of thioacetamide-induced acute hepatocellular necrosis was not reversed by (-)-naloxone, indicating that antinociception in this model is not opioid mediated. These findings provide support for the hypothesis that cholestasis is associated with increased opioidergic tone. © Journal of Hepatology. Key words." Antinociception; Cholestasis; Opioids; Rats; Tail-flick assay

It has been suggested that chronic cholestatic liver disease may be associated with increased neurotransmission mediated by the opioid system (1). Observations compatible with this hypothesis include precipitation of an opiate withdrawal-like syndrome in patients with chronic cholestatic liver disease by administration of an opiate antagonist (2) and a global down-regulation of mu-opioid receptors in the brain of rats with cholestasis due to bile-duct resection (BDR) (3). Morphine and related drugs with agonist properties are known to act at specific opioid receptors to which endogenous opioids also bind, and these drugs induce pruritus (1). Accordingly increased opioidcrgic tone (i.e. increased opioid-mediated neurotransmission) would be a potential mechanism of the pruritus of cholestatic liver disorders. Consistent with this concept,

and hence providing further support for the hypothesis that opioidergic tone is increased in cholestatic liver disease, are preliminary observations which suggest that opiate antagonists can induce ameliorations of the pruritus of cholestasis (2,4-6). One of the functions of the opioid system is the mediation of analgesia. It is well established that exogenously administered opiate agonists, such as morphine, mediate analgesia by binding to opioid receptors in the nervous system (7). A logical extension of this concept is that increased opiatergic tone secondary to a disease state would be associated with analgesia that is reversible by an opiate antagonist. To evaluate opioid neurotransmission in cholestasis, we have determined the effect of bileduct resection (BDR) in the rat on tail-flick latencies. The

(brrespondence to: Nora Valeria Bergasa, MD The Rockefeller University, Laboratory of the Biology of Addictive Diseases, Box H-28, 1230

"fork Avenue, New York, New York 10021, USA.

86 tail-flick assay is an extensively validated method for assaying the potency of opiates. Opiates that mediate analgesia, such as morphine, increase tail-flick latency (8-11). We postulated that if opioidergic tone is increased in cholestasis, rats with BDR would exhibit naloxone-reversible antinociception (decreased sensitivity to painful stimuli). However, the magnitude of such an effect mediated by endogenous opioids would be modest in relation to the profound antinociception induced by pharmacologic doses of potent opiate agonist drugs (8), since endogenous opioids have not been reported to mediate all of the effects of morphine.

Materials and Methods

Materials (-)-Naloxone was obtained from Dupont Pharmaceuticals, Inc. (Manati, P.R_) in vials containing 10 mg of naloxone, 0.4 mg/ml in 0.9 N saline. (+)-Naloxone was supplied by the Research Triangle Institute (Research Triangle, N_C.) in the form of a white anhydrous powder, which was dissolved in distilled water prior to injection. Thioacetamide (TAA) was obtained from Sigma (St. Louis, Mo.).

Anhnals Sprague-Dawley male rats weighing 150-200 g (Harlan Farms, Minneapolis, MN) were housed in standard cages and exposed to 12-h dark/light cycles. They were fed Puri-. na rat chow ad libitum and had free access to water_

Experh~Tental groups To study cholestasis, there were three experimental groups: unoperated control rats, sham-resected control rats and rats in which the main bile duct had been resected. Laparotomies were performed under general anesthesia induced by an intraperitoneal injection of 0.3 ml of an 8 : 1 (v/v) mixture of ketamine HCI, 50 mg/ml (ParkDavis, Morris Plains, N J), and xylazine HCI, 200 mg/ml (Fermenta Animal Health Company, Kansas City, MO). In the sham-resected group the main bile duct was identified, manipulated with forceps and left in situ. In the BDR group the main bile duct was first ligated using two ligatures approximately 0.5 cm apart and then transsected at the midpoint between the two ligatures (12). After closure of the abdominal wound in two layers, each shamresected and BDR animal received 4 ml 0.45 N saline 5% dextrose subcutaneously (s.c.). Operative mortality was less than 5%. Throughout the immediate postoperative period each animal was placed in a cage by itself to prevent wound dehiscence. Animals in each of the three experimental groups were studied in parallel.

N.V. BERGASA et al. To control for non-specific effects, a liver disease control group was also studied. This group consisted of rats 24 h after the intraperitoneal injection of TAA (600 mg/ kg). These rats constitute an uncomplicated model of acute hepatocellular necrosis characterized by serum alanine aminotransferase levels of >600 IU/I and pericentral hepatocellular necrosis (13). Controls for this group were rats 24 h after the intraperitoneal injection of saline.

Measurement of tail-flick latencies Tail-flick latencies (TFL) were measured in units of 0.1 s using a conventional tail-flick apparatus (8). A TFL is defined as the time interval between the application of a standardized beam of radiant light focused onto the tail and the abrupt removal of the tail from the nociceptive stimulus (tail-flick spinal reflex). In this study the beam was focused onto the tail 3 cm from its tip. The animal was allowed to acclimatize to the environment of the apparatus for at least 2 min before eliciting a tail flick.

Pilot studies Initially, pilot studies were conducted to standardize the nociceptive stimulus. Specifically, the precise intensity of the light beam focused on the tail was read from a Vernier scale on the apparatus and was adjusted by turning a control knob so that tail flicks occurred in normal rats after exposure to the beam for about 4 s (Vernier scale reading of 2.5 on the apparatus). This setting was subsequently kept constant throughout the conduct of these studies, except for a series of experiments in which the relationship between TFL and the intensity of nociceptive stimulus was specifically addressed. Pilot studies were also employed to determine optimal times after surgery at which to assess whether BDR increased TFL. Three days after surgery TFLs were moderately prolonged in the sham-resected group, consistent with postsurgical stress (14). By 5 days, prolongation of TFLs in the sham group had subsided.

Experimental design The operator of the tail-flick apparatus was unaware of the specific surgical or treatment group to which an animal belonged. Great care was taken to distinguish between pain-induced lash-like tail flicks, which were recorded, and spontaneous tail movements unrelated to its painful stimulation, which were rejected. Only the blinded operator made the judgement whether to accept or reject a given flick. Less than 2% of all flicks were rejected. When a flick was rejected, none of the data on that animal were subsequently used for analysis. a) Cholestasis study." On day 0 (i.e. before surgery in two of the experimental groups) and on days 5 and/or

INCREASED OPIATERGIC TONE IN CHOLESTASIS

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changes in T F L between days 0 and 7 (or 5) and those associated with ( - ) - n a l o x o n e administration were similar for unoperated and sham-resected control groups (p> 0.25), d a t a in these two groups were pooled and compared with corresponding d a t a in the B D R group using a t-test (43 df). Corresponding mean T F L changes during the 0-5-min interval after ( - ) or ( + ) naloxone administration were compared using a t-test.

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Fig. 1. Increased tail-flick latencies (TFL) in rats with acute cholestasis due to bile-duct resection (BDR) and their reversibility by an opiate antagonist. TFLs were significantly prolonged 7 days after BDR (n= 18), compared to unoperated (UNOP) (n= 13) and shamresected (SHAM) (n= 15) control groups. (-)-naloxone reversed the prolonged TFLs in the BDR group, but had no significant effect on TFLs in the control groups. TFLs were measured 5 min after treatment with (-)-naloxone. Data are means_+_SEM.

7, two baseline T F L s were obtained 15 min a p a r t before administration o f any opioid receptor ligand. Means o f these duplicate determinations were recorded. In one series o f experiments of days 5 and/or 7, T F L s were also obtained 5, 10 and 60 min after the s.c. administration o f ( - ) - n a l o x o n e (1 mg/kg). In another series o f experiments in which only B D R rats were studied, baseline T F L s were obtained at days 0 and 5 and at day 5, 5 min after the s.c. administration o f either ( + ) - n a l o x o n e (10 mg/kg) or ( - ) naloxone (1 mg/kg). In a further series of experiments T F L s were obtained in rats 5 days after B D R and in unoperated controls; each rat was exposed to the light beam at Vernier scale settings of 2.0, 3.0 and 4.0. The order of the exposures for each rat was 2.0, 4.0 and 3.0 to obviate order effects. b) Liver disease control study. T F L s were also obtained in rats with T A A - i n d u c e d acute hepatocellular necrosis and saline-injected controls before and 5 min after the s.c. administration o f ( - ) - n a l o x o n e (1 mg/kg).

Statistics Baseline mean T F L values o f the three main experimental groups were c o m p a r e d by a one-way analysis of variance. The mean changes in T F L over 7 (or 5) days for each experimental group were compared, i.e. for each rat the baseline T F L was subtracted from the T F L at 7 (or 5) days and the differences in each group were averaged. The effects o f ( - ) - n a l o x o n e and ( + ) - n a l o x o n e on T F L s were evaluated by comparing the mean T F L changes during the 0-5-min interval after drug administration, As the

In the first series o f experiments, baseline T F L s were similar for all three experimental groups (Fig. 1) (p>0.25). The mean T F L 7 days after B D R was significantly longer (18%) than that before surgery (p<0.05), whereas the corresponding means for unoperated and sham-resected groups were not significantly different from their respective baseline values (Fig. 1). By 5 min after administration o f the opiate antagonist, ( - ) - n a l o x o n e , a complete reversal of the increased T F L in the B D R group occurred. Following ( - ) - n a l o x o n e administration, the decrease in T F L that occurred in the B D R group was significantly greater than that in controls (p<0.01) (Fig. 1). This effect of ( - ) naloxone was transient; it was present 10 min after treatment but was undetectable when rats were tested 1 h after drug treatment (data not shown). These observations are consistent with naloxone being rapidly metabolized (15). Similar statistically significant findings were also obtained 5 days after surgery (data not shown). Thus, the model of cholestasis studied is associated with ( - ) - n a l o x o n e - r e v e r s ible antinociception. In a second series o f experiments, in which only B D R rats were studied, ( - ) - n a l o x o n e - m e d i a t e d reversal o f prolongation of T F L s 5 days after surgery was confirmed (Fig. 2). However, reversal o f the antinociception in BDR

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Fig. 2. Stereoselective reversal of antinociception in cholestatic rats by naloxone. Five days after BDR, prolonged TFLs were significantly decreased by (-)-naloxone (n=28), compared to an actual increase associated with its enantiomer, (+)-naloxone (n=29). TFLs were measured 5 min after injection of ( - ) or (+)-naloxone. Data are means-+SEM.

88

N.V. B E R G A S A et al.

animals was not induced by the enantiomer (+)-naloxone. In particular, the mean T F L change during the 0-5-min interval after (-)-naloxone was significantly greater than that after (+)-naloxone (p<0.001) (Fig. 2). Thus, antinociception in this model of cholestasis is stereoselectively reversed by naloxone. In a third series of experiments, TFLs were shown to vary inversely with the intensity of the nociceptive stimulus both in rats 5 days after BDR and in unoperated control rats. However, the percentage increase in mean T F L in BDR rats relative to that in controls was similar over a wide range of intensities of the nociceptive stimulus (Fig. 3). In a fourth series of experiments, TFLs were shown to be more prolonged in rats with TAA-induced acute hepatocellular necrosis (n= 15) than in controls (n= 14) (5.89-+ 0.209 vs 4.06+-0.187 s, respectively). The prolonged TFLs in the TAA-treated group 5 min after the administration of (-)-naloxone (5.98-+0.219 s) were similar to the values before (-)-naloxone. Thus, in contrast to the model of cholestasis, antinociception in this model of hepatocellular necrosis is not naloxone reversible.

Discussion A well-described function of the opioid neurotransmitter system is the mediation of analgesia, which is induced by the interaction between opioid agonist ligands and specific receptors in the central nervous system (7). I t

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Fig. 3. Inverse relationship between TFLs and the intensity of the nociceptive stimulus. Continuous line: rats 5 days after BDR; interrupted line: unoperated controls rats. The intensity of the nociceptive stimulus was varied by adjusting the Vernier scale setting on the tail-flick apparatus. Data are m e a n s _ S E M . Data obtained at the 2.5 setting were noncontemporaneous. Mean tail-flick latencies in the BDR group at settings of 2.0, 2.5, 3.0 and 4.0 were 18, 16, 17 and 15% more prolonged than corresponding means in the control group, respectively.

is well established that an increase in T F L in rodents is a manifestation of increased opiatergic tone induced by opiate agonist drugs, such as morphine (8-11)_ By analogy, increased opioidergic tone in the absence of exogenously administered opiates would be associated with an increase in TFL. In this study the tail-flick assay was applied for the novel purpose of determining whether antinociception mediated by the opioid system occurs in cholestasis. The observation made in this study that after BDR in the rat TFLs are prolonged indicates that antinociception occurs in a model of cholestasis, but alone this finding could be nonspecific. Indeed, antinociception was also found in a rat model of hepatocellular necrosis. However, the reversibility of the antinociception in BDR rats by the opiate antagonist (-)-naloxone suggests that this pathophysiologic state in cholestasis is opioid-receptor mediated (16). Further, the lack of effect of the pharmacologically inert enantiomer (+)-naloxone (17) on antinociception in BDR rats indicates that reversal of this phenomenon by naloxone is stereoselective and confirms the hypothesis that the antinociception in this model is opioid-receptor mediated. Thus, strong evidence that increased opioidergic tone contributes to the pathophysiology of cholestasis has been obtained in a classical model of this syndrome (12). The unequivocal increase in T F L associated with cholestasis in this study is not attributable to weight loss (18,19), is consistent with an effect mediated by endogenous opioids, and, when expressed as a percentage increase relative to that of appropriate controls, did not change appreciably over a wide range of intensities of nociceptive stimulation. In contrast to the BDR model of cholestasis, the antinociception observed in rats with hepatocellular necrosis was not reversed by (-)-naloxone, indicating that it is not opioid mediated. This finding demonstrates that cholestasis rather than hepatocellular injury in the BDR model is responsible for naloxone-reversible antinociception. These observations highlight the difference between the syndromes of cholestasis and hepatocellular necrosis with respect to opioid-mediated neurotransmission. Clinically, increased opioidergic tone is the pathophysiologic state associated with chronic opiate addiction in human beings. In this state an opiate withdrawal reaction can be abruptly induced, not only by discontinuing administration of opiates but also by giving an opiate antagonist, such as naloxone (20,21). An animal model of human opiate addiction is the morphine-dependent rat. Administration of (-)-naloxone to this model induces a florid opiate withdrawal syndrome (22,23). An opiate withdrawal reaction of the severity induced by an opiate antagonist in the morphine-dependent rat would not necessarily have been expected following (-)-naloxone

INCREASED OPIATERGIC TONE IN CHOLESTASIS

administration to B D R rats, because endogenous opioids have not been reported to induce either the same n u m b e r or the same intensity of opioid receptor-mediated effects as those induced by pharmacological doses of morphine. However, we observed both vigorous teeth chattering and head shaking in some of the B D R rats given ( - ) - n a l o x one. As these phenomena are components of the syndrome of opiate withdrawal in rats (22,23) these observations are consistent with ( - ) - n a l o x o n e inducing a mild opioid withdrawal syndrome in cholestatic rats. In addition to the results of this study, an increase in opioidergic tone in cholestasis is suggested by the precipitation of an opiate withdrawal-like syndrome in patients with chronic cholestasis by an opiate antagonist (2) and a global down-regulation of mu-opioid receptors in the brain in BDR rats (3). It has previously been shown that chronic cholestasis in h u m a n s is associated with increased serum levels of enkephalins, i.e. endogenous ligands which act as agonists at opioid receptors (2,24--28)_ Furthermore, serum metenkephalin and total opioid activity are significantly greater in the model of cholestasis studied than in sham-resected rats (29). However, a causal relationship between serum opioid levels and an altered status of the opioid system in cholestasis has not been established. Indeed, high serum levels of metenkephalin (comparable to those in B D R rats) have been found in the rat model of TAA-induced acute hepatocellular necrosis (30), without concomitant evidence of opioid-mediated antinociception in this model. Nevertheless, our demonstration that an opiate antagonist reverses a manifestation of cholestasis attributable to increased opioidergic tone (antinociception) suggests that opiate antagonists may modulate other manifestations of cholestasis which are also attributable to increased opioidergic tone. One such manifestation of cholestasis in humans may be pruritus. Preliminary reports of opiate-antagonist-induced ameliorations of the pruritus of cholestasis (2,4-6) suggest that increased opioidergic tone is a clinically important c o m p o n e n t of the syndrome of cholestasis. In summary, data are presented which indicate that antinociception mediated by the opioid system occurs in a rat model of cholestasis but not in a rat model of hepatocellular necrosis. These findings indicate that increased opioidergic tone occurs as a c o m p o n e n t of the pathophysiology of cholestasis.

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