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Cholestasis induced antinociception and decreased gene expression of MOR1 in rat brain
Neuroscience 284 (2015) 78–86
CHOLESTASIS INDUCED ANTINOCICEPTION AND DECREASED GENE EXPRESSION OF MOR1 IN RAT BRAIN S. AHMADI, * Z. KARAMI, A. MOHAMMADIAN, F. KHOSROBAKHSH AND J. ROSTAMZADEH
Georgiev et al., 2008). It has been shown that endogenous opioid levels increase during cholestasis (Nelson et al., 2006; Moser and Giesler, 2013), and has been reported that it should be associated with a decrease in nociception (Bergasa et al., 1994). Pruritus or increase in itch sensation is also a dominant symptom of cholestatic liver disease but is difficult to treat because the pathophysiological mechanisms of cholestatic pruritus have not completely been clarified (Jones and Bergasa, 1999; Seccareccia and Gebara, 2011; Bunchorntavakul and Reddy, 2013). Pruritus and a decrease in nociception that are induced by cholestasis had been attributed to the accumulation of endogenous opioids by other investigators (Swain et al., 1992; Ikoma et al., 2003). This theory was also supported by successful treatment of cholestasis pruritus with opioid antagonists (Bergasa et al., 1994; Lonsdale-Eccles and Carmichael, 2009). However, the pathogenesis of pruritus in cholestasis remains to be debated. According to research, there are many close associations in neural pathways of pruritus and pain (Ikoma et al., 2003; Moser and Giesler, 2013). Therefore, it is logical to hypothesize that the study of alteration in nociception induced by cholestasis is a way to further understand mechanisms underlying pruritus which is induced by cholestasis. Pain signal is initiated with activation of nociceptors by thermal, mechanical or chemical stimuli in peripheral tissues such as skin, and it is sent along sensory axons of the first-order neuron to dorsal horn of the spinal cord. The second-order neuron of the spinothalamic pathway of the pain originates from the spinal cord and conveys the pain signal to the thalamus, and from there a third-order neuron projects pain information to the somatosensory areas of the cortex (Millan, 1999; Almeida et al., 2004). In addition, pain information is sent to many areas including reticular formation of the brain stem and limbic areas such as the hypothalamus, prefrontal cortex (PFC), hippocampus, and motor areas including the striatum [for a review see (Almeida et al., 2004)]. It is thought these areas affect emotional-affective and cognitive components of pain sensations (Neugebauer et al., 2009). On the other hand, the above-mentioned limbic areas have been shown to express mu-opioid receptors (MOR) and also contribute in various rewarding, analgesic and side effects of opioids (Rozeske et al., 2009; George and Koob, 2010; Ikemoto, 2010; Le Marec et al., 2011; Trezza et al., 2011). Although there is some evidence of decreased MOR numbers in the brains of cholestatic rats (Bergasa et al., 1992), there is no exact report regarding the expression
Department of Biological Science and Biotechnology, Faculty of Science, University of Kurdistan, Sanandaj, Iran
Abstract—We examined antinociception and gene expression of mu-opioid receptor 1 (MOR1) in some brain areas of cholestatic rats, 21 days after common bile duct ligation (BDL). Cholestasis was induced in male Wistar rats during laparotomy and common BDL. Pain behavior was assessed on days 7, 14 or 21 of BDL using a hotplate test in control, sham and cholestatic groups. On day 21 of BDL, other groups of rats were sacrificed, whole brains were extracted, and the hypothalamus, prefrontal cortex (PFC), hippocampus and striatum in control, sham and cholestatic rats were dissected. We used a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) method for evaluating MOR1 gene expression. The results revealed that cholestatic rats showed significant antinociception on days 14 and 21 of ligation with the most significant effect on day 21, which was prevented by naloxone (1 mg/kg). On the other hand, the expression of MOR1 gene compared to the sham group was decreased by 42% in the hypothalamus, 41% in the PFC, and 67% in the hippocampus after 21 days of BDL, while no significant change in its expression in the striatum was observed. It can be concluded that a change in endogenous opioid levels and its subsequent influence on the gene expression of MOR in some areas of the rat brain may underlie the altered nociception and other possible pathological changes such as pruritus after induction of cholestasis. Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved.
Key words: cholestasis, antinociception, gene expression, semi-quantitative RT-PCR.
INTRODUCTION Cholestasis is a common pathological condition that can be reproduced in rodents by common bile duct ligation (BDL) during surgical laparotomy (Rodriguez-Garay, 2003; *Corresponding author. Address: Department of Biological Science and Biotechnology, Faculty of Science, University of Kurdistan, P.O. Box 416, Sanandaj, Iran. Tel: +98-871-33660075; fax: +98-87133622702. E-mail address: [email protected] (S. Ahmadi). Abbreviations: ANOVA, analysis of variance; BDL, bile duct ligation; MOR1, mu-opioid receptor 1; %MPE, percentage maximum possible effect; PCR, polymerase chain reaction; PFC, prefrontal cortex; RT-PCR, reverse transcription-polymerase chain reaction. http://dx.doi.org/10.1016/j.neuroscience.2014.08.063 0306-4522/Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved. 78
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of MOR at mRNA levels in the brains of cholestatic rats after significant changes in nociception. With regard to these backgrounds, we examined gene expression of MOR1 in the hypothalamus, hippocampus, PFC and striatum to further investigate their role in emotional and cognitive components of antinociception, and indirectly pruritus, induced by cholestasis.
EXPERIMENTAL PROCEDURES Animals Male Wistar rats weighing 300–350 g were used. The animals were kept in an animal house at a constant temperature (22 ± 2 °C) under a 12-h light/dark cycle (light beginning at 7:00 a.m.). They had free access to food and water except during the experiments. Behavioral tests were carried out during the light phase between 9:00 and 12:00 (a.m.). Each animal was used only once. Experimental groups consisted of either eight rats in biochemical analysis of the serum and hotplate test or four rats in gene expression studies. All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals (2011) prepared by the National Academy of Sciences’ Institute for Laboratory Animal Research.
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maximum possible effect (%MPE) using the following formula: %MPE = [(test latency baseline latency)/(cutoff time baseline latency)] 100 (Ossipov et al., 1990; Keil and Delander, 1995). Drug treatment Naloxone dihydrochloride was purchased from Ascent Scientific (Bristol, UK). The drug was dissolved in 0.9% (w/v) normal saline and administered at a volume of 1 ml/kg through i.p. route 20 min before examining test latency on days 7, 14 or 21 of ligation. Total RNA extraction and cDNA synthesis
Three experimental groups including control, sham and a group with BDL were used in each part of the study. Sham and BDL groups underwent laparotomies, which were performed under general anesthesia induced by intraperitoneal (i.p.) injection of a mixture of ketamine/ xylazine (50 and 5 mg/kg, respectively). The rats that were used for sham and BDL operations were food restricted for 12 h prior to the surgery. The sham operation consisted of laparotomy, bile duct identification and manipulation but without ligation and resection. In the group with BDL, common bile duct was completely exposed, ligated at two points with approximately 5 mm apart and then transected at midpoint between the ligatures. After closure of the abdominal wound, the rats did not receive any analgesics but each rat received 1 ml of saline (i.p.) and moved to a clean box until complete recovery. Mortality due to operation was under 5% during and/or after laparotomy.
Twenty-one days after laparotomy, rats were sacrificed, the whole brain quickly removed from the skull and the hypothalamus, PFC, hippocampus and striatum were immediately dissected on an ice-chilled sterile surface as per previously reported method (Chiu et al., 2007). After harvesting, each tissue was immediately moved into a tube, submerged in an RNAlater RNA Stabilization Reagent (QIAGEN) and incubated overnight in the reagent at 4 °C. The RNAlater solution was drained from the tubes after 24 h and tissues were stored at 80 °C until further analysis. Total RNA was extracted from 50 to 70 mg of each tissue using a Trizol method with some modification. In brief, each tissue sample was removed from 80 °C deep freezer, transferred to a new tube containing 1 ml of lysis buffer (RNX+ reagent, Cinnagen, Tehran, Iran), homogenized for 60 s with a high-speed homogenizer (Silent Crusher S., Heidolph, Germany), and then subjected to total RNA extraction by a highly pure tissue RNA extraction protocol according to the manufacturer’s manual (Cinnagen, Tehran, Iran). Quality of total RNA was assessed by electrophoresis on 1% agarose gel to visualize 28s and 18s ribosomal RNA. The concentrations of total RNA were also measured with a spectrophotometer (Specord210, Analytic Jena, Germany). Then, a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) method was used to assess gene expression of MOR1 (Marone et al., 2001). Reverse transcription (RT) reaction was performed using Viva 2-step RT-PCR Kit according to the manufacturer’s protocol (Vivantis Technologies, Selangor Darul Ehsan, Malaysia).
Measurement of nociception
Polymerase chain reaction (PCR)
We used a hotplate test to assess pain behavior. Each rat was placed on the hotplate (52 ± 0.1 °C) with a surrounding glass square (25 cm height) to prevent escaping of animals (Armaghan Co., Tabriz, Iran). Then, time elapse between placement of the animals on hotplate and licking one of the hind paws or first jumping was measured as an index of nociceptive response. First, baseline latency was measured 1 day before laparotomy. Second, different experimental groups (control, sham and BDL) on days 7, 14 or 21 of laparotomy were tested to measure test latency on the hotplate. A cutoff time of 80 s was set to prevent tissue damage. Finally, the two measured latencies were converted to percentage
PCR was used for amplification of b-actin (as a housekeeping gene) and MOR1 genes. Primers for both genes were designed at exon–exon junctions to prevent amplifications of genomic DNA fragments of the genes. Primers had the following sequences: b-actin forward primer, 50 -CTGGGTATGGAATCCTGTGGC-30 ; b-actin reverse primer, 50 -AGGAGGAGCAATGATCTTGATC-30 ; MOR1 forward primer, 50 -CAGGGGTCCATAGATTG CAC-30 ; MOR1 reverse primer, 50 -GAAGTGCCAGGAAA CGGTC-30 . PCR was carried out in a reaction volume of 20 ll consisting of 10 ll of PCR Master Mix (Thermo Scientific, Pittsburgh PA, USA), 2 ll of cDNA, 2 ll of upstream and
Surgical laparotomy
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downstream mix of MOR1 primers (10 lM), 0.5 ll of upstream and downstream mix of b-actin primers (10 lM), and nuclease free water up to 20 ll. Firstly, reaction parameters were adjusted to acquire conditions with a linear relation of RNA concentration used for cDNA synthesis, the number of PCR cycles and annealing temperature related to PCR product. According to PCR optimization, thermal cycling was initiated with a first denaturation step of 95 °C for 3 min, followed by 27 cycles of thermal cycling of 94 °C for 30 s, 59 °C for 30 s, 72 °C for 30 s, and finally followed by a 10-min final extension step at 72 °C and termination at 4 °C for 5 min (C1000 Thermal Cycler, BIO-RAD, Hercules CA, USA). The PCR products were subsequently analyzed on 2% agarose gel electrophoresis and bands were quantified by densitometry using Image J software. Experimental design Experiment 1: effect of cholestasis on nociception. Nine groups of rats (n = 8) were used. They were divided into three sets of three groups; each set included control, sham and BDL groups. Three groups of each set were submitted to the hotplate test at 7, 14 or 21 days after ligation. Then, the recorded pain reaction latencies were converted to MPE% to assess the effect of cholestasis on pain behavior. Experiment 2: effect of naloxone on antinociception induced by cholestasis after 21 days of BDL. Three groups of rats (n = 8) including control, sham and BDL were submitted to the hotplate test on day 21 of ligation. Each rat received naloxone (1 mg/kg) at 20 min before examining the test latency. Experiment 3: evaluating MOR1 gene expression after 21 days of BDL in the hypothalamus, PFC, hippocampus,
and striatum. Three groups of control, sham and cholestatic rats (n = 4) were used to assess the expression of MOR1 gene in the hypothalamus, PFC, hippocampus, and striatum 21 days after BDL. For this purpose, tissues were firstly submitted to the total RNA extraction and, cDNA synthesis as have been previously described. Then MOR1 and b-actin genes in each tissue were amplified using PCR. Thereafter, PCR products were evaluated on agarose gel and the band densities were quantified with an Image J program. Data of gene expression were shown as normalization to the control group, in which the ratio of MOR1 to b-actin was set at 100%. Statistical analysis Data from the hotplate test and gene expression passed normality and equal variance tests, therefore they were analyzed by a one-way analysis of variance (ANOVA), and after a significant F value, post hoc Holm–Sidak’s test was used for multiple pairwise comparisons. p < 0.05 was considered a statistically significant level throughout.
RESULTS Cholestasis caused antinociception that was prevented by naloxone As shown in Fig. 1, a one-way ANOVA revealed that pain behavior in rats with BDL on day 7 after BDL was not significantly altered [F (2, 21) = 2.11, P > 0.05]. The one-way ANOVA also revealed that on day 14 of BDL, pain behavior was significantly altered [F (2, 21) = 10.01, p < 0.01]. The post hoc test revealed that cholestatic rats compared to the sham group significantly showed an increase in %MPE (p < 0.05). In addition, the one-way ANOVA showed that antinociception after 21 days of BDL was more
Fig. 1. Effect of cholestasis on pain behavior. Twelve groups of rats (n = 8) were used. Three sets of three groups including control, sham and BDL in each set were submitted to hotplate test at 7, 14 or 21 days after ligation. Three groups of rats (n = 8) including control, sham and BDL were also submitted to hotplate test at 21 days after ligation but they also received naloxone (1 mg/kg) 20 min before recording test latency on the hotplate. Each bar represents mean ± S.E.M. of %MPE in each group. ⁄P < 0.05 compared to the sham group after 14 days of ligation, and +++P < 0.001 compared to the sham group after 21 days of ligation.
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Fig. 2. Effect of cholestasis on the expression of MOR1 gene in the hypothalamus on day 21 of BDL. Three groups of control, sham and cholestatic rats (n = 4) were used. Upper panel (panel A) shows the inverted black and white gel image indicating band densities of MOR1 and b-actin genes after amplification with PCR. Numbers 1 to 4 on top of the image show number of the animals in each group. (M) symbolizes 100-bp ladder on the gel image. Lower panel (panel B) shows bar graph of quantitative data related to gene expression. Each bar represents mean ± S.E.M. of MOR1 gene band density normalized to b-actin and set at 100%. ⁄⁄⁄P < 0.001 compared to the sham group.
significantly altered [F (2, 21) = 15.63, p < 0.001]. The post hoc test revealed that analgesia induced by BDL in cholestatic rats after 21 days was significant compared to the sham group (p < 0.01). The one-way ANOVA also revealed that naloxone, an opioid receptor antagonist, prevented the analgesia induced by cholestasis on day 21 after BDL, therefore no significant difference was observed in %MPE of the experimental groups [F (2, 21) = 0.16, p > 0.05] (Fig. 1). These results support that an increase in endogenous opioids and activation of MOR may underlie the antinociception induced by cholestasis. MOR1 gene expression decreased in the hypothalamus after 21 days of BDL Analysis of the gene expression results with a one-way ANOVA revealed that mRNA level of MOR1 gene after 21 days of BDL had decreased in the hypothalamus [F (2, 9) = 67.87, p < 0.001]. Post hoc Holm–Sidak’s test
revealed a significant decrease (P < 0.001) of MOR1 gene expression by 42% in the hypothalamus compared to the sham control group after 21 days of BDL (Fig. 2). MOR1 gene expression decreased in the PFC after 21 days of BDL A one-way ANOVA revealed that mRNA levels of MOR1 gene after 21 days of BDL had decreased in the PFC [F (2, 9) = 31.45, p < 0.001]. Post hoc Holm–Sidak’s test revealed a significant decrease (P < 0.001) of MOR1 gene expression by 41% in the PFC compared to the sham control group after 21 days of BDL (Fig. 3). MOR1 gene expression decreased in the hippocampus after 21 days of BDL A one-way ANOVA revealed that mRNA levels of MOR1 gene after 21 days of BDL had decreased in the hippocampus [F (2, 9) = 172.97, p < 0.001]. Post hoc
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Fig. 3. Effect of cholestasis on the expression of MOR1 gene in the PFC on day 21 of BDL. Three groups of control, sham and cholestatic rats (n = 4) were used. Upper panel (panel A) shows band densities of MOR1 and b-actin genes after amplification with PCR on the inverted black and white gel image. Numbers 1–4 on top of the image show number of the animals in each group. (M) symbolizes 100-bp ladder on the gel image. Lower panel (panel B) shows bar graph of quantitative data related to gene expression. Each bar represents mean ± S.E.M. of MOR1 gene band density normalized to b-actin and set at 100%. ⁄⁄⁄P < 0.001 compared to the sham group.
Holm–Sidak’s test showed a significant decrease (P < 0.001) in MOR1 gene expression by 67% in the hippocampus compared to the sham control group after 21 days of BDL (Fig. 4). MOR1 gene expression remained without significant change in the striatum after 21 days of BDL Analysis of the gene expression data with a one-way ANOVA showed that mRNA levels of MOR1 gene after 21 days of BDL remained without significant change in the striatum [F (2, 9) = 1.52, p > 0.05] (Fig. 5).
DISCUSSION It has been shown that cholestasis is associated with increased opioidergic tone (Bergasa et al., 1994; Davis, 2007). This study explored the expression of MOR at
mRNA levels in areas of the rat brain that participate in emotional and cognitive aspects of nociception in a model of cholestasis. First, the results revealed that cholestatic rats showed significant antinociception on days 14 and 21 of BDL with the most significant effect on day 21. Second, naloxone (1 mg/kg) prevented the antinociception that was induced by BDL on day 21 of BDL, which may confirm an increase in endogenous opioids and activation of MOR in this process. Third, the expression of MOR1 gene compared to the sham group decreased by 42% in the hypothalamus, 41% in the PFC, and 67% in the hippocampus after 21 days of BDL, while no significant change was observed for its expression in the striatum. It has been reported that acute and chronic liver failure alters brain functions and affects neurotransmitter systems (Lozeva et al., 2004; Garcia-Ayllon et al., 2008). In some studies, rats with BDL have been used as a model of liver failure (Magen et al., 2009; Wright
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Fig. 4. Effect of cholestasis on the expression of MOR1 gene in the hippocampus on day 21 of BDL. Upper panel (panel A) shows the inverted black and white gel image indicating band densities of MOR1 and b-actin genes related to three groups of control, sham and cholestatic rats (n = 4). Numbers 1–4 on top of the image show number of the animals in each group. (M) symbolizes 100-bp ladder on the gel image. Lower panel (panel B) shows bar graph of quantitative data related to gene expression. Each bar represents mean ± S.E.M. of MOR1 gene band density normalized to b-actin and set at 100%. ⁄⁄⁄P < 0.001 compared to the sham group.
et al., 2010). On the other hand, it has been shown that plasma total opioid levels in cholestatic liver disease is increased which may underlie pruritus and analgesia associated with cholestasis (Bergasa et al., 1995; Nicoll et al., 2005; Alemi et al., 2013). In line with the above reports, the result of the hotplate test in the present study revealed that nociceptive threshold in cholestatic rats increased with a maximum effect on day 21 of ligation, as revealed by an increase in %MPE on hotplate test. As has previously been reported in mice (Nelson et al., 2006), our results also confirmed that naloxone completely blocked the antinociception induced by cholestasis on day 21 of BDL in rats. However, since naloxone could readily enter the central nervous system, it has been reported that its peripheral injections would block opioid receptors not only located on peripheral nerve endings but also in the central pain pathways (Bergasa et al., 1994; Nelson et al., 2006). Naloxone, as a MOR antagonist, blocks these receptors and in the case of hotplate
test in the present study prevented the antinociception that was induced by increased opioid levels, and therefore reversed the behavioral changes in BDL rats. It has also been reported that inflammatory pathways could also contribute to pain perception (Okuse, 2007; Carr et al., 2014), and are also known to be involved in BDL (Noguchi et al., 2014). Therefore, their role in the changes of nociceptive threshold in cholestatic rats cannot be excluded. Taken together, complex peripheral and central actions may underlie antinociception and its blockade by naloxone in cholestatic rats. The results of gene expression in the present study revealed that the expression of MOR1 gene decreased in limbic areas including the hypothalamus, PFC and hippocampus, but remained without changes in the striatum on day 21 after BDL. To discuss the present results, one may propose that alteration in the expression of MOR1 gene could be a consequence of increased levels of endogenous opioids in cholestatic
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Fig. 5. Effect of cholestasis on the expression of MOR1 gene in the striatum on day 21 of BDL. Upper panel (panel A) shows the inverted black and white gel image indicating band densities of MOR1 and b-actin genes related to three groups of control, sham and cholestatic rats (n = 4) on agarose gel. Numbers 1 to 4 on top of the image show number of the animals in each group. (M) symbolizes 100-bp ladder on the gel image. Lower panel (panel B) shows bar graph of quantitative data related to gene expression. Each bar represents mean ± S.E.M. of MOR1 gene band density normalized to b-actin and set at 100%. ⁄⁄⁄P < 0.001 compared to the sham group.
rats. However, the expression of MOR1 gene in the striatum further indicates that cellular adaptation for an increase in opioids and/or changes in MOR1 gene in different brain areas may be a site-specific process. Nelson et al. (2006) reported that cholestasis in mice is associated with antinociception due to local effects of endogenous opioids at the level of sensory nerve endings (Nelson et al., 2006). However, these results support this idea that an increase in nociceptive threshold in cholestatic rats may result from changes in both peripheral and central opioid systems. The discrepancy between the two studies may result from using different subjects. Pain and pruritus are closely interrelated and opioids modulate both sensations (Nelson et al., 2006); therefore, the decreases in the expression of MOR1 gene may reflect an important mechanism of not only antinociception but also pathogenesis of the pruritus induced by cholestasis. In support of this suggestion, it has been shown that pain
afferent neurons in the spinothalamic pathway have spontaneous activity which can tonically suppress sending itch sensation to the thalamus [for a review see (Ikoma et al., 2003)]. Therefore, one may conclude that the increase of opioids during cholestasis prevents activity of pain neurons and induces antinociception, which in turn induces an increase of itch sensation. Furthermore, since activation of MOR induces antinociception, one may predict that their down-regulation should have increased nociception. However, pain perception is under the impact of many modulatory systems in the pain pathway, and it could not be predicted that down-regulation of MOR1 gene expression should have increased nociception. According to research, both opioidergic and serotonergic systems have been proposed as central regulators of pruritus (Bhargava and Gulati, 1990; Bergasa et al., 1992; Bunchorntavakul and Reddy, 2013). Other investigators have reported that opioids
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might also activate serotonin pathways, and analgesic effect of morphine may be mediated, at least partly, via activation of serotonergic inhibitory descending pain pathways originating from the brain stem, and terminating on neurons in the spinal cord gray matter (Ohsawa et al., 2000; Dogrul et al., 2009; Ozdemir et al., 2012). Furthermore, serotonergic pathways to the forebrain structures such as nucleus accumbens have been shown to be activated by opioids which in turn induced analgesia blocked by serotonin antagonists (Ohsawa et al., 2000). Magen et al. (2010) reported that BDL reduces 5-HT1A expression in the hippocampus. Since 5-HT1A receptors act as a regulator of serotonin release, its down-regulation could increase serotonin release (Magen et al., 2010). With regard to the above reports and the results of this study, an alternate mechanism for antinociception in cholestatic rats might be the activation of serotonin release as a result of the altered MOR and/or 5-HT1A receptor expression that could affect downstream neurons in the pain pathway. However, we did not explore 5-HT1A receptor expression and serotonin release, and this suggestion needs further investigation to be lucid.
CONCLUSIONS In summary, it can be concluded that cholestasis induced significant antinociception 21 days after BDL in rats which was prevented by naloxone. This result supports the idea that MOR might be the key elements of antinociception and possibly pruritus induced by cholestasis. In line with this suggestion, our results indicated that the expression of MOR1 gene decreased in the hypothalamus, PFC and hippocampus. However, its expression remains without significant changes in the striatum. Based on our results, it could be proposed that changes in the expression of MOR1 gene caused by cholestasis are site specific.
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Rodriguez-Garay EA (2003) Cholestasis: human disease and experimental animal models. Ann Hepatol 2:150–158. Rozeske RR, Der-Avakian A, Bland ST, Beckley JT, Watkins LR, Maier SF (2009) The medial prefrontal cortex regulates the differential expression of morphine-conditioned place preference following a single exposure to controllable or uncontrollable stress. Neuropsychopharmacology 34:834–843. Seccareccia D, Gebara N (2011) Pruritus in palliative care: getting up to scratch. Can Fam Physician 57(1010–1013):e1019–e1316. Swain MG, Rothman RB, Xu H, Vergalla J, Bergasa NV, Jones EA (1992) Endogenous opioids accumulate in plasma in a rat model of acute cholestasis. Gastroenterology 103:630–635. Trezza V, Damsteegt R, Achterberg EJ, Vanderschuren LJ (2011) Nucleus accumbens mu-opioid receptors mediate social reward. J Neurosci 31:6362–6370. Wright G, Soper R, Brooks HF, Stadlbauer V, Vairappan B, Davies NA, Andreola F, Hodges S, Moss RF, Davies DC, Jalan R (2010) Role of aquaporin-4 in the development of brain oedema in liver failure. J Hepatol 53:91–97.
(Accepted 21 August 2014) (Available online 5 October 2014)
CHOLESTASIS INDUCED ANTINOCICEPTION AND DECREASED GENE EXPRESSION OF MOR1 IN RAT BRAIN S. AHMADI, * Z. KARAMI, A. MOHAMMADIAN, F. KHOSROBAKHSH AND J. ROSTAMZADEH
Georgiev et al., 2008). It has been shown that endogenous opioid levels increase during cholestasis (Nelson et al., 2006; Moser and Giesler, 2013), and has been reported that it should be associated with a decrease in nociception (Bergasa et al., 1994). Pruritus or increase in itch sensation is also a dominant symptom of cholestatic liver disease but is difficult to treat because the pathophysiological mechanisms of cholestatic pruritus have not completely been clarified (Jones and Bergasa, 1999; Seccareccia and Gebara, 2011; Bunchorntavakul and Reddy, 2013). Pruritus and a decrease in nociception that are induced by cholestasis had been attributed to the accumulation of endogenous opioids by other investigators (Swain et al., 1992; Ikoma et al., 2003). This theory was also supported by successful treatment of cholestasis pruritus with opioid antagonists (Bergasa et al., 1994; Lonsdale-Eccles and Carmichael, 2009). However, the pathogenesis of pruritus in cholestasis remains to be debated. According to research, there are many close associations in neural pathways of pruritus and pain (Ikoma et al., 2003; Moser and Giesler, 2013). Therefore, it is logical to hypothesize that the study of alteration in nociception induced by cholestasis is a way to further understand mechanisms underlying pruritus which is induced by cholestasis. Pain signal is initiated with activation of nociceptors by thermal, mechanical or chemical stimuli in peripheral tissues such as skin, and it is sent along sensory axons of the first-order neuron to dorsal horn of the spinal cord. The second-order neuron of the spinothalamic pathway of the pain originates from the spinal cord and conveys the pain signal to the thalamus, and from there a third-order neuron projects pain information to the somatosensory areas of the cortex (Millan, 1999; Almeida et al., 2004). In addition, pain information is sent to many areas including reticular formation of the brain stem and limbic areas such as the hypothalamus, prefrontal cortex (PFC), hippocampus, and motor areas including the striatum [for a review see (Almeida et al., 2004)]. It is thought these areas affect emotional-affective and cognitive components of pain sensations (Neugebauer et al., 2009). On the other hand, the above-mentioned limbic areas have been shown to express mu-opioid receptors (MOR) and also contribute in various rewarding, analgesic and side effects of opioids (Rozeske et al., 2009; George and Koob, 2010; Ikemoto, 2010; Le Marec et al., 2011; Trezza et al., 2011). Although there is some evidence of decreased MOR numbers in the brains of cholestatic rats (Bergasa et al., 1992), there is no exact report regarding the expression
Department of Biological Science and Biotechnology, Faculty of Science, University of Kurdistan, Sanandaj, Iran
Abstract—We examined antinociception and gene expression of mu-opioid receptor 1 (MOR1) in some brain areas of cholestatic rats, 21 days after common bile duct ligation (BDL). Cholestasis was induced in male Wistar rats during laparotomy and common BDL. Pain behavior was assessed on days 7, 14 or 21 of BDL using a hotplate test in control, sham and cholestatic groups. On day 21 of BDL, other groups of rats were sacrificed, whole brains were extracted, and the hypothalamus, prefrontal cortex (PFC), hippocampus and striatum in control, sham and cholestatic rats were dissected. We used a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) method for evaluating MOR1 gene expression. The results revealed that cholestatic rats showed significant antinociception on days 14 and 21 of ligation with the most significant effect on day 21, which was prevented by naloxone (1 mg/kg). On the other hand, the expression of MOR1 gene compared to the sham group was decreased by 42% in the hypothalamus, 41% in the PFC, and 67% in the hippocampus after 21 days of BDL, while no significant change in its expression in the striatum was observed. It can be concluded that a change in endogenous opioid levels and its subsequent influence on the gene expression of MOR in some areas of the rat brain may underlie the altered nociception and other possible pathological changes such as pruritus after induction of cholestasis. Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved.
Key words: cholestasis, antinociception, gene expression, semi-quantitative RT-PCR.
INTRODUCTION Cholestasis is a common pathological condition that can be reproduced in rodents by common bile duct ligation (BDL) during surgical laparotomy (Rodriguez-Garay, 2003; *Corresponding author. Address: Department of Biological Science and Biotechnology, Faculty of Science, University of Kurdistan, P.O. Box 416, Sanandaj, Iran. Tel: +98-871-33660075; fax: +98-87133622702. E-mail address: [email protected] (S. Ahmadi). Abbreviations: ANOVA, analysis of variance; BDL, bile duct ligation; MOR1, mu-opioid receptor 1; %MPE, percentage maximum possible effect; PCR, polymerase chain reaction; PFC, prefrontal cortex; RT-PCR, reverse transcription-polymerase chain reaction. http://dx.doi.org/10.1016/j.neuroscience.2014.08.063 0306-4522/Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved. 78
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of MOR at mRNA levels in the brains of cholestatic rats after significant changes in nociception. With regard to these backgrounds, we examined gene expression of MOR1 in the hypothalamus, hippocampus, PFC and striatum to further investigate their role in emotional and cognitive components of antinociception, and indirectly pruritus, induced by cholestasis.
EXPERIMENTAL PROCEDURES Animals Male Wistar rats weighing 300–350 g were used. The animals were kept in an animal house at a constant temperature (22 ± 2 °C) under a 12-h light/dark cycle (light beginning at 7:00 a.m.). They had free access to food and water except during the experiments. Behavioral tests were carried out during the light phase between 9:00 and 12:00 (a.m.). Each animal was used only once. Experimental groups consisted of either eight rats in biochemical analysis of the serum and hotplate test or four rats in gene expression studies. All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals (2011) prepared by the National Academy of Sciences’ Institute for Laboratory Animal Research.
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maximum possible effect (%MPE) using the following formula: %MPE = [(test latency baseline latency)/(cutoff time baseline latency)] 100 (Ossipov et al., 1990; Keil and Delander, 1995). Drug treatment Naloxone dihydrochloride was purchased from Ascent Scientific (Bristol, UK). The drug was dissolved in 0.9% (w/v) normal saline and administered at a volume of 1 ml/kg through i.p. route 20 min before examining test latency on days 7, 14 or 21 of ligation. Total RNA extraction and cDNA synthesis
Three experimental groups including control, sham and a group with BDL were used in each part of the study. Sham and BDL groups underwent laparotomies, which were performed under general anesthesia induced by intraperitoneal (i.p.) injection of a mixture of ketamine/ xylazine (50 and 5 mg/kg, respectively). The rats that were used for sham and BDL operations were food restricted for 12 h prior to the surgery. The sham operation consisted of laparotomy, bile duct identification and manipulation but without ligation and resection. In the group with BDL, common bile duct was completely exposed, ligated at two points with approximately 5 mm apart and then transected at midpoint between the ligatures. After closure of the abdominal wound, the rats did not receive any analgesics but each rat received 1 ml of saline (i.p.) and moved to a clean box until complete recovery. Mortality due to operation was under 5% during and/or after laparotomy.
Twenty-one days after laparotomy, rats were sacrificed, the whole brain quickly removed from the skull and the hypothalamus, PFC, hippocampus and striatum were immediately dissected on an ice-chilled sterile surface as per previously reported method (Chiu et al., 2007). After harvesting, each tissue was immediately moved into a tube, submerged in an RNAlater RNA Stabilization Reagent (QIAGEN) and incubated overnight in the reagent at 4 °C. The RNAlater solution was drained from the tubes after 24 h and tissues were stored at 80 °C until further analysis. Total RNA was extracted from 50 to 70 mg of each tissue using a Trizol method with some modification. In brief, each tissue sample was removed from 80 °C deep freezer, transferred to a new tube containing 1 ml of lysis buffer (RNX+ reagent, Cinnagen, Tehran, Iran), homogenized for 60 s with a high-speed homogenizer (Silent Crusher S., Heidolph, Germany), and then subjected to total RNA extraction by a highly pure tissue RNA extraction protocol according to the manufacturer’s manual (Cinnagen, Tehran, Iran). Quality of total RNA was assessed by electrophoresis on 1% agarose gel to visualize 28s and 18s ribosomal RNA. The concentrations of total RNA were also measured with a spectrophotometer (Specord210, Analytic Jena, Germany). Then, a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) method was used to assess gene expression of MOR1 (Marone et al., 2001). Reverse transcription (RT) reaction was performed using Viva 2-step RT-PCR Kit according to the manufacturer’s protocol (Vivantis Technologies, Selangor Darul Ehsan, Malaysia).
Measurement of nociception
Polymerase chain reaction (PCR)
We used a hotplate test to assess pain behavior. Each rat was placed on the hotplate (52 ± 0.1 °C) with a surrounding glass square (25 cm height) to prevent escaping of animals (Armaghan Co., Tabriz, Iran). Then, time elapse between placement of the animals on hotplate and licking one of the hind paws or first jumping was measured as an index of nociceptive response. First, baseline latency was measured 1 day before laparotomy. Second, different experimental groups (control, sham and BDL) on days 7, 14 or 21 of laparotomy were tested to measure test latency on the hotplate. A cutoff time of 80 s was set to prevent tissue damage. Finally, the two measured latencies were converted to percentage
PCR was used for amplification of b-actin (as a housekeeping gene) and MOR1 genes. Primers for both genes were designed at exon–exon junctions to prevent amplifications of genomic DNA fragments of the genes. Primers had the following sequences: b-actin forward primer, 50 -CTGGGTATGGAATCCTGTGGC-30 ; b-actin reverse primer, 50 -AGGAGGAGCAATGATCTTGATC-30 ; MOR1 forward primer, 50 -CAGGGGTCCATAGATTG CAC-30 ; MOR1 reverse primer, 50 -GAAGTGCCAGGAAA CGGTC-30 . PCR was carried out in a reaction volume of 20 ll consisting of 10 ll of PCR Master Mix (Thermo Scientific, Pittsburgh PA, USA), 2 ll of cDNA, 2 ll of upstream and
Surgical laparotomy
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downstream mix of MOR1 primers (10 lM), 0.5 ll of upstream and downstream mix of b-actin primers (10 lM), and nuclease free water up to 20 ll. Firstly, reaction parameters were adjusted to acquire conditions with a linear relation of RNA concentration used for cDNA synthesis, the number of PCR cycles and annealing temperature related to PCR product. According to PCR optimization, thermal cycling was initiated with a first denaturation step of 95 °C for 3 min, followed by 27 cycles of thermal cycling of 94 °C for 30 s, 59 °C for 30 s, 72 °C for 30 s, and finally followed by a 10-min final extension step at 72 °C and termination at 4 °C for 5 min (C1000 Thermal Cycler, BIO-RAD, Hercules CA, USA). The PCR products were subsequently analyzed on 2% agarose gel electrophoresis and bands were quantified by densitometry using Image J software. Experimental design Experiment 1: effect of cholestasis on nociception. Nine groups of rats (n = 8) were used. They were divided into three sets of three groups; each set included control, sham and BDL groups. Three groups of each set were submitted to the hotplate test at 7, 14 or 21 days after ligation. Then, the recorded pain reaction latencies were converted to MPE% to assess the effect of cholestasis on pain behavior. Experiment 2: effect of naloxone on antinociception induced by cholestasis after 21 days of BDL. Three groups of rats (n = 8) including control, sham and BDL were submitted to the hotplate test on day 21 of ligation. Each rat received naloxone (1 mg/kg) at 20 min before examining the test latency. Experiment 3: evaluating MOR1 gene expression after 21 days of BDL in the hypothalamus, PFC, hippocampus,
and striatum. Three groups of control, sham and cholestatic rats (n = 4) were used to assess the expression of MOR1 gene in the hypothalamus, PFC, hippocampus, and striatum 21 days after BDL. For this purpose, tissues were firstly submitted to the total RNA extraction and, cDNA synthesis as have been previously described. Then MOR1 and b-actin genes in each tissue were amplified using PCR. Thereafter, PCR products were evaluated on agarose gel and the band densities were quantified with an Image J program. Data of gene expression were shown as normalization to the control group, in which the ratio of MOR1 to b-actin was set at 100%. Statistical analysis Data from the hotplate test and gene expression passed normality and equal variance tests, therefore they were analyzed by a one-way analysis of variance (ANOVA), and after a significant F value, post hoc Holm–Sidak’s test was used for multiple pairwise comparisons. p < 0.05 was considered a statistically significant level throughout.
RESULTS Cholestasis caused antinociception that was prevented by naloxone As shown in Fig. 1, a one-way ANOVA revealed that pain behavior in rats with BDL on day 7 after BDL was not significantly altered [F (2, 21) = 2.11, P > 0.05]. The one-way ANOVA also revealed that on day 14 of BDL, pain behavior was significantly altered [F (2, 21) = 10.01, p < 0.01]. The post hoc test revealed that cholestatic rats compared to the sham group significantly showed an increase in %MPE (p < 0.05). In addition, the one-way ANOVA showed that antinociception after 21 days of BDL was more
Fig. 1. Effect of cholestasis on pain behavior. Twelve groups of rats (n = 8) were used. Three sets of three groups including control, sham and BDL in each set were submitted to hotplate test at 7, 14 or 21 days after ligation. Three groups of rats (n = 8) including control, sham and BDL were also submitted to hotplate test at 21 days after ligation but they also received naloxone (1 mg/kg) 20 min before recording test latency on the hotplate. Each bar represents mean ± S.E.M. of %MPE in each group. ⁄P < 0.05 compared to the sham group after 14 days of ligation, and +++P < 0.001 compared to the sham group after 21 days of ligation.
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Fig. 2. Effect of cholestasis on the expression of MOR1 gene in the hypothalamus on day 21 of BDL. Three groups of control, sham and cholestatic rats (n = 4) were used. Upper panel (panel A) shows the inverted black and white gel image indicating band densities of MOR1 and b-actin genes after amplification with PCR. Numbers 1 to 4 on top of the image show number of the animals in each group. (M) symbolizes 100-bp ladder on the gel image. Lower panel (panel B) shows bar graph of quantitative data related to gene expression. Each bar represents mean ± S.E.M. of MOR1 gene band density normalized to b-actin and set at 100%. ⁄⁄⁄P < 0.001 compared to the sham group.
significantly altered [F (2, 21) = 15.63, p < 0.001]. The post hoc test revealed that analgesia induced by BDL in cholestatic rats after 21 days was significant compared to the sham group (p < 0.01). The one-way ANOVA also revealed that naloxone, an opioid receptor antagonist, prevented the analgesia induced by cholestasis on day 21 after BDL, therefore no significant difference was observed in %MPE of the experimental groups [F (2, 21) = 0.16, p > 0.05] (Fig. 1). These results support that an increase in endogenous opioids and activation of MOR may underlie the antinociception induced by cholestasis. MOR1 gene expression decreased in the hypothalamus after 21 days of BDL Analysis of the gene expression results with a one-way ANOVA revealed that mRNA level of MOR1 gene after 21 days of BDL had decreased in the hypothalamus [F (2, 9) = 67.87, p < 0.001]. Post hoc Holm–Sidak’s test
revealed a significant decrease (P < 0.001) of MOR1 gene expression by 42% in the hypothalamus compared to the sham control group after 21 days of BDL (Fig. 2). MOR1 gene expression decreased in the PFC after 21 days of BDL A one-way ANOVA revealed that mRNA levels of MOR1 gene after 21 days of BDL had decreased in the PFC [F (2, 9) = 31.45, p < 0.001]. Post hoc Holm–Sidak’s test revealed a significant decrease (P < 0.001) of MOR1 gene expression by 41% in the PFC compared to the sham control group after 21 days of BDL (Fig. 3). MOR1 gene expression decreased in the hippocampus after 21 days of BDL A one-way ANOVA revealed that mRNA levels of MOR1 gene after 21 days of BDL had decreased in the hippocampus [F (2, 9) = 172.97, p < 0.001]. Post hoc
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Fig. 3. Effect of cholestasis on the expression of MOR1 gene in the PFC on day 21 of BDL. Three groups of control, sham and cholestatic rats (n = 4) were used. Upper panel (panel A) shows band densities of MOR1 and b-actin genes after amplification with PCR on the inverted black and white gel image. Numbers 1–4 on top of the image show number of the animals in each group. (M) symbolizes 100-bp ladder on the gel image. Lower panel (panel B) shows bar graph of quantitative data related to gene expression. Each bar represents mean ± S.E.M. of MOR1 gene band density normalized to b-actin and set at 100%. ⁄⁄⁄P < 0.001 compared to the sham group.
Holm–Sidak’s test showed a significant decrease (P < 0.001) in MOR1 gene expression by 67% in the hippocampus compared to the sham control group after 21 days of BDL (Fig. 4). MOR1 gene expression remained without significant change in the striatum after 21 days of BDL Analysis of the gene expression data with a one-way ANOVA showed that mRNA levels of MOR1 gene after 21 days of BDL remained without significant change in the striatum [F (2, 9) = 1.52, p > 0.05] (Fig. 5).
DISCUSSION It has been shown that cholestasis is associated with increased opioidergic tone (Bergasa et al., 1994; Davis, 2007). This study explored the expression of MOR at
mRNA levels in areas of the rat brain that participate in emotional and cognitive aspects of nociception in a model of cholestasis. First, the results revealed that cholestatic rats showed significant antinociception on days 14 and 21 of BDL with the most significant effect on day 21. Second, naloxone (1 mg/kg) prevented the antinociception that was induced by BDL on day 21 of BDL, which may confirm an increase in endogenous opioids and activation of MOR in this process. Third, the expression of MOR1 gene compared to the sham group decreased by 42% in the hypothalamus, 41% in the PFC, and 67% in the hippocampus after 21 days of BDL, while no significant change was observed for its expression in the striatum. It has been reported that acute and chronic liver failure alters brain functions and affects neurotransmitter systems (Lozeva et al., 2004; Garcia-Ayllon et al., 2008). In some studies, rats with BDL have been used as a model of liver failure (Magen et al., 2009; Wright
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Fig. 4. Effect of cholestasis on the expression of MOR1 gene in the hippocampus on day 21 of BDL. Upper panel (panel A) shows the inverted black and white gel image indicating band densities of MOR1 and b-actin genes related to three groups of control, sham and cholestatic rats (n = 4). Numbers 1–4 on top of the image show number of the animals in each group. (M) symbolizes 100-bp ladder on the gel image. Lower panel (panel B) shows bar graph of quantitative data related to gene expression. Each bar represents mean ± S.E.M. of MOR1 gene band density normalized to b-actin and set at 100%. ⁄⁄⁄P < 0.001 compared to the sham group.
et al., 2010). On the other hand, it has been shown that plasma total opioid levels in cholestatic liver disease is increased which may underlie pruritus and analgesia associated with cholestasis (Bergasa et al., 1995; Nicoll et al., 2005; Alemi et al., 2013). In line with the above reports, the result of the hotplate test in the present study revealed that nociceptive threshold in cholestatic rats increased with a maximum effect on day 21 of ligation, as revealed by an increase in %MPE on hotplate test. As has previously been reported in mice (Nelson et al., 2006), our results also confirmed that naloxone completely blocked the antinociception induced by cholestasis on day 21 of BDL in rats. However, since naloxone could readily enter the central nervous system, it has been reported that its peripheral injections would block opioid receptors not only located on peripheral nerve endings but also in the central pain pathways (Bergasa et al., 1994; Nelson et al., 2006). Naloxone, as a MOR antagonist, blocks these receptors and in the case of hotplate
test in the present study prevented the antinociception that was induced by increased opioid levels, and therefore reversed the behavioral changes in BDL rats. It has also been reported that inflammatory pathways could also contribute to pain perception (Okuse, 2007; Carr et al., 2014), and are also known to be involved in BDL (Noguchi et al., 2014). Therefore, their role in the changes of nociceptive threshold in cholestatic rats cannot be excluded. Taken together, complex peripheral and central actions may underlie antinociception and its blockade by naloxone in cholestatic rats. The results of gene expression in the present study revealed that the expression of MOR1 gene decreased in limbic areas including the hypothalamus, PFC and hippocampus, but remained without changes in the striatum on day 21 after BDL. To discuss the present results, one may propose that alteration in the expression of MOR1 gene could be a consequence of increased levels of endogenous opioids in cholestatic
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Fig. 5. Effect of cholestasis on the expression of MOR1 gene in the striatum on day 21 of BDL. Upper panel (panel A) shows the inverted black and white gel image indicating band densities of MOR1 and b-actin genes related to three groups of control, sham and cholestatic rats (n = 4) on agarose gel. Numbers 1 to 4 on top of the image show number of the animals in each group. (M) symbolizes 100-bp ladder on the gel image. Lower panel (panel B) shows bar graph of quantitative data related to gene expression. Each bar represents mean ± S.E.M. of MOR1 gene band density normalized to b-actin and set at 100%. ⁄⁄⁄P < 0.001 compared to the sham group.
rats. However, the expression of MOR1 gene in the striatum further indicates that cellular adaptation for an increase in opioids and/or changes in MOR1 gene in different brain areas may be a site-specific process. Nelson et al. (2006) reported that cholestasis in mice is associated with antinociception due to local effects of endogenous opioids at the level of sensory nerve endings (Nelson et al., 2006). However, these results support this idea that an increase in nociceptive threshold in cholestatic rats may result from changes in both peripheral and central opioid systems. The discrepancy between the two studies may result from using different subjects. Pain and pruritus are closely interrelated and opioids modulate both sensations (Nelson et al., 2006); therefore, the decreases in the expression of MOR1 gene may reflect an important mechanism of not only antinociception but also pathogenesis of the pruritus induced by cholestasis. In support of this suggestion, it has been shown that pain
afferent neurons in the spinothalamic pathway have spontaneous activity which can tonically suppress sending itch sensation to the thalamus [for a review see (Ikoma et al., 2003)]. Therefore, one may conclude that the increase of opioids during cholestasis prevents activity of pain neurons and induces antinociception, which in turn induces an increase of itch sensation. Furthermore, since activation of MOR induces antinociception, one may predict that their down-regulation should have increased nociception. However, pain perception is under the impact of many modulatory systems in the pain pathway, and it could not be predicted that down-regulation of MOR1 gene expression should have increased nociception. According to research, both opioidergic and serotonergic systems have been proposed as central regulators of pruritus (Bhargava and Gulati, 1990; Bergasa et al., 1992; Bunchorntavakul and Reddy, 2013). Other investigators have reported that opioids
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might also activate serotonin pathways, and analgesic effect of morphine may be mediated, at least partly, via activation of serotonergic inhibitory descending pain pathways originating from the brain stem, and terminating on neurons in the spinal cord gray matter (Ohsawa et al., 2000; Dogrul et al., 2009; Ozdemir et al., 2012). Furthermore, serotonergic pathways to the forebrain structures such as nucleus accumbens have been shown to be activated by opioids which in turn induced analgesia blocked by serotonin antagonists (Ohsawa et al., 2000). Magen et al. (2010) reported that BDL reduces 5-HT1A expression in the hippocampus. Since 5-HT1A receptors act as a regulator of serotonin release, its down-regulation could increase serotonin release (Magen et al., 2010). With regard to the above reports and the results of this study, an alternate mechanism for antinociception in cholestatic rats might be the activation of serotonin release as a result of the altered MOR and/or 5-HT1A receptor expression that could affect downstream neurons in the pain pathway. However, we did not explore 5-HT1A receptor expression and serotonin release, and this suggestion needs further investigation to be lucid.
CONCLUSIONS In summary, it can be concluded that cholestasis induced significant antinociception 21 days after BDL in rats which was prevented by naloxone. This result supports the idea that MOR might be the key elements of antinociception and possibly pruritus induced by cholestasis. In line with this suggestion, our results indicated that the expression of MOR1 gene decreased in the hypothalamus, PFC and hippocampus. However, its expression remains without significant changes in the striatum. Based on our results, it could be proposed that changes in the expression of MOR1 gene caused by cholestasis are site specific.
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(Accepted 21 August 2014) (Available online 5 October 2014)