Morphine Preconditions Purkinje Cells against Cell Death under In Vitro  Simulated Ischemia–Reperfusion Conditions

The animal protocol was approved by the institutional Animal Care and Use Committee at the University of Virginia (Charlottesville, Virginia). All animal experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. 17All efforts were made to minimize the number of animals used and their suffering.

7-Benzylidenenaltrexone (BNTX) was purchased from Tocris Cookson (Ellisville, MO). All reagents, unless specified below, were obtained from Sigma Chemicals (St. Louis, MO).

As previously described, 18–20cerebellar brain slices were prepared from 2- to 3-month-old, 230- to 270-g male Sprague-Dawley rats (Hilltop, Scottsdale, PA). Rats were anesthetized with 40 mg/kg pentobarbital intraperitoneally and then decapitated. The cerebellum was removed rapidly and placed in ice-cold artificial cerebrospinal fluid (aCSF) bubbled with 5% CO²–95% O². The aCSF contained 116 mm NaCl, 26.2 mm NaHCO³, 5.4 mm KCl, 1.8 mm CaCl², 0.9 mm MgCl², 0.9 mm NaH²PO⁴, and 5.6 mm glucose at a pH of 7.4. The cerebellum was then sectioned with a tissue slicer into 400-μm transverse slices. After sectioning, slices were placed into a tissue holder (made of plastic and with small holes in it to allow free diffusion of gases and water; this holder also helps to avoid direct gas bubbling on slices) and remained in the oxygenated aCSF at room temperature for approximately 1 h for recovery of synaptic function. 18 

Ischemia was simulated in vitro  by OGD. As previously described, 18–20cerebellar slices were subjected to OGD by transferring them into a glass beaker containing 40 ml glucose-free aCSF (also containing 1 mm dithionite, an oxygen absorbent) bubbled with 95% N²–5% CO²(30 min of bubbling before the addition of brain slices was performed to reduce the oxygen content in the solution). The partial pressure of oxygen in the aCSF as measured by means of a Clark oxygen electrode (Cameron Instrument Co., Port Aransas, TX) was less than 0.1 mmHg. The beaker containing the slices was immersed in a water bath to keep the temperature of aCSF at 37 ± 0.2°C. After 20 min under the OGD condition, slices were transferred to oxygenated aCSF and remained in this aCSF for 5 h at 37°C before they were fixed for morphologic examination. This period was used to simulate reperfusion and also to allow cell injury and death that may not be evident immediately after the OGD episode to become apparent.

Morphine preconditioning was performed by incubating cerebellar brain slices with various concentrations (0.1–10 μm) of morphine at 37°C for 30 min. After 30 min in morphine-free aCSF at 37°C, they were then exposed to OGD. Naloxone (a non–type-selective opioid antagonist, 50 μm), β-funaltrexamine (β-FNA; a selective μ-opioid receptor antagonist, 10 μm), 21nor-binaltorphimine (nor-BNI; a selective κ-opioid receptor antagonist, 10 μm), 22naltrindole (a selective δ-opioid receptor antagonist, 10 μm), 23BNTX (a selective δ¹-opioid receptor antagonist, 0.5 μm), 23naltriben (a selective δ²-opioid receptor antagonist, 0.5 μm), 245-hydroxydecanoate (a selective mitochondrial K^ATPchannel blocker, 500 μm), 25or myxothiazol (a mitochondrial electron transport inhibitor, 1 μm) 10were present during the period from 30 min before the addition of morphine to the end of morphine pretreatment (total of 1 h). The concentration selection of these inhibitors was mainly based on previous studies. 6,8,10,11,24,25As a control study, these inhibitors were present for the same duration of time in the incubations of some brain slices that were not preconditioned with morphine before OGD.

After incubation, cerebellar slices were fixed in 4% paraformaldehyde in buffered saline overnight at 4°C. The slices were then paraffin embedded and sectioned into 4-μm-thick sections. The sections were cut from an interior region of the cerebellar slices (approximately 100 μm from the edge) to avoid areas subjected to slicing trauma during slice preparation. Morphologic examination was performed after staining the brain sections with hematoxylin and eosin. The sections were examined by an observer who was blinded to the group assignment to determine the percentage of undamaged Purkinje cells (survival rate). Damaged neurons were identified if the cells had one of the following characteristics: cell swelling; vacuolization; or presence of shrunken, darkened nuclei. 26Purkinje cells were recognized based on the morphology and the location (between the molecular and granular layers). At least 50 Purkinje cells were counted from each of the sections and cells in total three sections were counted for each experimental condition (> 150 Purkinje cells) to compute the percentages.

Results are presented as mean ± SEM. Each experiment included a control group and an OGD group. Each experimental condition was repeated with cerebellar slices from 9–14 rats. Statistical analysis was performed by means of Kruskal-Wallis test followed by Mann–Whitney rank sum test when appropriate. A P < 0.05 was considered significant.

Oxygen–glucose deprivation and reoxygenation induced Purkinje cell death (fig. 1). A 30-min preconditioning of the cerebellar brain slices with morphine increased the survival rate of Purkinje cells after OGD and reoxygenation (fig. 2). The protective effects of morphine preconditioning were concentration dependent and were maximal at concentrations higher than 0.3 μm (survival rates were 57 ± 4 and 39 ± 3% in slices preconditioned with 0.3 μm morphine before OGD and in slices subjected to OGD alone, respectively, n = 9, P < 0.05;fig. 2).

Morphine (3 μm) preconditioning–induced neuroprotection was abolished by the non–type-selective opioid receptor antagonist naloxone (naloxone reduced survival rates from 56 ± 2% in slices with morphine preconditioning plus OGD to 39 ± 3%, n = 11, P < 0.001, which was not different from that in slices subjected to OGD alone, 36 ± 3%;fig. 3). To identify the subtypes of opioid receptors that mediate morphine preconditioning–induced neuroprotection, selective antagonists for the μ-, κ-, or δ-opioid receptors were used in the study. When they were present with morphine during the preconditioning period, only naltrindole, a δ-opioid receptor antagonist, inhibited morphine preconditioning–induced neuroprotection (naltrindole reduced survival rates from 49 ± 2% in slices with morphine preconditioning plus OGD to 28 ± 2%, n = 11, P < 0.001, which was not different from that in slices subjected to OGD alone, 27 ± 2%;fig. 3). Either β-FNA or nor-BNI, antagonists for μ- or κ-opioid receptors, respectively, did not affect morphine preconditioning–induced neuroprotection (fig. 3). Additional data using selective δ¹- or δ²-opioid receptor antagonists showed that the neuroprotection induced by morphine preconditioning was abolished by the δ¹-opioid receptor antagonist BNTX (BNTX reduced survival rates from 56 ± 2% in slices with morphine preconditioning plus OGD to 38 ± 3%, n = 11, P < 0.001, which was not different from that in slices subjected to OGD alone, 36 ± 3%) and was not affected by the δ²-opioid receptor antagonist naltriben (fig. 4). The presence of naloxone, β-FNA, nor-BNI, naltrindole, BNTX, or naltriben alone did not change the survival rates of Purkinje neurons under the OGD and reoxygenation conditions (figs. 3 and 4).

Concomitant presence of 5-hydroxydecanoate or myxothiazol, inhibitors for mitochondrial K^ATPchannels and mitochondrial electron transport, respectively, during the 30-min exposure of cerebellar slices to morphine (3 μm) at least partially blocked morphine preconditioning–induced neuroprotection (5-hydroxydecanoate and myxothiazol reduced survival rates from 55 ± 2% in slices with morphine preconditioning plus OGD to 44 ± 3% and 44 ± 3%, respectively, n = 12, P < 0.05, which were also different from that in slices subjected to OGD alone, 34 ± 2%, n = 12, P < 0.05;fig. 5). 5-Hydroxydecanoate or myxothiazol alone had no effects on OGD-/reoxygenation-induced Purkinje cell death.

Our study demonstrated the following major findings: (1) morphine preconditioning induced neuroprotection; (2) this neuroprotection was mediated by δ¹-opioid receptors and may not depend on the activation of μ-, κ-, or δ²-opioid receptors; and (3) additional intracellular components, such as mitochondrial K^ATPchannels and electron transport system, may also be involved in morphine preconditioning–induced neuroprotection.

An earlier study showed that ischemic preconditioning–induced cardioprotection is mediated by δ¹-opioid receptors. 14In addition, pretreatment with morphine or δ¹-opioid receptor agonists also induced cardioprotection against ischemia. 8–11In isolated rabbit heart, morphine pretreatment and ischemic preconditioning were equally cardioprotective. 27In chick myocyte cultures, morphine or the selective δ-opioid receptor agonist BW373U86 attenuated ischemia–reperfusion injury. 8,10The protection induced by morphine was abolished by naloxone or BNTX. 9In isolated rat heart, activation of δ-opioid receptors also improved cardiac function. 28These findings led us to hypothesize that morphine preconditioning might also induce neuroprotection against ischemia. To avoid the presence of morphine in brain slices during OGD, we placed brain slices in morphine-free aCSF for 30 min before OGD. Our results indicate that morphine preconditioning induced a dose-dependent neuroprotective effect and the maximal protection was achieved at morphine concentrations higher than 0.3 μm, similar to the results of a previous study using chick myocyte cultures. 8Because the minimum effective concentrations in the blood for morphine to have analgesic effects are 0.06–0.6 μm, 29the concentrations for morphine to induce neuroprotection in our study are clinically relevant.

We then investigated which opioid receptors were involved in morphine preconditioning–induced neuroprotection. Three major opioid receptors, μ-, κ-, and δ-receptors, and two δ subtypes of opioid receptors, δ¹and δ², have been characterized. 5,30Previous studies have indicated that δ¹-opioid receptors are important for morphine preconditioning- and ischemic preconditioning–induced cardioprotection. 8–11,14In addition, it has been shown that activation of neuronal δ-opioid receptors during the application of injuries (not qualified as preconditioning) protected neurons from hypoxic injury 6or glutamate excitotoxicity 7in rat cortical neuron cultures. Inhibition of δ-opioid receptors but not μ- or κ-opioid receptors during hypoxia caused more severe neuronal injury than hypoxia alone. 6In whole animal experiments using [D-Pen,²D-Pen⁵]-enkephalin, an δ¹-opioid receptor agonist, and BNTX, it has been shown that the δ¹-opioid receptors mediated the increased survival time of mice to hypoxic environments. 16Consistent with these previous results, our data suggest that δ¹-opioid receptors play an important role in morphine preconditioning–induced neuroprotection because this neuroprotection was inhibited by naloxone, a non–type-selective opioid receptor antagonist, by naltrindole, a selective δ-opioid receptor antagonist, and by BNTX, a selective δ¹-opioid receptor antagonist. Our results also suggest that δ²-, μ-, or κ-receptors may not be important for morphine preconditioning–induced neuroprotection because naltriben, β-FNA, or nor-BNI, antagonists for these receptors, respectively, did not inhibit the neuroprotection. β-FNA, nor-BNI, and naltrindole have been shown to have extremely high binding affinities for their respective subtypes of opioid receptors (IC⁵⁰is in the nanomolar to picomolar range). 21,31–33In our study, we used 10 μm β-FNA, nor-BNI, and naltrindole. We assumed that such high concentrations of these antagonists would effectively block their respective receptors. In addition, 10 μm was the highest concentration used to indicate no involvement of μ or κ receptors in opioid-induced cardioprotection and neuroprotection in the previous studies. 6,11BNTX was reported to be approximately 10-fold less potent than naltriben to inhibit [³H]naltriben binding. 24In this study, although the same concentrations (0.5 μm) of naltriben and BNTX were used, naltriben did not inhibit morphine preconditioning–induced neuroprotection.

After activation of δ¹-opioid receptors in the cell membrane, what are the intracellular mediators for morphine preconditioning–induced neuroprotection? K^ATPchannels, especially mitochondrial K^ATPchannels, have been shown to mediate morphine preconditioning–induced cardioprotection. 8,9In addition, oxygen free radicals have also been found to be important in this cardioprotection. 9,10,34In fact, a positive feedback circle has been proposed for mitochondrial K^ATPchannels and free radicals: free radicals activate K^ATPchannels, and activation of K^ATPchannels can induce free radical production. 9,10We tested whether K^ATPchannels and free radicals played a role in morphine preconditioning–induced neuroprotection by using 5-hydroxydecanoate, a selective mitochondrial K^ATPchannel inhibitor with no effects on sarcolemmal K^ATPchannels, 25and myxothiazol, a mitochondrial site III electron transport inhibitor that can inhibit production of oxygen free radicals in mitochondria. 10,35Our results suggest that both K^ATPchannels and free radical production in mitochondria may be involved in morphine preconditioning–induced neuroprotection.

Therefore, our study suggests that the signaling pathway for morphine preconditioning–induced neuroprotection includes activation of δ¹-opioid receptors and mitochondrial K^ATPchannels and production of free radicals. Because it has been shown that inhibition of mitochondrial K^ATPchannels abolished free radical production induced by morphine and the selective δ-opioid receptor agonist BW373U86 in cultured myocytes, 9,10it is possible that mitochondrial K^ATPchannels may be an upstream event of free radical production in opioid preconditioning–induced neuroprotection. The positive feedback between K^ATPchannels and production of free radicals in mitochondria may then amplify the signals to induce neuroprotection.

Our experiments were performed using brain slices. Brain slices have an advantage over cultured neurons because neurons in brain slices are similarly sensitive to hypoxia and simulated ischemia as are neurons in the intact brain. 6,18–20The brain slice model also has an advantage over the intact animal model because it is easier to directly apply various drugs and to control variables such as oxygen tension, temperature, and chemical environments in brain slices. However, in vitro  models differ in many ways from intact brains. For example, the extracellular partial pressure of carbon dioxide, H⁺, glutamate, and lactic acid, which are important factors to determine cell death or survival after ischemia, might not change to the same degree as they would in vivo  because of diffusion away from the slice, even in the interior region of the slice that was used for our cell count. Therefore, it is not appropriate to extrapolate our results directly to in vivo  conditions. In addition, we evaluated cell death at 5 h after OGD because neuronal death in brain slices is manifest within 5 h after OGD, hypoxia, or overstimulation of glutamate receptors. 18–20However, delayed cell death can take a few days to develop 1,36–38and cannot be assessed by our slice model because neurons in the acutely prepared slices can be well preserved only for 5–10 h in vitro  in oxygenated buffer containing glucose. 26,39Therefore, our current study may have overestimated the morphine preconditioning–induced neuroprotection.

In summary, preconditioning brain slices with morphine at clinically relevant concentrations induced acute neuroprotection. This protection depends on activation of δ¹-opioid receptors. Activation of K^ATPchannels and free radical production in mitochondria may be important downstream events for morphine preconditioning–induced neuroprotection.