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OPIOID NEUROTOXICITY: COMPARISON OF MORPHINE AND TRAMADOL IN AN EXPERIMENTAL RAT MODEL
Sebnem Atici a; Leyla Cinel b; Ismail Cinel a; Nurcan Doruk a; Mustafa Aktekin c; Almila Akca a; Handan Camdeviren d; Ugur Oral a a Department of Anesthesiology & Reanimation, Mersin University School of Medicine, Mersin, Turkey. b Department of Pathology, Mersin University School of Medicine, Mersin, Turkey. c Department of Anatomy, Mersin University School of Medicine, Mersin, Turkey. d Department of Biostatistics, Mersin University School of Medicine, Mersin, Turkey. Online Publication Date: 01 August 2004 To cite this Article: Atici, Sebnem, Cinel, Leyla, Cinel, Ismail, Doruk, Nurcan, Aktekin, Mustafa, Akca, Almila, Camdeviren, Handan and Oral, Ugur (2004) 'OPIOID NEUROTOXICITY: COMPARISON OF MORPHINE AND TRAMADOL IN AN EXPERIMENTAL RAT MODEL', International Journal of Neuroscience, 114:8, 1001 - 1011 To link to this article: DOI: 10.1080/00207450490461314 URL: http://dx.doi.org/10.1080/00207450490461314
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Intern. J. Neuroscience, 114:1001–1011, 2004 Copyright Taylor & Francis Inc. ISSN: 0020-7454 / 1543-5245 online DOI: 10.1080/00207450490461314
OPIOID NEUROTOXICITY: COMPARISON OF MORPHINE AND TRAMADOL IN AN EXPERIMENTAL RAT MODEL SEBNEM ATICI Department of Anesthesiology & Reanimation Mersin University School of Medicine Mersin, Turkey
LEYLA CINEL Department of Pathology Mersin University School of Medicine Mersin, Turkey
ISMAIL CINEL NURCAN DORUK Department of Anesthesiology & Reanimation Mersin University School of Medicine Mersin, Turkey
MUSTAFA AKTEKIN Department of Anatomy Mersin University School of Medicine Mersin, Turkey
Received 15 September 2003. Presented at 8th European Society of Anaesthesiologists (ESA) Meeting, 6–9 April 2002, Nice, France. Address correspondence to Dr. Sebnem Atici, Menderes Mh. Bakanlik Cd., Liparis Sitesi Acelya Blok 10/22, 33280 Mezitli, Mersin, Turkey. E-mail:
[email protected]
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ALMILA AKCA Department of Anesthesiology & Reanimation Mersin University School of Medicine Mersin, Turkey
HANDAN CAMDEVIREN Department of Biostatistics Mersin University School of Medicine Mersin, Turkey
UGUR ORAL Department of Anesthesiology & Reanimation Mersin University School of Medicine Mersin, Turkey Histopathologic changes in rat brain due to chronic use of morphine and/or tramadol in progressively increased doses were investigated in this study. Thirty male Wistar rats (180–220 g) were included and divided into three groups. Normal saline (1 ml/kg) was given intraperitoneally as placebo in the control group (n = 10). Morphine group (n = 10) received morphine intraperitoneally at a dose of 4 mg/kg/day for the first 10 days, 8 mg/kg/day between 11–20 days, and 12 mg/kg/day between 21–30 days. The tramadol group (n = 10) received the drug intraperitoneally at doses of 20, 40, and 80 mg/kg/day in the first, second, and the third 10 days of the study, respectively. All rats were decapitated on the 30th day and the brain was removed intact for histology. The presence and the number of red neurons, which are a histologic marker of apoptosis, were investigated in the parietal, frontal, temporal, occipital, entorhinal, pyriform, and hippocampal CA1, CA2, CA3 regions. Red neurons were found in morphine and tramadol groups but not in the control group. The total number of red neurons was not different in morphine and tramadol groups, but the numbers of red neurons were significantly higher in the temporal and occipital regions in tramadol group as compared with the morphine group (p < .05). In conclusion, chronic use of morphine and/or tramadol in increasing doses is found to cause red neuron degeneration in the rat brain, which probably contributes to cerebral dysfunction. These findings should be taken into consideration when chrome use of opioids is indicated. Keywords apoptosis, morphine, opioid neurotoxicity, tramadol
The main goals of chronic non-cancer pain management in a population with normal life expectancy are pain relief and improvement
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in physical and social function (Dellemijn, 2001; Brena & Sander, 1991). Although it is generally accepted that the use of strong opioids is appropriate in the management of cancer pain, they have been used reluctantly for non-cancer pain. This is largely due to concerns about side-effects, physical tolerance, withdrawal reactions, and addiction (Turk et al., 1994; Bannwarth, 1999; Quang-Cantagrel et al., 2000). Experimental studies have shown that acute use of high-dose opioids may lead to neurotoxicity in animal models (Kofke et al., 1996: Sinz et al., 2000). Limbic cellular damage and formation of red neurons, which are brightly stained neurons with scanty eosinophilic cytoplasm, and nuclear pyknosis were the main histopathologic finding in these studies (Kofke et al., 1996). Tramadol, a synthetic opioid of the aminocyclohexanol group, is a centrally acting opiate (Lee et al., 1993). Tramadol has been found both in vivo and in vitro to have a low but preferential activity at µ opioid receptors, and also to inhibit both noradrenaline and 5-hydroxytryptamine (5-HT) neuronal reuptake, and to facilitate 5-HT release (Eggers & Power, 1995). Its analgesic potency is 5–10 times less than morphine and is indicated for management of moderate to severe pain. Tramadol, which has a low probability of tolerance, dependence, and abuse, has not been associated with clinically significant respiratory depression (Vikers et al., 1992; Preston et al., 1991). Although tramadol was reported to cause seizures in animals when given in toxic doses (Gasse et al., 2000; Spiller et al., 1997; Kahn et al., 1997), histopathologic changes in brain cells due to chronic use of tramadol have not been previously investigated. The aim of this study is to investigate histopathologic changes in rat brain with particular reference to formation of red neurons due to long-term use of morphine and/or tramadol in progressively increased doses.
MATERIALS AND METHODS The experiments described in this manuscript were performed in adherence to the National Institutes of Health Guidelines on the Care and Use of Laboratory Animals, and approval of the ethic committee of Mersin University School of Medicine was obtained before study.
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Experimental Protocol Thirty male Wistar rats, weighing between 180–220 g were used. As a standard protocol, all the rats were housed in a quiet nonstressful environment for one week before the study. Wistar rats were divided into three groups. All rats were given 45–50 kcal/day normal rat chows during the experimental period. The first group (morphine group, n = 10) received morphine intraperitoneally at a dose of 4 mg/kg/day for the first 10 days, 8 mg/kg/ day between 11–20 days, and 12 mg/kg/day between 21–30 days. The second group (tramadol group, n = 10) received tramadol intraperitoneally at doses of 20, 40, and 80 mg/kg/day on the first, second, and third ten days of the study, respectively. All morphine and tramadol doses were delivered in a volume of 1 ml of normal saline. It was planned to return to and continue with the previous dose if any complications related to morphine or tramadol such as apnea, convulsion were observed. As placebo, normal saline (0.9% NaCl, 1 ml) was given intraperitoneally in the control group (n = 10). All rats were decapitated on the 30th day under ketamine anesthesia and the brain was removed intact. Anatomic and Histopathologic Examination The brains were fixed in 10% buffered formalin. The brains were horizontally sliced at approximately 2–5 mm intervals, and the slices were embedded in paraffin using routine techniques. Six-micrometer sections of each brain block were than prepared, stained with hematoxylin and eosin, and evaluated by light microscopy. The presence and the number of red neurons (apoptotic neurons) were determined in the parietal, frontal, temporal, occipital, entorhinal, pyriform, and hippocampal CA1, CA2, CA3, regions by light microscopy. A red neuron was defined as a brightly stained neuron with scanty eosinophilic cytoplasm, nuclear pyknosis, and perineural retraction spaces. The slides were examined by a pathologist (LC) blinded to sample identity. Identified red neurons were counted in all aforementioned regions separately on high power field and then were summated for each rat.
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Statistical Analysis After applying square root transformation to cell numbers, statistical analysis was performed using analysis of variance (ANOVA), followed by post-hoc (Scheffe) least significant difference testing where p < .05 on ANOVA. Data were presented as mean ± SD and p < .05 was considered statistically significant. All calculations were made using SPSS for Windows 10.0 Program. RESULTS On the third day of 80 mg/kg/d of tramadol administration three rats expired due to convulsions and were excluded from the study. The dose of tramadol was reduced to 40 mg/kg/d for the remaining rats between 24–30th days of the study. There was no microscopic lesion in the brains of the control group (Figure 1A). Red neurons were found in morphine and tramadol groups (Figure 1B, 1C). Red neurons were observed primarily in the limbic system and its areas of association.
A
FIGURE 1. (A) High-power photomicrograph of normal-appearing hippocampal region (control group: hematoxylin-eosin ×200 original magnification). (See Color Plate I at end of issue.)
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B
C
FIGURE 1. (Continued) (B) High-power photomicrograph of the same portion of the hippocampal region. Arrows indicate red (apoptotic) neurons with scanty eosinophilic cytoplasm and pyknotic nuclei (morphine group: hematoxylin-eosin ×200 original magnification). (C) Photomicrograph depicting similar histopathologic findings as in morphine group. Arrows indicate red (apoptotic) neurons (tramadol group: hematoxylin-eosin ×200 original magnification). (See Color Plate I at end of issue.)
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The total number of red neurons were not different in morphine and tramadol groups (p > .05). However, the numbers of red neurons were significantly higher in the temporal and occipital regions in tramadol group as compared with in morphine group (p = .01 and p = .04, respectively) (Figures 2 and 3).
DISCUSSION Opioids are known to affect the neuronal survival in vivo. It is uncertain to which extent the opioids directly or indirectly affect the proliferation, differentiation, and death of neuronal precursors as well as the neuron (Hammer et al., 1989; Hauser et al., 2000; Eisch et al., 2000). Previous in vitro and in vivo studies have demonstrated that opioids effect the viability of neuronal and glial cells via an opioid receptor-mediated mechanism (Eisch et al., 2000; Polakiewicz et al., 1998; Kim et al., 2001; Hauser et al., 1996). Opioids may be involved not only in neuronal survival but also in programmed cell death, but there are conflicting results in the literature concerning the effects of opioids on apoptosis. Meriney et al. (1985) suggested
FIGURE 2. The numbers of red neurons were significantly higher in the occipital region in tramadol group as compared with morphine group ( #p = .04). Apoptotic neurons were not in the control group (*p < .01).
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FIGURE 3. The numbers of red neurons were significantly higher in the temporal region in tramadol group as compared with morphine group ( #p = .01). Apoptotic neurons were not in the control group (*p < .01).
that morphine might delay the neuronal cell death. Protective effects of morphine in peroxvnitrite-induced apoptosis in human neuroblastoma and rat neonatal astrocytes were also documented (Kim et al., 2001; Kanesaki et al., 1999). On the other hand, recent in vitro studies using embryonic chick brain and specific cell lines showed that opioids might induce or enhance apoptosis (Goswami et al., 1998; Dawson et al., 1997; Singhal et al., 1998). In concordance with these findings the present results indicate that chronic use of morphine or tramadol increased neuronal apoptosis in vivo. The molecular mechanisms of opioid-induced apoptosis have not been established yet, but various key proteins are involved in the regulation of programmed cell death (Sastry & Rao, 2000). Some members of the Bcl family, such as Bcl-2 and Bcl-xL are regulators of neuronal apoptosis, which are known to suppress apoptosis, whereas the expression of Bax and Bak, are pro-apoptotic. Bcl-2 protein localized mainly in the mitochondrial membrane has been shown to play an important role in protecting neurons from apoptotic cell death (Yuan & Yankner, 2000). Bax protein releases cytochrome c from the mitochondria and the subsequent activation of caspases-3/ 9 was shown to play the key role in apoptosis (Xiao et al., 2000). Morphine promotes the synthesis of Bax (Singhal et al., 1998). Another
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key element involved in the regulation of apoptosis is the Fas glycoprotein, a cell surface receptor that belongs to the tumor necrosis factor and nerve growth factor family. A recent in vitro study demonstrated that morphine caused increases of pro-apopototic receptor Fas expression (Yin et al., 1999). It can therefore be speculated that increased expression of Fas and/or promoted synthesis of Bax might have contributed to increased neuronal apoptosis due to chronic use of morphine and/or tramadol as shown in the present study. Neurotoxic effects of opioids were previously investigated by acute high dose alfentanil and fentanyl in vivo (Kofke et al., 1996; Kofke et al., 1992). The histopathologic changes were mainly shown in the limbic system and related areas. Because red neuron degeneration was found even seven days after the last dose of fentanyl, this neurotoxicity effect was thought to be permanent rather than a transient effect. On the other hand, Matthiessen et al. (1998) using tramadol at a dose of 40 mg/kg/day for two weeks were not able to prove any neurotoxic effect in the brain, brainstem, or the spinal cord. The present study was the first to show red neuron degeneration in various areas of brain when opioids were used in increasing doses for a period over four weeks. In conclusion, the authors believe that chronic use of opioids is an important factor in the occurrence of histopathologic changes related to neurons, which may be the cause of cerebral dysfunction after their prolonged use. This possibility should be taken into consideration when chronic use of opioids is indicated.
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