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orphanin FQ and nocistatin concentrations in rat chronic constriction nerve injury and diabetic neuropathic pain models
Neuroscience Letters 506 (2012) 104–106
Contents lists available at SciVerse ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Elevated prepronociceptin, nociceptin/orphanin FQ and nocistatin concentrations in rat chronic constriction nerve injury and diabetic neuropathic pain models Eugene H. Liu a,∗, Chunmei Li a, Malathi Govindasamy a, Hong Jye Neo a, Tat Leang Lee a, Chian Ming Low a,b , Shinro Tachibana a a b
Department of Anaesthesia, Yong Loo Lin School of Medicine, National University Health System, Singapore Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
a r t i c l e
i n f o
Article history: Received 19 September 2011 Received in revised form 22 October 2011 Accepted 24 October 2011 Keywords: Nociceptin/orphanin FQ Nocistatin Prepronociceptin Chronic constriction nerve injury Diabetic neuropathic pain
a b s t r a c t Nociceptin/orphanin FQ (N/OFQ) and nocistatin are derived from the same precursor peptide, prepronociceptin. N/OFQ and nocistatin have been postulated to participate in pain modulation. In this study, we investigated whether the prepronociceptin, N/OFQ and nocistatin concentrations in the brain and spinal cord would be altered in chronic constriction injury and diabetic rat neuropathic pain models. Total brain and spinal cord lysates as well as serum from rats that had undergone chronic constriction injury and streptozocin-induced diabetic neuropathy were used to determine the concentrations of three peptides using competitive radioimmunoassay. We found that N/OFQ and prepronociceptin concentrations were significantly raised in both rat neuropathic pain models. Nocistatin was raised in the brains of post traumatic neuropathy pain rats. Overall, our data have demonstrated for the first time that prepronociceptin, N/OFQ and nocistatin concentrations are significantly altered at different tissues of two rat neuropathy pain models. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Nociceptin (or nociceptin/orphanin FQ, N/OFQ) and nocistatin are derived from the same precursor peptide, termed prepronociceptin. N/OFQ and nocistatin have been postulated to have roles in pain modulation. N/OFQ was isolated and identified in 1995 as the endogenous ligand for the orphan opioid-like receptor (ORL1), a G protein coupled receptor [10,13]. Unlike other opioid ligand receptor systems, the administration of N/OFQ led to hyperalgesia and allodynia in some animal models. Nocistatin antagonizes the allodynia effect of N/OFQ, and can independently attenuate pain induced by prostaglandin E2 and can also attenuate anxiolysis induced by diazepam [6,12]. Recent work suggests that nocistatin has binding sites in the central nervous system and has agonist activity that is independent of its activity on the ORL1 receptor. Nocistatin’s endogenous receptor has been proposed to also be a G protein coupled receptor [3].
Although N/OFQ and nocistatin have several biological effects and may have opposing roles in nociception, their physiological roles remain unclear. Furthermore, the analgesic and allodynic effects by N/OFQ may vary depending on the animal used, route of administration or dose of peptide used [4]. N/OFQ and nocistatin may have biphasic effects that depend on the site of nociception – peripheral/spinal antinociceptive versus supraspinal pronociceptive effects [5,14,15]. Spinal administration of low dose nocistatin may also inhibit glycine-dependent NMDA receptor activation [11]. In this study, we studied the brain, spinal cord and serum levels of prepronociceptin, N/OFQ and nocistatin in two different rat models of neuropathic pain: pain in the chronic constriction nerve injury model, and the diabetic neuropathic pain model. 2. Materials and methods The animal work was approved by the Institutional Animal Care and Use Committee of the National University of Singapore. Ten-week-old Sprague-Dawley rats were kept in controlled conditions, two per cage at 24 ◦ C, with a 12 h light–dark cycle. Food and water were available ad libitum. 2.1. Neuropathy pain models
Abbreviations: N/OFQ, nociceptin/orphanin FQ; ORL1, orphan opioid-like receptor; NMDA, N-methyl-d-aspartate; CCI, chronic constriction nerve injury; RIA, radioimmunoassay. ∗ Corresponding author at: Department of Anaesthesia, National University Health System, 5 Lower Kent Ridge Road, Singapore 119074, Singapore. Tel.: +65 6772 4207; fax: +65 6777 5702. E-mail addresses: Eugene Liu [email protected], [email protected] (E.H. Liu). 0304-3940/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2011.10.059
The post traumatic neuropathic pain model was generated using the chronic constriction nerve injury (CCI) method as reported previously [1]. The diabetic neuropathic pain rat model was produced by i.p. injection streptozocin (60 mg/kg) [2]. Diabetes was confirmed at 7 days and 12 days post injection (glucose >12 mmol/L). Only rats with confirmed diabetes were then tested for mechanical allodynia on Day 14 using an automated von Frey’s anesthesiometer (Ugo Basile, Comerio, Italy).
E.H. Liu et al. / Neuroscience Letters 506 (2012) 104–106 Table 1 Significant elevations of prepronociceptin, N/OFQ and nocistatin concentrations in brain, spinal cord and serum of chronic constriction nerve injury (CCI) and diabetic neuropathic pain rats determined using competitive radioimmunoassay. Control (n = 8) Brain tissue Nociceptin (N/OFQ) Nocistatin Prepronociceptin Spinal cord Nociceptin (N/OFQ) Nocistatin Prepronociceptin Serum Nociceptin (N/OFQ) Nocistatin Prepronociceptin
CCI neuropathy (n = 8)
Diabetic neuropathy (n = 8)
151.8 (75.9) 19.6 (2.5) 202.9 (94.0)
842.3 (239.3)* 34.6 (9.2)* 252.9 (44.6)
540.1 (294.6)* 25.4 (4.3) 440.6 (207.7)*
36.3 (16.0) 25.6 (4.3) 70.3 (42.9)
167.9 (130.4)* 44.7 (42.9) 214.0 (189.9)*
169.5 (99.5)* 30.3 (6.0) 237.4 (125.7)*
1408.0 (512.8)* 53.4 (16.3) 763.9 (425.0)
1631.0 (531.4)* 47.9 (14.1) 667.9 (212.1)
829.8 (512.0) 37.3 (7.1) 490.1 (305.8)
Data are in fmol/mg protein for brain extract and spinal cord extract, and in fmol/ml for serum. * Indicates P < 0.05 (2-way ANOVA with post hoc Bonferroni tests compared between groups).
Briefly, all the rats were familiarized to the anesthesiometer for 30 min prior to starting the experiments. On the day of measurement, the rat was habituated to the anesthesiometer for 20 min. Baseline threshold for paw withdrawal in both feet prior to induction of the neuropathy and in the control rats were determined. A wire filament of the anesthesiometer was applied with gradated force to the mid plantar region of the feet (for 4 s) and we examined for foot withdrawal. Each foot was tested ten times at each force setting, beginning with 2.5 g and progressing in 2.5 g increments to a maximum of 35 g force. Threshold force was noted when the rat withdrew its foot during at least six out of ten applications of the filament. The baseline force threshold of control rats ranged between 25 and 30 g. For CCI rats to have developed post traumatic neuropathic pain, these animals displayed allodynia by a difference of ≥7.5 g in the threshold between the left foot (the side of sciatic nerve ligation) and the right foot. For diabetic neuropathic rats, these animals showed allodynia by a reduction of ≥7.5 g in the threshold between baseline and after induction of diabetes, in the left foot. Allodynia was tested at Day 14 and confirmed at Day 20. Untreated rats were used as control. Cerebral cortices, spinal cord (thoracic-to-lumbar) and serum (from right ventricle) were harvested from CCI neuropathic, diabetic neuropathic and control rats on Day 22 as described previously [8]. Briefly, tissues were homogenized in 0.1 M acetic acid, and the homogenates were kept at room temperature for 2 h with intermittent mixing. The supernatant was mixed with XAD-16, followed by washing with 1% acetonitrile containing trifluroacetic acid 0.1% three times. The bound proteins were eluted with 60% acetonitrile containing trifluroacetic acid 0.1% for subsequent competitive radioimmunoassay (RIA). Antisera against prepronociceptin and nocistatin that were previously raised in our laboratory and anti-N/OFQ (Peptide Institute Inc., Osaka, Japan) were mixed with each tissue samples for 48 h at 4 ◦ C. 125 I labelled synthetic peptide (all three unlabelled peptides were purchased from Peptide Institute Inc., Osaka, Japan) was subsequently incubated with the mixture for 24 h. The assay was stopped by adding 100 l of secondary antibody and 30% (w/v) PEG and incubated for another 2 h as reported previously [8].
3. Results In the brain, significantly higher levels of N/OFQ were observed in CCI and diabetic neuropathic rats than the control rats (Table 1). Nocistatin level was higher in CCI rats (P < 0.001) while prepronociceptin level was significantly higher in diabetic neuropathic rats (P = 0.013). In the spinal cord, N/OFQ and prepronociceptin levels were higher in CCI and diabetic neuropathic rats (P = 0.015 and 0.043, respectively) while there were no differences in nocistatin levels between neuropathic pain and control rats. In tests of serum, only N/OFQ levels were significantly higher in both neuropathic animal models (P = 0.016) compared to control.
105
normal rats. Nocistatin antagonizes the roles of N/OFQ on nociception/antinociception at the cellular and behavioural levels. Nocistatin has been previously demonstrated to antagonize N/OFQ induced hyperalgesic effect in mice using hotplate response time measurements [9]. Hence, we suggest that high N/OFQ levels may reflect both spinal and supraspinal effects on nociception. Our team had earlier shown greater increases in prepronociceptin, N/OFQ and nocistatin concentrations in a more severe neuropathic pain rat model – partial sciatic nerve ligation [8]. In this study, we detected significantly elevation of prepronociceptin concentrations in the spinal cord of both neuropathic pain models, with very high levels in the brains of diabetic neuropathic pain rats. Nocistatin concentration was only higher in the brain of chronic constriction injury neuropathic rats. As N/OFQ and nocistatin are derived from the same precursor prepronociceptin, one possible explanation for these changes in the tissues is that they could arise from corresponding increase in prepronociceptin levels and/or increased proteolytic cleavage of existing prepronociceptin to release these shorter peptides. However, the pharmacokinetic profiles (rate of metabolism and elimination) of each of these peptides are to-date unclear. The respective concentrations of the proteolyzed peptides may not intuitively linearly reflect prepronociceptin levels due to potentially varied pharmacokinetic parameters of each peptide. Despite the above, it is noteworthy that there is not only a general trend of higher concentrations of prepronociceptin, N/OFQ and nocistatin individually, but more importantly, the total peptide concentrations (i.e. summation of prepronocicpetin, N/OFQ and nocistatin) in the brain, spinal cord and serum were much higher in both neuropathic pain models when compared to control (Table 1). Our data cannot suggest that there may be some differences in the expression of the peptides between the two types of neuropathic pain and at different tissues. A limitation is that behavioral test responses in animals may not relate to perception of pain in human patients. It is also difficult to gradate the extent of neuropathic pain in rats. Although it is not possible to relate the severity of pain in the rat to the extent of change in peptide concentrations, our present data demonstrated chronic constriction injury and diabetic pain elicit specific significant increase in the levels of prepronociceptin, N/OFQ and nocistatin. Future work could correlate the level of peptides in human samples with the patients’ perception of chronic pain, and could be used to evaluate the success of chronic pain treatment. N/OFQ and its receptor may represent new therapeutic targets, with the potential advantage that modulating the N/OFQ system may mediate analgesia without affecting the potency of inhalational anaesthetics [7]. In conclusion, we found that N/OFQ concentrations are raised in the brain, spinal cord and serum of chronic constriction injury and diabetic neuropathy rats as compared to control rats.
Conflict of interests All authors declare no actual or potential conflict of interest including any financial, personal or other relationship with other people or organizations within three years of beginning the submitted work that could inappropriately influence, or be perceived to influence, this work.
4. Discussion
Contributors
It has been reported that both intracerebroventricular and intrathecal administration of N/OFQ may produce pain. For example, N/OFQ modulates allodynia in rats with neuropathic pain but does not alter mechanical allodynia thresholds in
EHL, CL, MG and HJN carried out the laboratory experiments and analyzed the data. EHL, TLL and CML contributed to writing this manuscript. ST conceived and managed the study. All authors approved the final version of the manuscript.
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Acknowledgement This work was supported by a grant from the National Medical Research Council in Singapore. References [1] D. Bridges, S.W. Thompson, A.S. Rice, Mechanisms of neuropathic pain, Br. J. Anaesth. 87 (2001) 12–26. [2] C. Courteix, A. Eschalier, J. Lavarenne, Streptozocin-induced diabetic rats: behavioural evidence for a model of chronic pain, Pain 53 (1993) 81–88. [3] M. Fantin, C. Fischetti, C. Trapella, M. Morari, Nocistatin inhibits 5hydroxytryptamine release in the mouse neocortex via presynaptic Gi/o protein linked pathways, Br. J. Pharmacol. 152 (2007) 549–555. [4] B. Fioravanti, T.W. Vanderah, The ORL-1 receptor system: are there opportunities for antagonists in pain therapy? Curr. Top. Med. Chem. 8 (2008) 1442–1451. [5] X. Fu, Y.Q. Wang, G.C. Wu, Involvement of nociceptin/orphanin FQ and its receptor in electroacupuncture-produced anti-hyperalgesia in rats with peripheral inflammation, Brain Res. 1078 (2006) 212–218. [6] E.C. Gavioli, F.S. Duarte, R. Guerrini, G. Calo, G.A. Rae, M.D. Lima TC, GABA(A) signalling is involved in N/OFQ anxiolytic-like effects but not in nocistatin anxiogenic-like action as evaluated in the mouse elevated plus maze, Peptides 29 (2008) 1404–1412. [7] S. Himukashi, Y. Miyazaki, H. Takeshima, S. Koyanagi, K. Mukaida, T. Shichino, H. Uga, K. Fukuda, Nociceptin system does not affect MAC of volatile anaesthetics, Acta Anaesthesiol. Scand. 49 (2005) 771–773.
[8] T. Joseph, T.L. Lee, C. Li, C. Siau, Y. Nishiuchi, T. Kimura, S. Tachibana, Levels of neuropeptides nocistatin, nociceptin/orphanin FQ and their precursor protein in a rat neuropathic pain model, Peptides 28 (2007) 1433–1440. [9] E.H. Liu, Y. Nishiuchi, T. Kimura, S. Tachibana, Supraspinal nocistatin and its amide derivative antagonize the hyperalgesic effects of nociceptin in mice, Neurosci. Lett. 397 (2006) 59–63. [10] J.C. Meunier, C. Mollereau, L. Toll, C. Suaudeau, C. Moisand, P. Alvinerie, J.L. Butour, J.C. Guillemot, P. Ferrara, B. Monsarrat, H. Mazarguil, G. Vassart, M. Parmentier, J. Costentin, Isolation and structure of the endogenous agonist of opioid receptor-like Orl(1) receptor, Nature 377 (1995) 532–535. [11] U. Muth-Selbach, E. Dybek, K. Kollosche, J.U. Stegmann, H. Holthusen, P. Lipfert, H.U. Zeilhofer, The spinal antinociceptive effect of nocistatin in neuropathic rats is blocked by d-serine, Anesthesiology 101 (2004) 753–758. [12] E. Okuda-Ashitaka, T. Minami, S. Tachibana, Y. Yoshihara, Y. Nishiuchi, T. Kimura, S. Ito, Nocistatin, a peptide that blocks nociceptin action in pain transmission, Nature 392 (1998) 286–289. [13] R.K. Reinscheid, H.P. Nothacker, A. Bourson, A. Ardati, R.A. Henningsen, J.R. Bunzow, D.K. Grandy, H. Langen, F.J. Monsma, O. Civelli, Orphanin-Fq – a neuropeptide that activates an opioid-like G-protein-coupled receptor, Science 270 (1995) 792–794. [14] A. Rizzi, C. Nazzaro, G.G. Marzola, S. Zucchini, C. Trapella, R. Guerrini, H.U. Zeilhofer, D. Regoli, G. Calo’, Endogenous nociceptin/orphanin FQ signalling produces opposite spinal antinociceptive and supraspinal pronociceptive effects in the mouse formalin test: pharmacological and genetic evidences, Pain 124 (2006) 100–108. [15] M. Sandrini, G. Vitale, L.A. Pini, G. Lopetuso, P. Romualdi, S. Candeletti, Nociceptin/orphanin FQ prevents the antinociceptive action of paracetamol on the rat hot plate test, Eur. J. Pharmacol. 507 (2005) 43–48.
Contents lists available at SciVerse ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Elevated prepronociceptin, nociceptin/orphanin FQ and nocistatin concentrations in rat chronic constriction nerve injury and diabetic neuropathic pain models Eugene H. Liu a,∗, Chunmei Li a, Malathi Govindasamy a, Hong Jye Neo a, Tat Leang Lee a, Chian Ming Low a,b , Shinro Tachibana a a b
Department of Anaesthesia, Yong Loo Lin School of Medicine, National University Health System, Singapore Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
a r t i c l e
i n f o
Article history: Received 19 September 2011 Received in revised form 22 October 2011 Accepted 24 October 2011 Keywords: Nociceptin/orphanin FQ Nocistatin Prepronociceptin Chronic constriction nerve injury Diabetic neuropathic pain
a b s t r a c t Nociceptin/orphanin FQ (N/OFQ) and nocistatin are derived from the same precursor peptide, prepronociceptin. N/OFQ and nocistatin have been postulated to participate in pain modulation. In this study, we investigated whether the prepronociceptin, N/OFQ and nocistatin concentrations in the brain and spinal cord would be altered in chronic constriction injury and diabetic rat neuropathic pain models. Total brain and spinal cord lysates as well as serum from rats that had undergone chronic constriction injury and streptozocin-induced diabetic neuropathy were used to determine the concentrations of three peptides using competitive radioimmunoassay. We found that N/OFQ and prepronociceptin concentrations were significantly raised in both rat neuropathic pain models. Nocistatin was raised in the brains of post traumatic neuropathy pain rats. Overall, our data have demonstrated for the first time that prepronociceptin, N/OFQ and nocistatin concentrations are significantly altered at different tissues of two rat neuropathy pain models. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Nociceptin (or nociceptin/orphanin FQ, N/OFQ) and nocistatin are derived from the same precursor peptide, termed prepronociceptin. N/OFQ and nocistatin have been postulated to have roles in pain modulation. N/OFQ was isolated and identified in 1995 as the endogenous ligand for the orphan opioid-like receptor (ORL1), a G protein coupled receptor [10,13]. Unlike other opioid ligand receptor systems, the administration of N/OFQ led to hyperalgesia and allodynia in some animal models. Nocistatin antagonizes the allodynia effect of N/OFQ, and can independently attenuate pain induced by prostaglandin E2 and can also attenuate anxiolysis induced by diazepam [6,12]. Recent work suggests that nocistatin has binding sites in the central nervous system and has agonist activity that is independent of its activity on the ORL1 receptor. Nocistatin’s endogenous receptor has been proposed to also be a G protein coupled receptor [3].
Although N/OFQ and nocistatin have several biological effects and may have opposing roles in nociception, their physiological roles remain unclear. Furthermore, the analgesic and allodynic effects by N/OFQ may vary depending on the animal used, route of administration or dose of peptide used [4]. N/OFQ and nocistatin may have biphasic effects that depend on the site of nociception – peripheral/spinal antinociceptive versus supraspinal pronociceptive effects [5,14,15]. Spinal administration of low dose nocistatin may also inhibit glycine-dependent NMDA receptor activation [11]. In this study, we studied the brain, spinal cord and serum levels of prepronociceptin, N/OFQ and nocistatin in two different rat models of neuropathic pain: pain in the chronic constriction nerve injury model, and the diabetic neuropathic pain model. 2. Materials and methods The animal work was approved by the Institutional Animal Care and Use Committee of the National University of Singapore. Ten-week-old Sprague-Dawley rats were kept in controlled conditions, two per cage at 24 ◦ C, with a 12 h light–dark cycle. Food and water were available ad libitum. 2.1. Neuropathy pain models
Abbreviations: N/OFQ, nociceptin/orphanin FQ; ORL1, orphan opioid-like receptor; NMDA, N-methyl-d-aspartate; CCI, chronic constriction nerve injury; RIA, radioimmunoassay. ∗ Corresponding author at: Department of Anaesthesia, National University Health System, 5 Lower Kent Ridge Road, Singapore 119074, Singapore. Tel.: +65 6772 4207; fax: +65 6777 5702. E-mail addresses: Eugene Liu [email protected], [email protected] (E.H. Liu). 0304-3940/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2011.10.059
The post traumatic neuropathic pain model was generated using the chronic constriction nerve injury (CCI) method as reported previously [1]. The diabetic neuropathic pain rat model was produced by i.p. injection streptozocin (60 mg/kg) [2]. Diabetes was confirmed at 7 days and 12 days post injection (glucose >12 mmol/L). Only rats with confirmed diabetes were then tested for mechanical allodynia on Day 14 using an automated von Frey’s anesthesiometer (Ugo Basile, Comerio, Italy).
E.H. Liu et al. / Neuroscience Letters 506 (2012) 104–106 Table 1 Significant elevations of prepronociceptin, N/OFQ and nocistatin concentrations in brain, spinal cord and serum of chronic constriction nerve injury (CCI) and diabetic neuropathic pain rats determined using competitive radioimmunoassay. Control (n = 8) Brain tissue Nociceptin (N/OFQ) Nocistatin Prepronociceptin Spinal cord Nociceptin (N/OFQ) Nocistatin Prepronociceptin Serum Nociceptin (N/OFQ) Nocistatin Prepronociceptin
CCI neuropathy (n = 8)
Diabetic neuropathy (n = 8)
151.8 (75.9) 19.6 (2.5) 202.9 (94.0)
842.3 (239.3)* 34.6 (9.2)* 252.9 (44.6)
540.1 (294.6)* 25.4 (4.3) 440.6 (207.7)*
36.3 (16.0) 25.6 (4.3) 70.3 (42.9)
167.9 (130.4)* 44.7 (42.9) 214.0 (189.9)*
169.5 (99.5)* 30.3 (6.0) 237.4 (125.7)*
1408.0 (512.8)* 53.4 (16.3) 763.9 (425.0)
1631.0 (531.4)* 47.9 (14.1) 667.9 (212.1)
829.8 (512.0) 37.3 (7.1) 490.1 (305.8)
Data are in fmol/mg protein for brain extract and spinal cord extract, and in fmol/ml for serum. * Indicates P < 0.05 (2-way ANOVA with post hoc Bonferroni tests compared between groups).
Briefly, all the rats were familiarized to the anesthesiometer for 30 min prior to starting the experiments. On the day of measurement, the rat was habituated to the anesthesiometer for 20 min. Baseline threshold for paw withdrawal in both feet prior to induction of the neuropathy and in the control rats were determined. A wire filament of the anesthesiometer was applied with gradated force to the mid plantar region of the feet (for 4 s) and we examined for foot withdrawal. Each foot was tested ten times at each force setting, beginning with 2.5 g and progressing in 2.5 g increments to a maximum of 35 g force. Threshold force was noted when the rat withdrew its foot during at least six out of ten applications of the filament. The baseline force threshold of control rats ranged between 25 and 30 g. For CCI rats to have developed post traumatic neuropathic pain, these animals displayed allodynia by a difference of ≥7.5 g in the threshold between the left foot (the side of sciatic nerve ligation) and the right foot. For diabetic neuropathic rats, these animals showed allodynia by a reduction of ≥7.5 g in the threshold between baseline and after induction of diabetes, in the left foot. Allodynia was tested at Day 14 and confirmed at Day 20. Untreated rats were used as control. Cerebral cortices, spinal cord (thoracic-to-lumbar) and serum (from right ventricle) were harvested from CCI neuropathic, diabetic neuropathic and control rats on Day 22 as described previously [8]. Briefly, tissues were homogenized in 0.1 M acetic acid, and the homogenates were kept at room temperature for 2 h with intermittent mixing. The supernatant was mixed with XAD-16, followed by washing with 1% acetonitrile containing trifluroacetic acid 0.1% three times. The bound proteins were eluted with 60% acetonitrile containing trifluroacetic acid 0.1% for subsequent competitive radioimmunoassay (RIA). Antisera against prepronociceptin and nocistatin that were previously raised in our laboratory and anti-N/OFQ (Peptide Institute Inc., Osaka, Japan) were mixed with each tissue samples for 48 h at 4 ◦ C. 125 I labelled synthetic peptide (all three unlabelled peptides were purchased from Peptide Institute Inc., Osaka, Japan) was subsequently incubated with the mixture for 24 h. The assay was stopped by adding 100 l of secondary antibody and 30% (w/v) PEG and incubated for another 2 h as reported previously [8].
3. Results In the brain, significantly higher levels of N/OFQ were observed in CCI and diabetic neuropathic rats than the control rats (Table 1). Nocistatin level was higher in CCI rats (P < 0.001) while prepronociceptin level was significantly higher in diabetic neuropathic rats (P = 0.013). In the spinal cord, N/OFQ and prepronociceptin levels were higher in CCI and diabetic neuropathic rats (P = 0.015 and 0.043, respectively) while there were no differences in nocistatin levels between neuropathic pain and control rats. In tests of serum, only N/OFQ levels were significantly higher in both neuropathic animal models (P = 0.016) compared to control.
105
normal rats. Nocistatin antagonizes the roles of N/OFQ on nociception/antinociception at the cellular and behavioural levels. Nocistatin has been previously demonstrated to antagonize N/OFQ induced hyperalgesic effect in mice using hotplate response time measurements [9]. Hence, we suggest that high N/OFQ levels may reflect both spinal and supraspinal effects on nociception. Our team had earlier shown greater increases in prepronociceptin, N/OFQ and nocistatin concentrations in a more severe neuropathic pain rat model – partial sciatic nerve ligation [8]. In this study, we detected significantly elevation of prepronociceptin concentrations in the spinal cord of both neuropathic pain models, with very high levels in the brains of diabetic neuropathic pain rats. Nocistatin concentration was only higher in the brain of chronic constriction injury neuropathic rats. As N/OFQ and nocistatin are derived from the same precursor prepronociceptin, one possible explanation for these changes in the tissues is that they could arise from corresponding increase in prepronociceptin levels and/or increased proteolytic cleavage of existing prepronociceptin to release these shorter peptides. However, the pharmacokinetic profiles (rate of metabolism and elimination) of each of these peptides are to-date unclear. The respective concentrations of the proteolyzed peptides may not intuitively linearly reflect prepronociceptin levels due to potentially varied pharmacokinetic parameters of each peptide. Despite the above, it is noteworthy that there is not only a general trend of higher concentrations of prepronociceptin, N/OFQ and nocistatin individually, but more importantly, the total peptide concentrations (i.e. summation of prepronocicpetin, N/OFQ and nocistatin) in the brain, spinal cord and serum were much higher in both neuropathic pain models when compared to control (Table 1). Our data cannot suggest that there may be some differences in the expression of the peptides between the two types of neuropathic pain and at different tissues. A limitation is that behavioral test responses in animals may not relate to perception of pain in human patients. It is also difficult to gradate the extent of neuropathic pain in rats. Although it is not possible to relate the severity of pain in the rat to the extent of change in peptide concentrations, our present data demonstrated chronic constriction injury and diabetic pain elicit specific significant increase in the levels of prepronociceptin, N/OFQ and nocistatin. Future work could correlate the level of peptides in human samples with the patients’ perception of chronic pain, and could be used to evaluate the success of chronic pain treatment. N/OFQ and its receptor may represent new therapeutic targets, with the potential advantage that modulating the N/OFQ system may mediate analgesia without affecting the potency of inhalational anaesthetics [7]. In conclusion, we found that N/OFQ concentrations are raised in the brain, spinal cord and serum of chronic constriction injury and diabetic neuropathy rats as compared to control rats.
Conflict of interests All authors declare no actual or potential conflict of interest including any financial, personal or other relationship with other people or organizations within three years of beginning the submitted work that could inappropriately influence, or be perceived to influence, this work.
4. Discussion
Contributors
It has been reported that both intracerebroventricular and intrathecal administration of N/OFQ may produce pain. For example, N/OFQ modulates allodynia in rats with neuropathic pain but does not alter mechanical allodynia thresholds in
EHL, CL, MG and HJN carried out the laboratory experiments and analyzed the data. EHL, TLL and CML contributed to writing this manuscript. ST conceived and managed the study. All authors approved the final version of the manuscript.
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