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Cerebrolysin and morphine decrease glutathione and 5-hydroxyindole acetic acid levels in fasted rat brain
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www.sciencedirect.com Biomedicine & Pharmacotherapy 63 (2009) 517e521
Original article
Cerebrolysin and morphine decrease glutathione and 5-hydroxyindole acetic acid levels in fasted rat brain c David Caldero´n Guzma´n a, Norma Osnaya Brizuela b, Raquel Garcı´a Alvarez , c a c,d,* ´ ´ ´ ´ ´ ´ Ernestina Hernandez Garcıa , Gerardo Barragan Mejıa , Hugo Juarez Olguın a
Laboratorio de Neuroquı´mica, Instituto Nacional de Pediatrı´a (INP), Mexico b Laboratorio de Patologı´a, Instituto Nacional de Pediatrı´a (INP), Mexico c Laboratorio de Farmacologı´a, Instituto Nacional de Pediatrı´a (INP), Mexico d Departamento de Farmacologı´a, Facultad de Medicina, Universidad Nacional Auto´noma de Mexico, Mexico Received 27 January 2008; accepted 26 September 2008 Available online 23 October 2008
Abstract Purpose: The aim was to evaluate if morphine sulphate combined with cerebrolysin enhances the risk of oxidative damage in the presence of moderate hypoglycaemia. Methods: Wistar rats under starvation for 48 h received a single dose of 215 mg/kg cerebrolysin or 4 mg/kg morphine sulphate. Glutathione (GSH) and 5-hydroxyindoleacetic acid (5-HIAA) levels were measured in brain tissue, as well as lipid peroxidation, NaþeKþ ATPase and total ATPase enzymatic activities, by fluorescence and spectrophotometric methods. Results: GSH and 5-HIAA levels decreased significantly ( p < 0.05) in animals which received cerebrolysin and morphine alone or combined. TBARS levels increased in all groups, but the values were statistically significant only in those animals that received cerebrolysin combined with morphine ( p < 0.05). NaþeKþ ATPase and total ATPase activities decreased significantly in rats treated only with morphine, but the cerebrolysin and morphine groups showed a significant increase in these enzymatic activities. Conclusions: Results suggest that cerebrolysin as well as morphine induced changes in cellular regulation and biochemical responses to oxidative stress induced by moderate hypoglycaemia in brain. Ó 2008 Elsevier Masson SAS. All rights reserved. Keywords: Morphine; Cerebrolysin; Starved; 5-HIAA; Glutathione; Brain; Neurology; Malnutrition; Drug abuse; Nutrients
1. Background Opioid analgesics, mainly represented by morphine, are the most potent pain-relieving drugs currently available to treat severe and chronic postoperative pain and cancer pain management [1]. While low-dose spinal opioid administration is safe [2], the long-term use of these drugs is associated with tolerance, related to cellular adaptability [3], so that increasing dosages are required to maintain their analgesic effect along * Corresponding author. Laboratorio de Farmacologı´a, Instituto Nacional de Pediatrı´a. Avenida Ima´n No 1, 3rd piso Colonia Cuicuilco, CP 04530, Mexico City, Mexico. Tel./fax: þ5255 1084 3883. E-mail address: [email protected] (H. Jua´rez Olguı´n). 0753-3322/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biopha.2008.09.013
the period of treatment. Tolerance represents one of the major complications to be overcome in the clinical management of these drugs due to untreated side effects [4], and abuse among young and adult people in urban zones with recreational purposes, according to the National Questionnaire of Addictions in Mexico [5]. The mechanisms by which opioids induce tolerance are not completely understood [6], but their abuse has been correlated with central nervous system’s oxidative stress and free radical production [7]. Administration of antioxidants may be employed in pain treatment to decrease the doses of analgesics and to prevent the negative impact of reactive oxygen species on nociception [8]. One of the main naturally occurring free-radical scavengers capable of protecting the body from oxidative stress
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is the tripeptide known as reduced glutathione, or GSH, which is also an ubiquitous reducing agent whose absence induces severe oxidative stress (OS) [9]. It has been suggested that GSH interacts with nitric oxide (NO) producing S-nitrosoglutathione (GNSO) in the presence of endogenously produced oxygen [10], as part of normal NO catabolism, in which GSH is necessary to facilitate its diffusion. GSH is the main redox equilibrium regulator and it plays an important role in the protection of tissues that suffer damage by oxidative agents [11]. Recent studies indicate that the use of neuroprotective agents induces defensive mechanisms in the brain by diminishing the extent of the oxygen-derived free radical-induced lipid peroxidation [12], and some drugs, such as morphine, provoke changes in amino acid levels in the brain, which may participate in the pharmacological action of a number of compounds [13]. Cerebrolysin is a drug consisting of small, porcine brainderived peptides, similar or identical to those endogenously produced; it is capable of crossing the bloodebrain barrier partially, because the drug contains 25% of low molecular weight peptides [14], and has been successfully used in animal models with induced hypoglycaemia, in which it has proved to decrease oxidative stress substantially [15]. Hypoglycaemia reflects a pathophysiological situation. It is a condition that arises from diverse metabolic impairments, which may promote cell death [16], and it may also alter the body’s response to the effect of morphine [17], although its mode of action remains unknown. The central nervous system is particularly susceptible to oxidative damage [18] causing age-related brain dysfunction and neurodegenerative disorders [9], and the membrane lipids are the main target [19]. Among individuals who use large amounts of opioids lipid peroxidation occurs due to the imbalance between oxidation by free radicals and antioxidant activity by free radical scavengers [20]. Membrane lipids are mainly represented by phospholipids, and they are known to strongly interact with the lipid bilayer structural proteins [21], such as the NaþeKþ ATPase, which is responsible of ion interchange across the membrane necessary for neuronal excitability [22]. The inhibition of the NaþeKþ ATPase promotes the release of excitatory amino acids in the CNS, such as serotonin and its main metabolite 5-HIAA, through a mechanism similar to that exerted by brain ischemia. This effect is also induced by morphine, since it alters serotonin brain metabolism [23]. Based on the above information, the present study was conducted to evaluate a possible protective effect of cerebrolysin and morphine on the brain of female rats, by determining the levels of TBARS, GSH, 5-HIAA, as well as total and NaþeKþ ATPases. 2. Methods Forty female Wistar rats, weighing 250 g, were divided into four groups of 10 animals each, according to the administered drug as follows: group I, control (NaCl 0.9%); group II, cerebrolysin; group III, morphine sulphate, and group IV,
cerebrolysin þ morphine sulphate. A single intraperitoneal (i.p.) dose, previously evaluated in our lab, of cerebrolysin (215 mg/kg) and morphine sulphate (4 mg/kg) was each administered to the rats which had a previous 48-h starvation period. An hour after the medication, the animals were decapitated, the brain was immediately placed in saline (NaCl 0.9%) at 4 C, and blood glucose concentration was determined at the same time. A sagittal section of the brain was made; the left portion was homogenized in 5 volumes of 0.05 M TriseHCl, pH 7.4, and used to determine lipid peroxidation and NaþeKþ ATPase activity. The right portion was homogenized in 5 volumes of 0.1 M perchloric acid (HClO4) and GSH and 5HIAA concentrations were determined. The homogenized tissue was kept at 20 C until assayed. The animals were kept in closed boxes throughout the study, 48 h, with a photoperiod of 12/12 h light/darkness, under starvation, and drinking water ad libitum. All experimental procedure were done with authorization from the Laboratory Animals Use and Care Committee of our institution. 2.1. Blood glucose concentration In order to evaluate the hypoglycaemic status in the experimental groups, blood glucose concentration was determined before and after the 48-h starvation period. Twenty microliters of circulating and anticoagulant-free blood was obtained from rats by cutting a 2 mm tail slice, and then introducing a reactive strip (Accu-Chek active, Roche Mannheim, Germany) that detects blood glucose directly in mg/ dL. 2.2. Lipid peroxidation (TBARS) Oxidative damage to membrane lipids was determined by the modified assay of Gutteridge and Halliwell [24], as follows: 2 mL of a 250 mL solution containing thiobarbituric acid (1.25 g), trichloroacetic acid (40 g) and 6.25 mL HCl was added to 1 mL of homogenized rat tissue in 5 mL phosphate buffer, pH 7.4. This sample was then heated to boiling for 30 min (Thermomix 1420), placed on ice for 5 min, and then centrifuged at 3000 g for 15 min (Sorvall RC-5B Dupont). Supernatant absorbance was read spectrophotometrically (Helios-a, UNICAM) at 532 nm and the concentration of thiobarbituric acid reactive substances (TBARS) was expressed as mM of malondialdehyde (MDA) per gram of wet tissue. 2.3. NaþeKþ ATPase and total ATPase activities This parameter was analyzed based on the method reported by Caldero´n-Guzma´n et al. [25]. One hundred microliters of 10% homogenates of brain in 0.05 M Trise HCl, pH 7.4, was incubated for 15 min in a solution containing 3 mM MgCl2, 7 mM KCl, 100 mM NaCl and added or not with 0.06 mM ouabain. Afterwards, 4 mM TriseATP was added and the mixture was incubated for another
D. Caldero´n Guzma´n et al. / Biomedicine & Pharmacotherapy 63 (2009) 517e521
30 min at 37 C in a shaking water bath (Dubnoff Labconco) and the reaction was stopped with 100 mL of 5% trichloroacetic acid. Samples were centrifuged at 3500 g for 5 min at 4 C and inorganic phosphate (Pi) was determined in an aliquot from the supernatant by the Fiske and Subbarrow method [26]. Absorbances from the oubain-containing and oubain-free solutions were determined spectrophotometrically at 660 nm (Helios-a, UNICAM) and the difference was taken as the Naþ/Kþ ATPase activity, expressed as mM Pi/g wet tissue/ min. Total ATPase activity was measured without using ouabain. 2.4. Reduced glutathione (GSH) concentration Perchloric acid homogenized tissue was centrifuged at 8000 g for 5 min (microcentrifuge Mikro 12-42, Germany) and GSH levels were measured from the supernatant as described by Hissin and Hif [27]. To 1.8 mL of phosphate buffer (0.2% EDTA), pH 8.0, an aliquot of perchloric acid homogenate and 100 mL of o-phthaldialdehyde (1 mg/mL, m/v in methanol) were added. This reaction mixture was kept from the light, incubated at room temperature for 15 min, and analyzed in a Perkin Elmer LS 55 spectrofluorometer, 350 nm/ 420 nm excitation/emission using FL Win Lab software, version 4.00.02. Results are reported according to a previously constructed standard curve. 2.5. Levels of 5-hydroxyindole acetic acid (5-HIAA) The level of 5-HIAA was measured in the perchloric acid homogenate, as described for GSH, following the method proposed by Beck et al. [28]. A vial with 1.9 mL of 0.01 M acetate buffer, pH 5.5, containing a tissue homogenate aliquot, was incubated at room temperature for 5 min, keeping it away from light. Samples were analyzed in a spectrofluorometer with excitation/emission of 296 nm/333 nm, as previously described.
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Table 1 Blood levels of glucose in rats before and after 48 h under starvation period. Control
Cerebrolysin
Morphine
Cerebrolysin þ morphine
74.83 6.0
87.29 12.1
98.14 9.1
101.86 11.9
Average values are expressed as mg/dL S.D. Control (NaCl 0.9%), dose given: cerebrolysin 215 mg/kg, morphine 4 mg/kg. Blood levels of glucose in rats before starvation 114.83 4.4 mg/dL.
Glucose concentration increased by 15e30% in animals with combined treatment or with cerebrolysin or morphine alone, compared to the control starvation group. Fig. 1 shows the levels of lipid peroxidation products in the brain of hypoglycaemic rats treated with cerebrolysin and morphine sulphate. All of the experimental groups showed slightly higher TBARS levels than the control group, regardless of the treatment received. TBARS levels in the combined cerebrolysin- and morphine-treated group showed a significant increase (ANOVA; p ¼ 0.0372). GSH levels also showed a statistically significant decrease (ANOVA; p ¼ 0.0001), regardless of treatment type (Fig. 2). As shown in Fig. 3, the animals treated with cerebrolysin or morphine alone had lower NaþeKþ ATPase activity than the control group, but these values were statistically significant only in morphine-treated rats (ANOVA; p ¼ 0.0009). The cerebrolysin/ morphine combination induced NaþeKþ ATPase activity slightly higher than the control and significantly different from the morphine-treated group. Total ATPase activity showed a similar behaviour as the NaþeKþ ATPase among experimental groups as displayed in Fig. 4. 5-HIAA levels also decreased significantly in all experimental groups (ANOVA; p ¼ 0.0001) (Fig. 5). 4. Discussion The central nervous system (CNS) is extremely sensitive to hypoglycaemic damage, because of the properties of the hematoencephalic barrier and because of the lack of other substrates in the CNS. In the present study, moderate hypoglycaemic condition was shown by the low blood glucose levels [30], ranging from 4 to 5.5 mmol/L in all fasted rats,
2.6. Statistical analysis Data were analyzed by ANOVA and the contrasts by the Dunnett’s test, after proving the variance homogeneity. Values were considered statistically significant to a p < 0.05 level [29], using the Statistical Discovery Software (for academic use only) JMP IN 5.1 version 2003. 3. Results Table 1 shows blood glucose levels before and after starvation, and in cerebrolysin- and/or morphine-treated Wistar rats. As shown in Table 1, blood glucose levels decreased significantly in fasted animals, with respect to non-starved animals.
Fig. 1. Lipid peroxidation levels in moderate hypoglycaemic rat brain treated with cerebrolysin and morphine sulphate. Mean S.D. *p < 0.05, Ctrl ¼ control, Cer ¼ cerebrolysin, Mor ¼ morphine, Cer þ Mor ¼ cerebrolysin þ morphine.
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Fig. 2. Glutathione levels in moderate hypoglycaemic rat brain treated with cerebrolysin and morphine sulphate. Mean S.D. *p < 0.05, Ctrl ¼ control, Cer ¼ cerebrolysin, Mor ¼ morphine, Cer þ Mor ¼ cerebrolysin þ morphine.
compared to 6.3 mmol/L in non-fasted rats [31]. These results suggest that starvation caused changes in intracellular ion availability, such as Naþ, in fasted rat groups. As expected, lipid peroxidation products increased in morphine-treated animals, probably due to free radical production induced by this drug in brain tissue [32], which is in agreement with the fact that brain neurons synthesize nitric oxide (NO) in response to exposure to morphine, leading to oxidative damage [33]; the lack of food did not change this effect [34]. Cerebrolysin did not influence the morphine effect, since the group treated with both drugs showed an increase in TBARS as well. This effect suggests that cerebrolysin failed to have a GSH-like antioxidant effect. With respect to the first proposal, morphine-treated animals showed decreased GSH levels and, although not statistically significant, this effect was less evident in the presence of cerebrolysin, which suggests that the small cerebrolysin peptides may interact with NO metabolism, as has been shown for GSH and, therefore, a protective effect. These results strongly suggest that morphine induces oxidative brain damage by decreasing GSH, as previously stated by Goudas et al. [35], who reported a decline in GSH and enhanced susceptibility to oxidative stress in morphine-treated cancer patients.
Fig. 4. Total ATPase in moderate hypoglycaemic rat brain treated with cerebrolysin and morphine sulphate. Mean S.D. *p < 0.05, Ctrl ¼ control, Cer ¼ cerebrolysin, Mor ¼ morphine, Cer þ Mor ¼ cerebrolysin þ morphine.
Further studies should be conducted in order to rule out that morphine induces lipid peroxidation in an NO-independent manner, and to find the other drugs that interfere with the NO production induced by morphine and improve its clinical use, as was suggested by Pryor et al. [36]. Changes in intracellular Naþ availability or membrane depolarization provoked by morphine-induced tolerance, may be the cause of decreased NaþeKþ ATPase activity [37] in the morphinetreated group and cerebrolysin almost completely abolished the morphine effect by inducing the recovery of enzymatic activity. A possible mechanism of this enzyme activity recovery may be that cerebrolysin alters the intracellular sodium content, increasing the competence of the enzyme for sodium, and thus provoking a different effect than that of morphine. This suggests that the NaþeKþ ATPase activity may play an important role in the analgesic effect [38,39]. This result has clinically important implications, since the analgesic effect of morphine seems to be NaþeKþ ATPase inactivation. Another reason for the cerebrolysin beneficial effect could be its antioxidant properties, which may permit the remodelling of membrane composition. This phenomenon requires further investigation. 5. Conclusions Cerebrolysin showed a protective effect due morphine decreased total ATPase and NaþeKþ ATPase activity, probably
Fig. 3. NaþeKþ ATPase activity in moderate hypoglycaemic rat brain treated with cerebrolysin and morphine sulphate. Mean S.D. *p < 0.05, Ctrl ¼ control, Cer ¼ cerebrolysin, Mor ¼ morphine, Cer þ Mor ¼ cerebrolysin þ morphine.
Fig. 5. 5-HIAA levels in moderate hypoglycaemic rat brain treated with cerebrolysin and morphine sulphate. Mean S.D. *p < 0.05, Ctrl ¼ control, Cer ¼ cerebrolysin, Mor ¼ morphine, Cer þ Mor ¼ cerebrolysin þ morphine.
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to its interaction with morphine’s opioid receptors [40]. The levels of 5-HIAA decreased in animals under starvation conditions, treated with both morphine and cerebrolysin, which maybe due to the presence of NO, since this compound has been implicated in meal intake regulation and in the central serotoninergic system [41]. On the other hand, studies conducted by Enrico et al. [42] suggest that morphine increases serotoninergic metabolism, since its by-products lower the risk of brain oxidative damage. Present results suggest that cerebrolysin as well as morphine induce changes in cell regulation and in the brain’s biochemical response to oxidative stress caused by moderate hypoglycaemia. References [1] Lundorff L, Peuckmann V, Sjogren P. Pain management of opioid-treated cancer patients in hospital settings in Denmark. Acta Anaesthesiol Scand 2008;52:137e42. [2] Giovannelli M, Bedforth N, Aitkenhead A. Survey of intrathecal opioid usage in the UK. Eur J Anaesthesiol 2008;25:118e22. [3] Ossipov MH, Lai J, King T, Vanderah TW, Porreca F. Underlying mechanisms of pronociceptive consequences of prolonged morphine exposure. Biopolymers 2005;80:319e24. [4] Puppala BL, Matwyshyn G, Bhalla S, Gulati A. Evidence that morphine tolerance may be regulated by endothelin in the neonatal rat. Biol Neonate 2004;86:138e44. [5] Encuesta Nacional de Adicciones. SSA, CONADIC, INPRFM, DGE, INEGI; 2002. [6] Nishikawa K, Tanobe K, Hinohara H, Okamoto T, Saito S, Goto F. Molecular mechanism of morphine tolerance and biological approaches to resolve tolerance. Masui 2004;53:502e7. [7] Caldero´n GD, Espitia VI, Osnaya BN, Garcı´a AR, Barraga´n MG, Herna´ndez GE, et al. Assessment of oxidative damage induced by acute doses of morphine sulfate in postnatal and adult rat brain. Neurochem Res 2006;31:549e54. [8] Rokyta R, Holecek V, Pekarkova I, Krejcova J, Racek J, Trefil L, et al. Free radicals after painful stimulation are influenced by antioxidants and analgesics. Neuro Endocrinol Lett 2003;24:304e9. [9] Driver AS, Kodavanti PR, Mundy WR. Age-related changes in reactive oxygen species production in rat brain homogenates. Neurotoxicol Teratol 2000;22:175e81. [10] Benuck M, Banay-Schwartz M, DeGuzman T, Lajtha A. Effect of food deprivation on glutathione and amino acid levels in brain and liver of young and aged rats. Brain Res 1995;678:259e64. [11] Parris MK. Glutathione: systemic protectant against oxidative and free radical damage. Altern Med Rev 1997;2:155e76. [12] Ladurner G, Kalvach P, Moessler HCerebrolisyn Study Group. Neuroprotective treatment with cerebrolysin in patients with acute stroke: a randomized controlled trial. J Neural Transm 2005;112:415e28. [13] Toth J, Lajtha A. Drug-induced changes in the composition of the cerebral free amino acid pool. Neurochem Res 1981;6:3e12. [14] Hartbauer M, Hutter-Paier B, Skofitsch G. Antiapoptotic effects of the peptidergic drug cerebrolysin on primary cultures of embryonic chick cortical neurons. J Neural Transm 2001;108:459e73. [15] Patockova´ J, Krsiak M, Marhol P, Tumova´ E. Cerebrolysin inhibits lipid peroxidation induced by insulin hypoglycemia in the brain and heart of mice. Physiol Res 2003;52:455e60. [16] Cavaliere F, D‘Ambrosi N, Sancesario G, Bernardi G, Volonte C. Hypoglycaemia-induced cell death: features of neuroprotection by the P2 receptor antagonist basilen blue. Neurochem Int 2001;38:199e207. [17] Simon GS, Dewey WL. Narcotics and diabetes. I. The effects of streptozotocin-induced diabetes on the antinociceptive potency of morphine. J Pharmacol Exp Ther 1981;218:318e23. [18] Philipp E, Pirke KM. Effect of starvation on hypothalamic tyrosine hydroxylase activity in adult male rats. Brain Res 1987;413:53e9.
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www.sciencedirect.com Biomedicine & Pharmacotherapy 63 (2009) 517e521
Original article
Cerebrolysin and morphine decrease glutathione and 5-hydroxyindole acetic acid levels in fasted rat brain c David Caldero´n Guzma´n a, Norma Osnaya Brizuela b, Raquel Garcı´a Alvarez , c a c,d,* ´ ´ ´ ´ ´ ´ Ernestina Hernandez Garcıa , Gerardo Barragan Mejıa , Hugo Juarez Olguın a
Laboratorio de Neuroquı´mica, Instituto Nacional de Pediatrı´a (INP), Mexico b Laboratorio de Patologı´a, Instituto Nacional de Pediatrı´a (INP), Mexico c Laboratorio de Farmacologı´a, Instituto Nacional de Pediatrı´a (INP), Mexico d Departamento de Farmacologı´a, Facultad de Medicina, Universidad Nacional Auto´noma de Mexico, Mexico Received 27 January 2008; accepted 26 September 2008 Available online 23 October 2008
Abstract Purpose: The aim was to evaluate if morphine sulphate combined with cerebrolysin enhances the risk of oxidative damage in the presence of moderate hypoglycaemia. Methods: Wistar rats under starvation for 48 h received a single dose of 215 mg/kg cerebrolysin or 4 mg/kg morphine sulphate. Glutathione (GSH) and 5-hydroxyindoleacetic acid (5-HIAA) levels were measured in brain tissue, as well as lipid peroxidation, NaþeKþ ATPase and total ATPase enzymatic activities, by fluorescence and spectrophotometric methods. Results: GSH and 5-HIAA levels decreased significantly ( p < 0.05) in animals which received cerebrolysin and morphine alone or combined. TBARS levels increased in all groups, but the values were statistically significant only in those animals that received cerebrolysin combined with morphine ( p < 0.05). NaþeKþ ATPase and total ATPase activities decreased significantly in rats treated only with morphine, but the cerebrolysin and morphine groups showed a significant increase in these enzymatic activities. Conclusions: Results suggest that cerebrolysin as well as morphine induced changes in cellular regulation and biochemical responses to oxidative stress induced by moderate hypoglycaemia in brain. Ó 2008 Elsevier Masson SAS. All rights reserved. Keywords: Morphine; Cerebrolysin; Starved; 5-HIAA; Glutathione; Brain; Neurology; Malnutrition; Drug abuse; Nutrients
1. Background Opioid analgesics, mainly represented by morphine, are the most potent pain-relieving drugs currently available to treat severe and chronic postoperative pain and cancer pain management [1]. While low-dose spinal opioid administration is safe [2], the long-term use of these drugs is associated with tolerance, related to cellular adaptability [3], so that increasing dosages are required to maintain their analgesic effect along * Corresponding author. Laboratorio de Farmacologı´a, Instituto Nacional de Pediatrı´a. Avenida Ima´n No 1, 3rd piso Colonia Cuicuilco, CP 04530, Mexico City, Mexico. Tel./fax: þ5255 1084 3883. E-mail address: [email protected] (H. Jua´rez Olguı´n). 0753-3322/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biopha.2008.09.013
the period of treatment. Tolerance represents one of the major complications to be overcome in the clinical management of these drugs due to untreated side effects [4], and abuse among young and adult people in urban zones with recreational purposes, according to the National Questionnaire of Addictions in Mexico [5]. The mechanisms by which opioids induce tolerance are not completely understood [6], but their abuse has been correlated with central nervous system’s oxidative stress and free radical production [7]. Administration of antioxidants may be employed in pain treatment to decrease the doses of analgesics and to prevent the negative impact of reactive oxygen species on nociception [8]. One of the main naturally occurring free-radical scavengers capable of protecting the body from oxidative stress
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is the tripeptide known as reduced glutathione, or GSH, which is also an ubiquitous reducing agent whose absence induces severe oxidative stress (OS) [9]. It has been suggested that GSH interacts with nitric oxide (NO) producing S-nitrosoglutathione (GNSO) in the presence of endogenously produced oxygen [10], as part of normal NO catabolism, in which GSH is necessary to facilitate its diffusion. GSH is the main redox equilibrium regulator and it plays an important role in the protection of tissues that suffer damage by oxidative agents [11]. Recent studies indicate that the use of neuroprotective agents induces defensive mechanisms in the brain by diminishing the extent of the oxygen-derived free radical-induced lipid peroxidation [12], and some drugs, such as morphine, provoke changes in amino acid levels in the brain, which may participate in the pharmacological action of a number of compounds [13]. Cerebrolysin is a drug consisting of small, porcine brainderived peptides, similar or identical to those endogenously produced; it is capable of crossing the bloodebrain barrier partially, because the drug contains 25% of low molecular weight peptides [14], and has been successfully used in animal models with induced hypoglycaemia, in which it has proved to decrease oxidative stress substantially [15]. Hypoglycaemia reflects a pathophysiological situation. It is a condition that arises from diverse metabolic impairments, which may promote cell death [16], and it may also alter the body’s response to the effect of morphine [17], although its mode of action remains unknown. The central nervous system is particularly susceptible to oxidative damage [18] causing age-related brain dysfunction and neurodegenerative disorders [9], and the membrane lipids are the main target [19]. Among individuals who use large amounts of opioids lipid peroxidation occurs due to the imbalance between oxidation by free radicals and antioxidant activity by free radical scavengers [20]. Membrane lipids are mainly represented by phospholipids, and they are known to strongly interact with the lipid bilayer structural proteins [21], such as the NaþeKþ ATPase, which is responsible of ion interchange across the membrane necessary for neuronal excitability [22]. The inhibition of the NaþeKþ ATPase promotes the release of excitatory amino acids in the CNS, such as serotonin and its main metabolite 5-HIAA, through a mechanism similar to that exerted by brain ischemia. This effect is also induced by morphine, since it alters serotonin brain metabolism [23]. Based on the above information, the present study was conducted to evaluate a possible protective effect of cerebrolysin and morphine on the brain of female rats, by determining the levels of TBARS, GSH, 5-HIAA, as well as total and NaþeKþ ATPases. 2. Methods Forty female Wistar rats, weighing 250 g, were divided into four groups of 10 animals each, according to the administered drug as follows: group I, control (NaCl 0.9%); group II, cerebrolysin; group III, morphine sulphate, and group IV,
cerebrolysin þ morphine sulphate. A single intraperitoneal (i.p.) dose, previously evaluated in our lab, of cerebrolysin (215 mg/kg) and morphine sulphate (4 mg/kg) was each administered to the rats which had a previous 48-h starvation period. An hour after the medication, the animals were decapitated, the brain was immediately placed in saline (NaCl 0.9%) at 4 C, and blood glucose concentration was determined at the same time. A sagittal section of the brain was made; the left portion was homogenized in 5 volumes of 0.05 M TriseHCl, pH 7.4, and used to determine lipid peroxidation and NaþeKþ ATPase activity. The right portion was homogenized in 5 volumes of 0.1 M perchloric acid (HClO4) and GSH and 5HIAA concentrations were determined. The homogenized tissue was kept at 20 C until assayed. The animals were kept in closed boxes throughout the study, 48 h, with a photoperiod of 12/12 h light/darkness, under starvation, and drinking water ad libitum. All experimental procedure were done with authorization from the Laboratory Animals Use and Care Committee of our institution. 2.1. Blood glucose concentration In order to evaluate the hypoglycaemic status in the experimental groups, blood glucose concentration was determined before and after the 48-h starvation period. Twenty microliters of circulating and anticoagulant-free blood was obtained from rats by cutting a 2 mm tail slice, and then introducing a reactive strip (Accu-Chek active, Roche Mannheim, Germany) that detects blood glucose directly in mg/ dL. 2.2. Lipid peroxidation (TBARS) Oxidative damage to membrane lipids was determined by the modified assay of Gutteridge and Halliwell [24], as follows: 2 mL of a 250 mL solution containing thiobarbituric acid (1.25 g), trichloroacetic acid (40 g) and 6.25 mL HCl was added to 1 mL of homogenized rat tissue in 5 mL phosphate buffer, pH 7.4. This sample was then heated to boiling for 30 min (Thermomix 1420), placed on ice for 5 min, and then centrifuged at 3000 g for 15 min (Sorvall RC-5B Dupont). Supernatant absorbance was read spectrophotometrically (Helios-a, UNICAM) at 532 nm and the concentration of thiobarbituric acid reactive substances (TBARS) was expressed as mM of malondialdehyde (MDA) per gram of wet tissue. 2.3. NaþeKþ ATPase and total ATPase activities This parameter was analyzed based on the method reported by Caldero´n-Guzma´n et al. [25]. One hundred microliters of 10% homogenates of brain in 0.05 M Trise HCl, pH 7.4, was incubated for 15 min in a solution containing 3 mM MgCl2, 7 mM KCl, 100 mM NaCl and added or not with 0.06 mM ouabain. Afterwards, 4 mM TriseATP was added and the mixture was incubated for another
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30 min at 37 C in a shaking water bath (Dubnoff Labconco) and the reaction was stopped with 100 mL of 5% trichloroacetic acid. Samples were centrifuged at 3500 g for 5 min at 4 C and inorganic phosphate (Pi) was determined in an aliquot from the supernatant by the Fiske and Subbarrow method [26]. Absorbances from the oubain-containing and oubain-free solutions were determined spectrophotometrically at 660 nm (Helios-a, UNICAM) and the difference was taken as the Naþ/Kþ ATPase activity, expressed as mM Pi/g wet tissue/ min. Total ATPase activity was measured without using ouabain. 2.4. Reduced glutathione (GSH) concentration Perchloric acid homogenized tissue was centrifuged at 8000 g for 5 min (microcentrifuge Mikro 12-42, Germany) and GSH levels were measured from the supernatant as described by Hissin and Hif [27]. To 1.8 mL of phosphate buffer (0.2% EDTA), pH 8.0, an aliquot of perchloric acid homogenate and 100 mL of o-phthaldialdehyde (1 mg/mL, m/v in methanol) were added. This reaction mixture was kept from the light, incubated at room temperature for 15 min, and analyzed in a Perkin Elmer LS 55 spectrofluorometer, 350 nm/ 420 nm excitation/emission using FL Win Lab software, version 4.00.02. Results are reported according to a previously constructed standard curve. 2.5. Levels of 5-hydroxyindole acetic acid (5-HIAA) The level of 5-HIAA was measured in the perchloric acid homogenate, as described for GSH, following the method proposed by Beck et al. [28]. A vial with 1.9 mL of 0.01 M acetate buffer, pH 5.5, containing a tissue homogenate aliquot, was incubated at room temperature for 5 min, keeping it away from light. Samples were analyzed in a spectrofluorometer with excitation/emission of 296 nm/333 nm, as previously described.
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Table 1 Blood levels of glucose in rats before and after 48 h under starvation period. Control
Cerebrolysin
Morphine
Cerebrolysin þ morphine
74.83 6.0
87.29 12.1
98.14 9.1
101.86 11.9
Average values are expressed as mg/dL S.D. Control (NaCl 0.9%), dose given: cerebrolysin 215 mg/kg, morphine 4 mg/kg. Blood levels of glucose in rats before starvation 114.83 4.4 mg/dL.
Glucose concentration increased by 15e30% in animals with combined treatment or with cerebrolysin or morphine alone, compared to the control starvation group. Fig. 1 shows the levels of lipid peroxidation products in the brain of hypoglycaemic rats treated with cerebrolysin and morphine sulphate. All of the experimental groups showed slightly higher TBARS levels than the control group, regardless of the treatment received. TBARS levels in the combined cerebrolysin- and morphine-treated group showed a significant increase (ANOVA; p ¼ 0.0372). GSH levels also showed a statistically significant decrease (ANOVA; p ¼ 0.0001), regardless of treatment type (Fig. 2). As shown in Fig. 3, the animals treated with cerebrolysin or morphine alone had lower NaþeKþ ATPase activity than the control group, but these values were statistically significant only in morphine-treated rats (ANOVA; p ¼ 0.0009). The cerebrolysin/ morphine combination induced NaþeKþ ATPase activity slightly higher than the control and significantly different from the morphine-treated group. Total ATPase activity showed a similar behaviour as the NaþeKþ ATPase among experimental groups as displayed in Fig. 4. 5-HIAA levels also decreased significantly in all experimental groups (ANOVA; p ¼ 0.0001) (Fig. 5). 4. Discussion The central nervous system (CNS) is extremely sensitive to hypoglycaemic damage, because of the properties of the hematoencephalic barrier and because of the lack of other substrates in the CNS. In the present study, moderate hypoglycaemic condition was shown by the low blood glucose levels [30], ranging from 4 to 5.5 mmol/L in all fasted rats,
2.6. Statistical analysis Data were analyzed by ANOVA and the contrasts by the Dunnett’s test, after proving the variance homogeneity. Values were considered statistically significant to a p < 0.05 level [29], using the Statistical Discovery Software (for academic use only) JMP IN 5.1 version 2003. 3. Results Table 1 shows blood glucose levels before and after starvation, and in cerebrolysin- and/or morphine-treated Wistar rats. As shown in Table 1, blood glucose levels decreased significantly in fasted animals, with respect to non-starved animals.
Fig. 1. Lipid peroxidation levels in moderate hypoglycaemic rat brain treated with cerebrolysin and morphine sulphate. Mean S.D. *p < 0.05, Ctrl ¼ control, Cer ¼ cerebrolysin, Mor ¼ morphine, Cer þ Mor ¼ cerebrolysin þ morphine.
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Fig. 2. Glutathione levels in moderate hypoglycaemic rat brain treated with cerebrolysin and morphine sulphate. Mean S.D. *p < 0.05, Ctrl ¼ control, Cer ¼ cerebrolysin, Mor ¼ morphine, Cer þ Mor ¼ cerebrolysin þ morphine.
compared to 6.3 mmol/L in non-fasted rats [31]. These results suggest that starvation caused changes in intracellular ion availability, such as Naþ, in fasted rat groups. As expected, lipid peroxidation products increased in morphine-treated animals, probably due to free radical production induced by this drug in brain tissue [32], which is in agreement with the fact that brain neurons synthesize nitric oxide (NO) in response to exposure to morphine, leading to oxidative damage [33]; the lack of food did not change this effect [34]. Cerebrolysin did not influence the morphine effect, since the group treated with both drugs showed an increase in TBARS as well. This effect suggests that cerebrolysin failed to have a GSH-like antioxidant effect. With respect to the first proposal, morphine-treated animals showed decreased GSH levels and, although not statistically significant, this effect was less evident in the presence of cerebrolysin, which suggests that the small cerebrolysin peptides may interact with NO metabolism, as has been shown for GSH and, therefore, a protective effect. These results strongly suggest that morphine induces oxidative brain damage by decreasing GSH, as previously stated by Goudas et al. [35], who reported a decline in GSH and enhanced susceptibility to oxidative stress in morphine-treated cancer patients.
Fig. 4. Total ATPase in moderate hypoglycaemic rat brain treated with cerebrolysin and morphine sulphate. Mean S.D. *p < 0.05, Ctrl ¼ control, Cer ¼ cerebrolysin, Mor ¼ morphine, Cer þ Mor ¼ cerebrolysin þ morphine.
Further studies should be conducted in order to rule out that morphine induces lipid peroxidation in an NO-independent manner, and to find the other drugs that interfere with the NO production induced by morphine and improve its clinical use, as was suggested by Pryor et al. [36]. Changes in intracellular Naþ availability or membrane depolarization provoked by morphine-induced tolerance, may be the cause of decreased NaþeKþ ATPase activity [37] in the morphinetreated group and cerebrolysin almost completely abolished the morphine effect by inducing the recovery of enzymatic activity. A possible mechanism of this enzyme activity recovery may be that cerebrolysin alters the intracellular sodium content, increasing the competence of the enzyme for sodium, and thus provoking a different effect than that of morphine. This suggests that the NaþeKþ ATPase activity may play an important role in the analgesic effect [38,39]. This result has clinically important implications, since the analgesic effect of morphine seems to be NaþeKþ ATPase inactivation. Another reason for the cerebrolysin beneficial effect could be its antioxidant properties, which may permit the remodelling of membrane composition. This phenomenon requires further investigation. 5. Conclusions Cerebrolysin showed a protective effect due morphine decreased total ATPase and NaþeKþ ATPase activity, probably
Fig. 3. NaþeKþ ATPase activity in moderate hypoglycaemic rat brain treated with cerebrolysin and morphine sulphate. Mean S.D. *p < 0.05, Ctrl ¼ control, Cer ¼ cerebrolysin, Mor ¼ morphine, Cer þ Mor ¼ cerebrolysin þ morphine.
Fig. 5. 5-HIAA levels in moderate hypoglycaemic rat brain treated with cerebrolysin and morphine sulphate. Mean S.D. *p < 0.05, Ctrl ¼ control, Cer ¼ cerebrolysin, Mor ¼ morphine, Cer þ Mor ¼ cerebrolysin þ morphine.
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