As a consequence of thoracic and thoracoabdominal aortic aneurysm repair surgery, some patients develop paraparesis or paraplegia resulting from spinal cord ischemia (SCI) injury. Hypothermia, cerebro-spinal fluid drainage, N-methyl-d-aspartate receptor antagonists, calcium channel blockers, and free radical scavengers have been used to protect the spinal cord against ischemic injury (1–3). However, because efficacy is limited, pursuit of novel therapies to protect against SCI remains important.
δ-Opioid receptor agonism prolonged the cell survival of various organs in animal models, including the lung, heart, liver, and kidney (4). In the myocardium, stimulation of the δ1-opioid receptor contributes to cardioprotection, similar to ischemic preconditioning (5). Su (4) demonstrated in the brain that treatment with [D-Ala2, D-Leu5]enkephalin (DADLE), a peptidic δ-opioid receptor agonist, attenuated infarct volume after transient middle cerebral artery occlusion. Tsao et al. (6) also reported that DADLE reduced methamphetamine (METH)-induced neuronal damage in the striatum. These findings indicate that δ-opioid agonists may have neuroprotective efficacy in the central nervous system. However, there are no data on the efficacy of δ-opioid receptor agonism on neuronal injury after SCI. It has been reported that the δ-opioid receptor is located in the region of motoneurons in the spinal cord (7). We hypothesized that treatment with a δ-opioid receptor agonist would attenuate neuronal damage after SCI.
SNC80, (+)-4-[(αR)-α((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N, N-diethylbenzamide, is a highly selective, nonpeptidic δ-opioid agonist. Although peptidic opioids may rapidly degrade, nonpeptidic opioids are proteolytically stable and have enhanced bioavailability (8,9). This study was undertaken to investigate the effects of intrathecal administration of the δ-opioid agonist SNC80 on hind-limb motor function and neuronal injury in rats subjected to SCI. To assess the influence of severity of SCI on the neuroprotective efficacy of SNC80, 9 and 11 min of SCI were used as models of mild and moderate to severe ischemia, respectively.
Sprague-Dawley rats (Japan SLC, Inc., Shizuoka, Japan) weighing 350 to 450 g were used in this study. They were housed and maintained on a 12-h light/dark cycle with free access to food and water. The Animal Experiment Committee of Nara Medical University (Nara, Japan) approved this study.
Implantation of an intrathecal catheter was performed as previously reported (10), with modification. In brief, 58 rats were anesthetized in an acrylic box with 5% isoflurane in an air/oxygen mixture. After induction, the anesthesia was maintained with 1.5% isoflurane via a snout cone. The heads were mounted in a stereotaxic frame. Polyethylene tubing (PE-10) was inserted and advanced 8.5 cm caudal to the level of lumbar enlargement of the spinal cord via the atlantooccipital membrane. We performed this implantation carefully by using microscopy. The rats were returned to their cages and housed for 4–7 days. SNC80 (Alexis Japan, Tokyo, Japan) was prepared in its vehicle (20% dimethyl sulfoxide and 100 mM HCl) so that 10 μL of volume contained 400 nmol of SNC80.
SCI was induced as reported by Taira and Marsala (11). Briefly, the rats that had been implanted with intrathecal catheters and showed no neurological deficits were anesthetized again in an acrylic box with 5% isoflurane in an air/oxygen mixture. After induction, the tracheas were intubated and lungs ventilated mechanically with 1.5% isoflurane in an air/oxygen mixture. Rectal temperature was continuously monitored and maintained between 37.2°C and 37.8°C with an under-body heating pad and heat lamps. For monitoring distal arterial blood pressure and collecting blood specimens, a PE-50 catheter was inserted into the tail artery. A 2F Fogarty balloon catheter was inserted via the left femoral artery to the descending thoracic aorta so that the tip of the catheter reached the level of the left subclavian artery (10.8 to 11.4 cm from the site of insertion). A PE-60 catheter was inserted into the left carotid artery and connected to an external blood reservoir positioned at 54 cm above the rat’s body to reduce arterial blood pressure above the occlusion site during aortic occlusion to a target of 40 mm Hg. Immediately after all cannulations, the rats received 200 U of heparin injected into the tail artery. After the above preparation, the rats were randomly allocated to one of the following five groups: 1) vehicle before 9 min of SCI (group V-9; n = 12), 2) SNC80 before 9 min of SCI (group SNC-9; n = 12), 3) vehicle before 11 min of SCI (group V-11; n = 12), 4) SNC80 before 11 min of SCI (group SNC-11; n = 10), or 5) vehicle in sham (sham group; n = 12). SNC80 or vehicle was given 15 min before the induction of SCI followed by 20 μL of saline to flush the catheter. The investigator was blinded to the content of the syringes.
Fifteen minutes after intrathecal administration, the Fogarty balloon catheter was inflated with 0.05 mL of saline to occlude the descending aorta. The efficiency of the occlusion was assessed by an immediate and sustained loss of any detectable pulsation and a decrease in pressure below the level of aortic occlusion. After 9 or 11 min of SCI, the balloon was deflated, and blood was reinfused for 30 s. The catheters were then removed and incisions closed. Protamine sulfate (4 mg) was administered subcutaneously before animals recovered from anesthesia. Blood gases, pH, and hematocrit were measured 10 min before and 10 min after the ischemia. In the sham group, all catheters were inserted in the same manner, but the balloon was not inflated.
An observer who was blinded to the experimental procedures carefully examined hind-limb motor function of the rats 48 h after reperfusion. Hind-limb motor function was assessed with the Basso, Beattie, Bresnahan (BBB) Locomotor Rating Scale (12). In brief, the scale has 22 levels, which range from 0 (total paralysis) to 21 (normal locomotion). Values in the range of 1 to 8 are scored for small or large movements of the three hind-limb joints without plantar weight support or dorsal stepping. A score of 9 involves attainment of plantar weight support or dorsal stepping. Scores of 10 to 20 are given for progressive improvements in coordinated walking ability. The scores for both hind-limbs were averaged to obtain the score for each measurement.
Forty-eight hours after reperfusion, rats were anesthetized with 5% isoflurane, and thiopental 3 mg/kg was given intraperitoneally. The rats were then transcardially perfused with 100 mL of heparinized saline, followed by 150 mL of 3.7% formaldehyde. Twenty-four hours after perfusion, the spinal cords were removed and postfixed in the same fixative for 1–2 days. After this period, L4 and L5 spinal segments were dissected, embedded in paraffin, and cut transversely to attain a thickness of 5 μm. These cuttings were stained with hematoxylin and eosin for quantitative evaluation. Cells that contained Nissl substance in the cytoplasm, loose chromatin, and prominent nucleoli were considered to be normal neurons. The number of normal neurons per 0.5 mm2 of tissue in Rexed’s laminae VII, VIII, and IX was counted in both sides of spinal cord gray matter under high-power microscopic magnification (200×) in a blinded fashion. The total number of normal neurons in 6 microscopic fields with total areas of 3 mm2 was obtained for each section at L4 and L5. The total number of normal neurons of both sides at L4 and L5 was divided by 4 to obtain a unilateral average.
Physiological variables and the number of normal neurons were considered as parametric data. Values (expressed as mean ± s.d.) were compared with one-factor analysis of variance followed by the Student-Newman-Keuls test for multiple comparisons. BBB scores were considered to be nonparametric data and were compared by using the Kruskal-Wallis test followed by the Mann-Whitney U-test. Non-parametric values are presented as median (interquartile range). A probability value < 0.05 was considered significant.
Physiological values are shown in Table 1. Before ischemia, there were no significant differences in pH, PaCO2, PaO2, hematocrit, glucose, mean arterial blood pressure, or rectal temperature among the groups. During ischemia, distal mean arterial blood pressure values in the ischemic groups were lower than those in the sham group (P < 0.05). After reperfusion, pH values in the ischemic groups were lower than those in the sham group (P < 0.05). PaO2 values in the SNC-9 group and hematocrit in the V-11 and SNC-11 groups were higher than those in the sham group (P < 0.05). There were no significant differences among groups in rectal temperature during ischemia or after reperfusion (Tables 1 and 2).
The median BBB scores (interquartile ranges) 48 h after reperfusion were 21 (21–21) in the sham group, 0.8 (0–11) in the V-9 group, 13.8 (11–16) in the SNC-9 group, 0 (0–0) in the V-11 group, and 0.5 (0–18) in the SNC-11 group (Fig. 1). BBB scores in the V-9, V-11, SNC-9, and SNC-11 groups were lower than those in the sham group (P < 0.05). BBB scores in the SNC-9 group were higher than those in the V-9 group (P < 0.05), whereas there were no statistical differences in BBB scores between the SNC-11 and V-11 groups.
There were fewer normal neurons in the V-9, V-11, and SNC-11 groups than in the sham group (P < 0.05) (Fig. 2). The number of normal neurons was similar in the sham and SNC-9 groups. There were more normal neurons in the SNC-9 and SNC-11 groups than in the V-9 and V-11 groups (P < 0.05).
The results of this study show that intrathecal treatment with the δ-opioid agonist SNC80 15 min before the induction of ischemia attenuated hind-limb motor dysfunction after 9 minutes of SCI and neuronal injury after both 9 and 11 minutes of SCI in rats. These data suggest that intrathecal SNC80 may have neuroprotective efficacy in a rat model of SCI.
This is the first report to investigate the effects of SNC80 on neuronal injury after SCI. There have been several reports regarding the effects of another δ-opioid agonist, DADLE, on the survival of neurons in the central nervous system (4,6,13). Su (4) investigated the effects of DADLE on infarct volume in rats subjected to 90 minutes of middle cerebral artery occlusion and demonstrated reduced infarct volume in the striatum. Tsao et al. (6) demonstrated that DADLE protected the terminal membranes of dopaminergic neurons against METH-induced neuronal damage in mice. Borlongan et al. (13) also reported that DADLE enhanced in vitro and in vivo survival of rat dopaminergic neurons. Zhang et al. (14) investigated the effects of DADLE on glutamate-induced excitotoxic injury of neocortical neurons in vitro and demonstrated that DADLE reduced injury; naltrindole, a δ-opioid receptor antagonist, completely blocked the neuroprotective efficacy of DADLE. Hayashi et al. (15) examined the action of DADLE in serum-deprived pheochromocytoma cells (PC12) and showed that DADLE at a small concentration had antiapoptotic efficacy in those cells. These findings are consistent with the results obtained in the present study.
The mechanisms by which the δ-opioid agonist protects the spinal cord against ischemia/reperfusion injury after transient SCI are unknown. However, possible explanations are as follows. First, mitochondrial adenosine triphosphate-sensitive potassium (KATP) channel activation might be involved. A number of investigators have suggested a role of mitochondrial KATP channel activation in cardioprotection mediated by ischemic preconditioning and δ-opioid receptor agonists (16–18). δ-Opioid receptor activation has been shown to contribute to the acquisition of ischemic tolerance after ischemic preconditioning via a mitochondrial KATP channel (18). Second, opioid receptors can stimulate extracellular signal-regulated protein kinase (ERK), a member of the mitogen-activated protein kinase family. This ERK kinase (MEK)/ERK pathway might be involved in cell survival after SCI. Hayashi et al. (15) demonstrated that activation of δ-opioid receptors by DADLE at small concentrations attenuated apoptotic cell death via the MEK/ERK pathway in serum-derived PC12 cells. Third, opioid receptor agonists may act as free radical scavengers, thus resulting in neuroprotective efficacy. Tsao et al. (6) suggested that the protective property of DADLE on METH-induced dopaminergic terminal damage may involve reactive oxygen species. Finally, other mechanisms, including protein kinase C activation and modulation of voltage-dependent Ca2+ channels, may be also involved in δ-opioid-mediated cellular protection (19,20). However, these are speculative mechanisms. In addition, we did not measure the spinal blood flow in this study. Further study is required to clarify the mechanisms of SNC80-mediated neuroprotection.
There are several limitations to this study. First, although the results showed neuroprotective efficacy of SNC80, we did not confirm whether this effect was mediated by δ-opioid receptor activation. However, others have clearly shown the involvement of δ-opioid receptor activation in δ-opioid receptor agonist-mediated neuroprotection (14). Second, although SNC80 attenuated neuronal injury after both 9 and 11 minutes of SCI, SNC80 was effective for hind-limb motor dysfunction only after 9 minutes of SCI, but not after 11 minutes of SCI. Larger doses of SNC80 or repetitive treatment might have affected hind-limb motor dysfunction after 11 minutes of SCI. In this study, we determined the doses of SNC80 on the basis of data from previous studies (21,22). Our pilot work showed that 400 nmol of SNC had maximal efficacy for neuroprotection. In fact, larger doses of SNC80 had less efficacy for neuroprotection. Hayashi et al. (15) also reported that although DADLE is antiapoptotic at small concentrations, it can be cytotoxic at large concentrations. With respect to the timing of drug treatment, further study may be required. Third, we assessed hind-limb motor function and histopathology 48 hours after reperfusion, because work examining long-term outcome showed the most spinal cord injury at 48 hours after SCI in rats (23). However, recent evidence suggests that long-term assessment may be required to evaluate the efficacy of the drugs on neuronal injury after ischemia (23–25). Further study of SNC80 in SCI with long-term outcome assessment is required.
In summary, we investigated the effects of intrathecal treatment with the δ-opioid agonist SNC80 on hind-limb motor function and neuronal injury in rats subjected to SCI. The results indicate that intrathecal administration of SNC80 can attenuate neuronal injury, measured 48 hours after SCI, in this rat model. These findings suggest that a δ-opioid agonist can be used to prevent neuronal injury when the spinal cord is at risk for ischemic injury. Further studies with other SNC80 treatment paradigms, such as repetitive and delayed treatment and use of the IV route, will improve our understanding of the neuroprotective properties of δ-opioid receptor agonists.
The authors thank Drs. Piyush M. Patel, John C. Drummond, and Osamu Kakinohana (Department of Anesthesiology, University of California, San Diego); Drs. Manabu Kakinohana and Joho Tokumine (Department of Anesthesiology, University of the Ryukyus); and Dr. Tomoki Nishiyama (Department of Anesthesiology, The University of Tokyo) for their support.
1. de Haan P, Kalkman CJ, Jacobs MJ. Pharmacologic neuroprotection in experimental spinal cord ischemia: a systematic review. J Neurosurg Anesthesiol 2001;13:3–12.
2. Wan IYP, Angelini GD, Bryan AJ, et al. Prevention of spinal cord ischaemia during thoracic and thoracoabdominal aortic surgery. Eur J Cardiothorac Surg 2001;19:203–13.
3. Juvonen T, Biancari F, Rimpiläinen J, et al. Strategies for spinal cord protection during descending thoracic and thoracoabdominal aortic surgery: up-to-date experimental and clinical results—a review. Scand Cardiovasc J 2002;36:136–60.
4. Su TP. Delta opioid peptide [D-Ala2
]enkephalin promotes cell survival. J Biomed Sci 2000;7:195–9.
5. Schultz JJ, Hsu AK, Nagase H, Gross GJ. TAN-67, a delta 1-opioid receptor agonist, reduces infarct size via activation of Gi/o proteins and KATP channels. Am J Physiol 1998;274:H909–14.
6. Tsao LI, Ladenheim B, Andrews A, et al. Delta opioid peptide [D-Ala2
]enkephalin blocks the long-term loss of dopamine transporter induced by multiple administration of methamphetamine: involvement of opioid receptors and reactive oxygen species. J Pharmacol Exp Ther 1998;287:322–31.
7. Mailly P, Gustard M, Cupo A. Subcellular distribution of delta-opioid receptors in the rat spinal cord: an approach using a three-dimensional reconstruction of confocal series of immunolabelled neurons. J Neurosci Methods 1999;87:17–24.
8. Gomez-Flores R, Rice KC, Zhang X, Weber RJ. Increased tumor necrosis factor-alpha and nitric oxide production by rat macrophages following in vitro stimulation and intravenous administration of the delta-opioid agonist SNC 80. Life Sci 2001;68:2675–84.
9. Brandt MR, Furness MS, Mello NK, et al. Antinociceptive effects of delta-opioid agonists in Rhesus monkeys: effects on chemically induced thermal hypersensitivity. J Pharmacol Exp Ther 2001;296:939–46.
10. LoPachin RM, Rudy TA, Yaksh TL. An improved method for chronic catheterization of the rat spinal subarachnoid space. Physiol Behav 1981;27:559–61.
11. Taira Y, Marsala M. Effect of proximal arterial perfusion pressure on function, spinal cord blood flow, and histopathologic changes after increasing intervals of aortic occlusion in the rat. Stroke 1996;27:1850–8.
12. Baso DM, Beattie MS, Bresnahan JC. A sensitive and reliable locomotor rating scale for open field testing in rats. J Neurotrauma 1995;12:1–21.
13. Borlongan CV, Su TP, Wang Y. Treatment of delta opioid peptide enhances in vitro and in vivo survival of rat dopaminergic neurons. Neuroreport 2000;11:923–6.
14. Zhang J, Haddad GG, Xia Y. Delta-, but not mu- and kappa-, opioid receptor activation protects neocortical neurons from glutamate-induced excitotoxic injury. Brain Res 2000;885:143–53.
15. Hayashi T, Tsao LI, Su TP. Antiapoptotic and cytotoxic properties of delta opioid peptide [D-Ala2
]enkephalin in PC12 cells. Synapse 2002;43:86–94.
16. Fryer RM, Hsu AK, Eells JT, et al. Opioid-induced second window of cardioprotection, potential role of mitochondrial KATP
channels. Circ Res 1999;84:846–51.
17. Schultz J, Hsu A, Gross G. Ischemic preconditioning in the intact rat heart is mediated by delta 1- but not mu- or kappa- opioid receptors. Circulation 1998;97:1282–9.
18. Cohen MV, Baines CP, Downey JM. Ischemic preconditioning: from adenosine receptor to KATP channel. Annu Rev Physiol 2000;62:79–109.
19. Miki T, Cohen M, Downey J. Opioid receptor contributes to ischemic preconditioning through protein kinase C activation in rabbits. Mol Cell Biochem 1998;186:3–12.
20. Acosta CG, Lopez HS. Delta opioid receptor modulation of several voltage-dependent Ca2+
currents in rat sensory neurons. J Neurosci 1999;19:8337–48.
21. Fraser GL, Pradhan AA, Clarke PB, Wahlestedt C. Supraspinal antinociceptive response to [D-Pen(2,5)]-enkephalin (DPDPE) is pharmacologically distinct from that to other delta-agonists in the rat. J Pharmacol Exp Ther 2000;295:1135–41.
22. Fraser GL, Gaudreau GA, Clarke PB, et al. Antihyperalgesic effects of delta opioid agonists in a rat model of chronic inflammation. Br J Pharmacol 2000;129:1668–72.
23. Sakamoto T, Kawaguchi M, Kurita N, et al. Long-term assessment of hind limb motor function and neuronal injury following spinal cord ischemia in rats. J Neurosurg Anesthesiol 2003;15:104–9.
24. Du C, Hu R, Csenansky CA, et al. Very delayed infarction after mild focal cerebral ischemia: a role for apoptosis? J Cereb Blood Flow Metab 1996;16:195–201.
© 2004 International Anesthesia Research Society
25. Kawaguchi M, Kimbro JR, Drummond JC, et al. Isoflurane delays but does not prevent cerebral infarction in rats subjected to focal ischemia. Anesthesiology 2000;92:1335–42.