Neuroprotective role of delta -opioid receptors in cortical neurons

Junhui Zhang*, Geoffrey Thomas Gibney*, Peng Zhao, and Ying Xia

Department of Pediatrics, Yale University, New Haven, Connecticut 06520


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We recently demonstrated that delta -opioid receptor (DOR) activation protects cortical neurons against glutamate-induced injury. Because glutamate is a mediator of hypoxic injury in neurons, we hypothesized that DOR is involved in neuroprotection during O2 deprivation and that its activation/inhibition may alter neuronal susceptibility to hypoxic stress. In this work, we tested the effect of opioid receptor activation and inhibition on cultured cortical neurons in hypoxia (1% O2). Cell injury was assessed by lactate dehydrogenase release, morphology-based quantification, and live/dead staining. Our results show that 1) immature neurons (days 4 and 6) were not significantly injured by hypoxia until 72 h of exposure, whereas day 8 neurons were injured after only 24-h hypoxia; 2) DOR inhibition (naltrindole) caused neuronal injury in both day 4 and day 8 normoxic cultures and further augmented hypoxic injury in these neurons; 3) DOR activation ([D-Ala2,D-Leu5]enkephalin) reduced neuronal injury in day 8 cultures after 24 h of normoxic or hypoxic exposure and attenuated naltrindole-induced injury with prolonged exposure; and 4) µ- or kappa -opioid receptor inhibition (beta -funaltrexamine or nor-binaltorphimine) had little effect on neurons in either normoxic or hypoxic conditions. Collectively, these data suggest that DOR plays a crucial role in neuroprotection in normoxic and hypoxic environments.

cortex; hypoxia; injury; protection; opioids


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

MAMMALIAN CORTICAL NEURONS are highly sensitive to hypoxic stress. Depending on severity and duration, hypoxia can lead to major alterations in neuronal function, metabolism, and morphology (18, 25). For example, prolonged hypoxic exposure may cause irreversible neuronal injury or even cell death, thus leading to serious neurological disorders (18). However, the cellular and molecular mechanisms of hypoxic injury and adaptation in neurons are not clearly understood.

Our recent studies (50) suggest that activation of the delta -opioid receptor (DOR) system protects cortical neurons against glutamate-induced stress, whereas activation of µ- or kappa -opioid receptors offers no significant neuroprotection. Because glutamate is a mediator of hypoxic/ischemic injury (8, 16) and endogenous opioids are released during hypoxia (2, 3, 15, 46), we predicted that DOR might also play a role in neuroprotection during hypoxic stress. However, there is no direct evidence showing the effect of DOR on neuronal responses to O2 deprivation. In whole animal experiments, systemic administration of DOR agonists prolonged the survival period of mice during hypoxia (5, 23, 24). Because of the complex effects of drug administration in the whole body, the targets of DOR-activated protection are not clearly defined. For example, systemic DOR agonists may act on cardiac DOR, which is protective against ischemic stress (1, 32).

On the basis of our previous work, we hypothesized that neuronal DOR is involved in self-protection and that its inhibition leads to increased neuronal injury in response to hypoxia. To test this hypothesis, we exposed cultured neurons to hypoxia and determined the effect of DOR activation and inhibition on neuronal responses to O2 deprivation. The specificity of DOR was also examined by assessing the effect of µ- and kappa -opioid receptor antagonists on the same neuronal cultures. Because our past investigations (44, 45) and those of others (41) demonstrated a high density of DORs in the mammalian cortex, we focused the present investigation on cortical neurons.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Pregnant (embryonic day 16-17) Sprague-Dawley rats were purchased from Charles River Laboratories (Wilmington, MA). All animal procedures were performed in accordance with the guidelines of the Animal Care Committee of Yale University School of Medicine, which is accredited by the American Association for Accreditation of Laboratory Animal Care.

Preparation of neuronal cultures. Primary cultures of cortical neurons from embryonic day 16-17 rats were used as previously described (50). In brief, fetuses were decapitated and cortical tissue was collected under sterile conditions. The tissue was dispersed with a 1-ml pipette and then passed through an 80-µm nylon mesh with a Teflon pestle. Cells were resuspended in neuron-defined, serum-free Neurobasal medium (GIBCO-BRL, Grand Island, NY), supplemented with B-27, glutamine (0.5 mM), glutamate (25 µM), and a combination of penicillin (100 IU/ml) and streptomycin (100 µg/ml). The cells were plated onto poly-D-lysine (100 µg/ml; Sigma, St. Louis, MO)-coated 35-mm culture dishes at 1 × 106 cells/ml. The culture dishes were then kept in a humidified atmosphere of 95% air and 5% CO2 at 37°C.

Hypoxic induction of rat cortical neurons. Neurons were exposed to hypoxia beginning at day 4, 6, or 8 for durations of 0.5, 4, 8, 24, 48, and 72 h. Culture dishes were randomly divided into two groups, one in a normoxic incubator and the other in an incubator maintained at 1% O2, 5% CO2, and 94% N2.

Cell treatment. Culture dishes were treated with [D-Ala2,D-Leu5]enkephalin (DADLE, a highly selective DOR agonist; Refs. 20, 50), naltrindole (NTI, a highly selective DOR antagonist; Ref. 28), beta -funaltrexamine (FNA, a selective µ-opioid receptor antagonist; Refs. 38, 43), or nor-binaltorphimine (BNI, a selective kappa -opioid receptor antagonist; Ref. 6). DADLE, NTI, FNA, or BNI was added to cultures at a final concentration of 10 µM, and then they were immediately exposed to either normoxia or hypoxia. For long-term exposures (72 h), supplemental doses were added to the culture medium after 36 h. NTI, FNA, and BNI were purchased from RBI (Natick, MA), and DADLE was purchased from Sigma. After 24-72 h of exposure, neurons were assessed for neuronal injury via assay of lactate dehydrogenase (LDH) release, morphology-based quantification, and/or viability/cytotoxicity assay (Molecular Probes, Eugene, OR; see Live/dead assay).

LDH assay. LDH activity in the culture medium was measured with an LDH kit (Sigma Diagnostics Procedure no. 228-UV) and a Beckman DU-640 spectrophotometer system (Beckman Instruments, Fullerton, CA). Culture medium was sampled and centrifuged to remove cellular debris from the supernatant. Subsequently, 100 µl of the sample was added to a polystyrene cuvette containing 1 ml of LDH reagent (50 mM lactate and 7 mM NAD+ in 0.05% sodium azide buffer, pH 8.9). The cuvette was placed immediately into the spectrophotometer, maintained at 30°C. After stabilization for 30 s, absorbance at 340 nm was recorded at 30-s intervals for 2 min. The change in absorbance was then expressed in concentration units (U) per liter and converted to percentage of control levels.

Morphological studies. For the qualitative assessment of viable and injured neurons, a "same-field" quantification method, developed in our laboratory (50), was used with a computer-based image analysis system. In brief, microphotographs of cultured cells were taken before experimental treatment, using a phase-contrast microscope to establish baseline viability. After experimental treatment, the same field of each culture dish was reexamined and photomicrographs were taken again. Viable and injured neurons were compared in the same field before and after experiments to assess cell injury. The criteria for neuronal injury were the same as in our previous work (50).

Live/dead assay. Neuronal survival was quantified using a live/dead viability/cytotoxicity kit (L-3224) from Molecular Probes. In accordance with the manufacturer's protocol, neurons were exposed to cell-permeant calcein AM (3 µM), which is hydrolyzed by intracellular esterases, and to ethidium homodimer-1 (4 µM), which binds to nucleic acids. (The cleavage product of calcein AM, calcein, produces a green fluorescence when exposed to 494-nm light and is used to identify live cells. Bound ethidium homodimer-1 produces a red fluorescence when exposed to 528-nm light, allowing the identification of dead cells.) Culture dishes were dually stained and examined under a fluorescence microscope system (Zeiss Axiovert 25; Sony Progressive 3CCD and Camera Adapter CMA-D2; blue excitation filter: 488/515 nm, green excitation filter: 514/550 nm). Neuronal viability was determined from five random fields per dish, averaged and expressed as percent cell survival, i.e., [live cells/(dead + live cells)] × 100.

Data analysis. The data are expressed as means ± SE and were subjected to statistical analysis via nonpaired, two-tailed Student's t-test with GraphPad Prism 3.0 software (GraphPad Software, San Diego, CA). The level of statistical significance was set at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Hypoxic susceptibility increases with neuronal maturation and stress duration. Because responses to hypoxic stress are dependent on hypoxic duration and neuronal maturation (18), we determined the effect of these factors on neuronal injury in our culture system. We exposed the cortical neurons at day 4, 6, or 8 to hypoxia (1% oxygen) for 0.5-72 h and assessed hypoxia-induced injury by measuring LDH release into the medium. The method of LDH assay provided the possibility of studying dynamic changes in the same culture dish. In day 4 neurons, no substantial injury was observed during the first 48 h of hypoxic exposure, although a significant increase in medium LDH occurred after 72 h of exposure (30% increase; n = 16; Fig. 1). A slightly different pattern was observed in day 6 neurons. After the first 24 h of exposure, a small increase in LDH levels was observed, and by 72 h LDH activity increased by 50% (n = 14; Fig. 1). In contrast, day 8 neurons experienced elevated medium LDH levels after only 8 h of hypoxia, and these levels rose significantly after 24 h (30% increase; n = 9; Fig. 1). The greatest increase in LDH leakage in these neurons was observed after 72-h hypoxia (100% increase; n = 15), which was 2.5-fold greater than that of day 4 neurons exposed to the same hypoxic duration. Furthermore, our same-field morphological data (see Figs. 4C-C' and 6C-C') demonstrated more observable injury due to hypoxic stress in day 8 cultures than in day 4 cultures. These observations confirm our LDH results in that day 8 neurons are more susceptible to hypoxic insult than younger neurons.


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Fig. 1.   Time course of hypoxia-induced injury in cultured cortical neurons. Hypoxia (1% O2) was applied to neurons starting at day 4, 6, or 8 in culture (n = 7-16 for all groups). Medium lactate dehydrogenase (LDH) activities were measured immediately after hypoxic exposure. Values are expressed as % of control levels from sister cultures maintained in normoxia for the same durations. Each group represents mean ± SE. star P < 0.05, day 8 vs. day 4 and day 6; P < 0.05, day 6 vs. day 4. Note that LDH activity did not substantially change in day 4 and day 6 neurons exposed to hypoxia for up to 48 h, whereas hypoxia induced a significant increase in LDH level after only 24 h in day 8 neurons. More prolonged exposure resulted in greater increases in LDH levels, especially in day 6 and day 8 neurons. LDH activity dramatically increased after 72 h of hypoxia in all age groups.

DOR activation reduces neuronal injury in hypoxic conditions. Our previous work (50) showed that DOR activation greatly reduces glutamate-induced injury in day 8 cortical neurons. Because hypoxia induced significant injury in day 8 neurons after 24 h of exposure, as shown in Hypoxic susceptibility increases with neuronal maturation and stress duration, we used the same method of DOR activation (50) to test whether DOR protects these neurons from hypoxic stress. First, we determined the effect of DOR activation on neuronal survival under hypoxia with a live/dead staining assay. Because of the accumulation of cell death beginning with neuronal preparation and plating through day 9 in culture, an average base level of 67% neuronal viability was seen in a given field in control cultures (day 9, Figs. 2A-A' and 3A). Neuronal cultures exposed to hypoxia (1% O2) for 24 h significantly decreased survival to 59% (Figs. 2B-B' and 3A). DADLE treatment before hypoxic exposure completely abolished the hypoxia-induced neuronal injury (Figs. 2C-C' and 3A; P < 0.05 compared with hypoxia alone). To further ascertain the protective effects of DOR activation, we measured LDH release in day 8 cultures exposed to 24-h hypoxia and 24-h hypoxia plus DADLE. As shown in Fig. 3B, LDH release increased significantly after 24-h hypoxia. This hypoxic elevation of LDH release was abolished by concurrent treatment with DADLE. These data strongly suggest that DOR activation protects cortical neurons against hypoxic stress. It is interesting to note that addition of DADLE to the same neuronal cultures exposed to prolonged hypoxia (48-72 h) provided no appreciable neuroprotection.


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Fig. 2.   Effect of delta -opioid receptor (DOR) activation on neuronal survival with hypoxia. Day 8 neurons were exposed to normoxia (control), hypoxia (1% O2), or hypoxia + DOR activation [1% O2 and 10 µM [D-Ala2,D-Leu5]enkephalin (DADLE)] for 24 h. Culture dishes were subsequently stained with Molecular Probes live/dead assay and viewed under a fluorescence microscope system. A, B, and C: control, hypoxic and hypoxic + DADLE cultures, respectively, with intracellular calcein fluorescence signifying viable neurons. A', B', and C': ethidium homodimer fluorescence of the same fields as A, B, and C, respectively, which demonstrate injured and nonviable neurons. The number of injured/dead neurons included two parts: 1) a base level of natural or nonexperimental accumulation of neuronal injury/death from culture day 1 to day 8 (base level) and 2) experimental change from culture day 8 to day 9. Note that hypoxia increased neuronal injury/death after 24 h, which was diminished by DOR activation.



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Fig. 3.   DOR protection against hypoxia-induced injury. Day 8 neurons were exposed to normoxia (C), hypoxia (H; 1% O2), or hypoxia + DOR activation (H + DADLE; 1% O2 and 10 µM DADLE) for 24 h. A: neuronal survival determined with live/dead assay (Molecular Probes). Live/dead neuron counts were made in 5 random fields for each culture dish, averaged, and expressed as % of viable neurons {[live/(live + dead)] × 100; n = 6}. B: neuronal injury was determined by measuring LDH release after exposure. Data were normalized to control levels (normoxia) and are presented as means ± SE (n = 12-13). Note that significant neuronal injury resulted from hypoxic exposure, as is evident from the decreased neuronal viability and increased LDH release (star P < 0.05 for control vs. hypoxia). Treatment with DADLE markedly eliminated the hypoxia-induced release of LDH and decrease in neuronal viability (black-lozenge P < 0.005 for hypoxia vs. hypoxia + DADLE).

DOR inactivation increases hypoxia-induced neuronal injury. Because DOR is neuroprotective against hypoxia, we further asked whether its inhibition leads to an increase in neuronal injury during hypoxia. We also questioned whether DOR protection is dependent on neuronal age, because DOR density is much greater in day 8 than in day 4 cortical neurons (49). Therefore, we compared day 4 and day 8 neurons in terms of their responses to DOR inhibition. Day 4 neurons demonstrated observable morphological changes with same-field microscopy after 72 h of normoxia with NTI administration (Fig. 4). Furthermore, hypoxia-induced neuronal injury was amplified with NTI treatment. These findings were confirmed by LDH release measurements. As depicted in Fig. 5 A, medium LDH levels increased significantly in normoxic day 4 cultures after NTI administration for 72 h (50% increase; n = 15; P < 0.0001). In addition, exposure of NTI-treated day 4 neurons to 72-h hypoxia further raised medium LDH levels by 75% (n = 14; P < 0.0001; Fig. 5B), a 1.5-fold greater increase than in normoxia.


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Fig. 4.   Same-field morphology in day 4 neurons exposed to hypoxia, DOR inhibition, or both. Photomicrographs of the same fields were taken before (A-D) and after (A'-D') 72 h of treatment. A-A': normoxia. B-B': normoxia + 10 µM naltrindole (NTI). C-C': hypoxia (1% O2). D-D': hypoxia + 10 µM NTI. Note that after 72 h of exposure, hypoxia (C') as well as NTI (B') induced minor neuronal injury, whereas no appreciable damage was present in normoxic cells (A'). Included in this injury are the disappearance of neurons, swelling of cell bodies, and loss of dendrite/axon integrity among other morphological changes. Moreover, exposure to both NTI and hypoxia (D') induced more severe injury than either hypoxia or NTI alone.



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Fig. 5.   Effect of delta -, µ-, and kappa -opioid receptor inhibition on day 4 neurons. Day 4 cultured cortical neurons were treated with opioid receptor antagonist [delta : NTI; µ: beta -funaltrexamine (FNA); kappa : nor-binaltorphimine (BNI)] at a final concentration of 10 µM. Dishes were then simultaneously exposed to normoxic or hypoxic (1% O2) conditions for 72 h. Medium LDH activities were measured immediately after treatment and are expressed as % of control levels. A: both control and antagonist groups were exposed to normoxia during treatment. B: both control and antagonist groups were exposed to hypoxia during treatment. Data are presented as means ± SE of 14-15 samples from 8 different cultures. star P < 0.05 vs. control group; black-lozenge P < 0.05 NTI vs. FNA and BNI groups. Note that DOR inhibition (NTI) substantially increased LDH release in both normoxia and hypoxia, whereas µ- and kappa -opioid receptor inhibition resulted in slight changes in LDH levels.

In more mature neurons (day 8), NTI caused severe injury in both normoxic and hypoxic conditions as well. Same-field microscopy revealed that 72-h exposure to either NTI or hypoxia alone caused conspicuous neuronal damage and simultaneous exposure to both treatments further increased the observed injury (Fig. 6). LDH release measurements confirmed these findings and also demonstrated that day 8 neurons were much more susceptible to NTI-induced injury. Administration of NTI to normoxic day 8 neurons caused a 175% increase in medium LDH levels (n = 19; P < 0.0001; Fig. 7 A), a 3.5-fold greater increase than that in day 4 neurons. Similarly, NTI treatment resulted in a further increase in medium LDH in day 8 neurons exposed to 72-h hypoxia (125% increase, P < 0.0001; n = 19; Fig. 7B), which is a 1.7-fold greater increase than day 4 neurons exposed to both hypoxia and NTI.


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Fig. 6.   Same-field morphology in day 8 neurons exposed to hypoxia, DOR inhibition, or both. Photomicrographs of the same fields were taken before (A-D) and after (A'-D') 72 h of treatment. A-A': normoxia. B-B': normoxia + 10 µM NTI. C-C': hypoxia (1% O2). D-D': hypoxia + 10 µM NTI. Note that significant neuronal injury was present after exposure to either NTI (B') or hypoxia (C') for 72 h. Such morphological damage includes the loss of dendrite/axon integrity, swelling of cell bodies, and disappearance/disintegration of neurons. Furthermore, more severe injury was observed in hypoxic neurons that were simultaneously treated with NTI (D').



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Fig. 7.   Effect of delta -, µ-, and kappa -opioid receptor inhibition on day 8 neurons. Day 8 cultured cortical neurons were treated with opioid receptor antagonist (delta : NTI; µ: FNA; kappa : BNI) at a final concentration of 10 µM. Dishes were then simultaneously exposed to normoxic or hypoxic (1% O2) conditions for 72 h. Medium LDH activities were measured immediately after treatment and are expressed as % of control levels. A: both control and antagonist groups were exposed to normoxia during treatment. B: both control and antagonist groups were exposed to hypoxia during treatment. Data are presented as means ± SE of 14-19 samples from 8 different cultures. star P < 0.05 vs. control group; black-lozenge P < 0.05 for NTI vs. FNA and BNI groups. Note that DOR inhibition (NTI) dramatically increased LDH release in both normoxic and hypoxic conditions, whereas µ- and kappa -opioid receptor inhibition resulted in slight LDH changes.

Collectively, all these data suggest that DOR inhibition induces serious injury in either normoxic or hypoxic conditions. It is also noteworthy that more prolonged DOR inhibition caused more severe neuronal injury.

DOR agonists reduce neuronal injury induced by DOR inactivation. To further clarify the role of DOR in neuroprotection, we questioned whether the NTI effect could be attenuated by an increase in DOR agonists in culture medium. Again, we took advantage of the LDH assay, i.e., dynamic studies of neuronal injury with hypoxic time from the same neuronal culture. As shown in Fig. 8 A, DOR inhibition in normoxic conditions induced a significant rise in medium LDH levels after only 24 h (15% increase; n = 12; P < 0.05). Although continued exposure for 48 h resulted in considerably more LDH release (40% increase; n = 9; P < 0.05), prolonged exposure for 72 h showed the most dramatic rise in LDH. In hypoxic neurons, exposure to NTI greatly elevated medium LDH levels (in comparison to hypoxic controls) after only 24 h of treatment (30% increase; n = 10; P < 0.05) and further increased levels with more prolonged exposure durations (Fig. 8B).


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Fig. 8.   Time course of neuronal injury with DOR activation and inhibition. Day 8 neurons were exposed to DADLE (10 µM), NTI (10 µM), or DADLE + NTI (10 µM each) for 24-72 h. Medium LDH activities were measured immediately after exposure and expressed as % of control levels. Results are represented as means ± SE (n = 6-14 from 3-8 cultures). A: normoxia. B: hypoxia. star P < 0.05 for control vs. treatment. black-lozenge P < 0.05 for NTI vs. DADLE + NTI. Note that after 24 h of either normoxia or hypoxia, LDH release was significantly reduced in DADLE-treated dishes, whereas exposure to NTI substantially increased LDH release. With more prolonged treatments, NTI caused greater escalations in LDH release, which were partially blocked by the coadministration of DADLE.

As described in DOR activation reduces neuronal injury in hypoxic conditions, 24-h treatment with DADLE significantly reduced LDH release in both normoxic and hypoxic neurons (15% decrease for both conditions; P < 0.05; Fig. 8). However, longer durations in either normoxia or hypoxia offered no such protection. To examine the effects of the DOR agonist DADLE on NTI-induced neuronal injury, NTI and DADLE were coadministered to neuronal cultures. Treatment of normoxic neurons with DADLE tended to reduce NTI-induced injury in all time points, although no statistically significant change was present. More interestingly, NTI-induced injury in hypoxic neurons was substantially reduced by DOR activation, specifically in 48- and 72-h hypoxic neurons [P < 0.05; decrease of 40% (48 h) and 50% (72 h); n = 6].

µ- and kappa -opioid receptor inactivation causes minor neuronal injury. To test the specificity of DOR with respect to its role in neuronal responses to hypoxia, µ- and kappa -opioid receptor antagonists (FNA and BNI, respectively) were also used in the treatment of cultured neurons in both normoxic and hypoxic conditions for 72 h. LDH measurements revealed that treatment with either FNA or BNI at 10 µM induced a very slight increase in medium LDH levels in both normoxic day 4 (Fig. 5) and day 8 (Fig. 7) neurons. Treatment with FNA yielded a 9% and 13% increase in LDH levels in day 4 and day 8 neurons, respectively, whereas BNI exposure resulted in somewhat smaller rises in LDH for both groups. Furthermore, a comparable pattern was seen under hypoxic conditions. As demonstrated in Figs. 5 and 7, exposure to FNA produced a 25-30% increase in LDH release over hypoxia alone in day 4 and day 8 neurons and BNI yielded a 15-25% increase in these neurons. All changes were significantly smaller than those seen in NTI-treated neurons.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The major findings of the present study are 1) hypoxia-induced injury is dependent on hypoxic duration and neuronal age in cultured cortical neurons; 2) DOR activation is neuroprotective in both normoxic and hypoxic neurons; and 3) DOR inhibition, but not µ- or kappa -opioid receptor inhibition, induces major neuronal injury in normoxic conditions and further increases injury during hypoxia, which is attenuated by DOR agonist.

We used three independent approaches to determine neuronal injury in this work, i.e., LDH assay, morphology-based same-field quantification, and live/dead staining. Among these approaches, LDH assay has a unique advantage, i.e., the use of a small sample for dynamic study of the same culture dish, which provides relatively more accurate time course data for either hypoxic duration- or DOR treatment-based experiments. The release of intracellular LDH into extracellular fluids has been documented as a reliable indicator of neuronal injury (19, 22, 50). In past investigations (50), we observed that glutamate injury in cultured cortical neurons was determined equally by assaying LDH release or same-field morphology. Other works have also demonstrated that the results of LDH release assays parallel those of phase-contrast microscopy, trypan blue staining, and fluorescent staining (19, 22). However, the use of LDH release as an index of neuronal injury does not provide direct assessment of the percentage of neurons injured and/or dead in culture. Therefore, we conducted additional experiments, including morphological assessment and live/dead staining, to assess neuronal injury more directly. The consistency of our results for all assays further confirms the reliability of our data in terms of hypoxia and the effect of opioid receptor activation/inhibition on neuronal injury.

The present study draws a detailed time course for hypoxia-induced injury in cultured cortical neurons based on neuronal age and hypoxic duration, demonstrating that cultured cortical neurons become less tolerant to hypoxic stress with further maturation. This is similar to what is seen in newborn and adult brain tissues (9, 17, 18). Interestingly, our results indicate a major increase in hypoxic sensitivity between culture day 6 and day 8 in cortical neurons. This is also true in cultured hippocampal neurons, which exhibit increased hypoxic susceptibility after 7 days in vitro (13). It seems, therefore, that a critical developmental transition occurs in cultured neurons after 7 days in terms of neuronal vulnerability to stress. A variety of factors are believed to be related to this phenomenon (18, 25), including membrane protein expression, metabolism, and release of excitatory amino acids. One of the major factors that influence neuronal susceptibility may be attributed to glutamate release and receptor expression. During hypoxic exposure, glutamate is expelled from neurons (8, 47), resulting in the overstimulation of glutamate receptors and subsequent cell injury or death (7, 18). Because glutamate receptor expression increases during development (31) and sensitivity to glutamate excitotoxicity increases with neuronal maturation (50), the observed differences in hypoxic susceptibility between neuronal ages may be associated with the developmental increase in glutamate toxicity.

In this work, we demonstrated that stimulation of DOR reduces neuronal injury after 24-h treatments in either normoxic or hypoxic conditions but does not yield any substantial benefit with prolonged exposure durations. A possible explanation for this phenomenon is that prolonged hypoxia may cause a significant accumulation of opioid release, which saturates DORs in these neurons. Past investigations showed that opioids are present at significant levels in bovine glial cultures and cerebral spinal fluid (2, 3). In response to short-term hypoxia, the level of enkephalins, which are endogenous agonists for delta - and µ-opioid receptors, sharply increases. Other works have shown similar findings with different models (15, 46). From these findings, it appears that cortical neurons may release opioids during normal function and in response to hypoxic stress as a mechanism of self-protection against injury. Because high levels of endogenous opioids may already have been present in the culture medium after prolonged hypoxia, adding more DOR agonist may not have further increased DOR protection. On the other hand, desensitization of DOR may also have occurred because of the prolonged treatment with DOR agonist in conjunction with endogenous opioid release during chronic hypoxia. Indeed, past investigations showed that chronic DOR agonist treatment of NG108 and C6 gliomal cell lines results in DOR downregulation via expression and activity (29, 48).

On the basis of the above discussion, it is reasonable to predict that cortical neurons are deeply susceptible to DOR inhibition, which causes serious neuronal injury, especially during hypoxic stress. In fact, our results strongly support this prediction. Interestingly, greater injury was observed in day 8 neurons than in day 4 neurons in both normoxic and hypoxic conditions, which suggests that maturational differences exist between these age groups. We previously observed (44, 49) in the brain and in cultured neurons that DOR expression increases significantly with development. Because DOR density of day 8 neurons is more than twofold that of day 4 neurons (49), these more mature neurons may have a greater dependence on this pathway to maintain neuronal function and, therefore, may be more susceptible to neuronal injury with DOR inhibition. Another noteworthy point is that DADLE did not increase protection on neurons subjected to prolonged hypoxia but significantly attenuated NTI-induced injury in the same neurons. This observation suggests that the increase in DOR agonist may compete with DOR antagonist in terms of DOR binding and thus reduce neuronal injury induced by DOR inhibition during prolonged hypoxia.

The question arises of why delta -, but not µ- or kappa -, opioid receptors are involved in neuroprotection. In addressing this issue, the inhibitory efficacies of the three antagonists used should first be considered. Previous reports showed that NTI, FNA, and BNI have extremely high binding affinities, with dissociation constant (Kd) and IC50 values in the nanomolar to picomolar range, to their respective receptors (6, 14, 38, 43). Because a final concentration of 10 µM was used for each antagonist, it may be assumed that each type of opioid receptor was sufficiently saturated by antagonist, suggesting that differences in neuroprotective effects are less likely associated with inhibition efficacy. However, we could not rule out the possibility that FNA or BNI binds to DOR at the concentration used in this study. In fact, it seems possible that these µ- and kappa -specific antagonists may have yielded a slight inhibition of DOR, an occurrence that has been observed in other works (37, 43). If these antagonists did partially block DOR, it would account for the minor neuronal injury that was observed in FNA- and BNI-treated neurons. A second issue to address is whether differences in expression levels of the various opioid receptors account for the observed phenomenon in cortical neurons. Past studies demonstrated that µ-opioid receptors are present at similar or even higher densities than DOR in the mammalian cortex, although kappa -opioid receptor density is slightly lower (33, 41, 44, 45). This implies that the relative distribution/expression levels of opioid receptor subtypes within the cortex, as a whole, may not be a key factor in the observations shown in this work. In light of our previous work (45, 50), we are confident that DOR, but not µ- or kappa -opioid receptors, plays an important role in neuronal survival in normoxic conditions and during environmental stress.

The mechanism(s) of DOR-mediated neuroprotection is unknown at present. It may involve the regulation of specific G proteins, ion channels (e.g., Ca2+ and K+ channels), and excitatory neurotransmitter release. For instance, studies at the cellular level showed that DOR activation inhibits calcium currents by restricting N-type Ca2+ channels in NG108-15 cells (40) and can also inhibit voltage-gated L-type Ca2+ channels in GH3 pituitary cells (27). Because intracellular Ca2+ levels are elevated during hypoxic exposure, leading to irreversible cell injury (18, 34), the inhibition of Ca2+ currents by DOR stimulation may serve as a neuroprotective mechanism in prevention of Ca2+ overload. In addition, DOR regulation of glutamate signaling may also be involved in normal function and protection of neurons. Immunolabeling studies have shown that DOR is localized at presynaptic and postsynaptic terminals in a variety of neurons, including those of the mammalian cortex (4, 10, 36, 42). At the axon terminal, it has been proposed that blockage of Ca2+ currents by DOR stimulation prevents the release of glutamate from presynaptic vesicles, thereby reducing glutamate excitability (26, 39). Alternatively, past studies also found that DOR may interact with glutamate receptors on the postsynaptic membrane and suppress neuronal signaling (35, 39). In either situation, DOR apparently has the ability to reduce neuronal overstimulation via inhibition of glutamate excitation. Such regulation may be utilized during normal cell functioning and in response to environmental stresses. Therefore, one of the reasons why DOR inhibition induced substantial injury in normoxic neurons may be the loss of inhibitory regulation of excitatory neurotransmitter release and/or receptor excitation.

In general, delta -, µ- and kappa -opioid receptors have many similarities, such as consisting of seven transmembrane domains, existing as ~60% identical sequences, and being coupled to G proteins (20). Similarities also exist in several of their regulatory targets, including adenylyl cyclases, protein kinases, and certain Ca2+ and K+ channels. However, DOR seems to play a unique role in neuroprotection. One possible explanation for the difference in neuroprotective capabilities is that individual opioid receptors regulate different effectors, thereby eliciting different responses. Because of the common features of opioid receptors, it was proposed by Connor and Christie (11) that the selectivity of these receptors for eliciting specific pathways does not lie in differences between each opioid receptor subtype but in their association with divergent types of G proteins. Included in this statement are observations that demonstrate that each opioid receptor subtype preferentially couples to specific G proteins, such as where DOR is more efficiently coupled to the G16 protein than either µ- or kappa -opioid receptors (21). Whatever the mechanistic pathway is, it apparently yields specialized differences between receptor subtypes and the regulation of effectors. For example, it was shown that µ-opioid receptors, but not delta - or kappa -opioid receptors, are involved in the regulation of Ca2+ channels in neurons isolated from the nucleus tractus solitarii of rats and in mouse periaqueductal gray neurons (12, 30).

In conclusion, the present data clearly show that DOR modulation, but not that of µ- and kappa -opioid receptors, plays a major role in neuroprotection in both normoxic and hypoxic environments. Although the related mechanisms are unknown at present, we speculate that this phenomenon may be linked to the role of DOR in selective regulation of G proteins, excitatory neurotransmitter release, glutamate receptor stimulation, and Ca2+ homeostasis. Because the activation of DOR may have a wide range of clinical applications in treating acute hypoxia-related impairments, further research is warranted to develop a better understanding of opioid receptors and the pathways involved in neuroprotection.


    ACKNOWLEDGEMENTS

This work was supported by March of Dimes (FY00-722) and National Institute of Child Health and Human Development (R01-HD-34852) grants to Y. Xia.


    FOOTNOTES

* J. Zhang and G. T. Gibney contributed equally to this work.

Address for reprint requests and other correspondence: Y. Xia, Yale Univ. School of Medicine, Dept. of Pediatrics, 333 Cedar St., LMP 3107, New Haven, CT 06520 (E-mail: ying.xia{at}yale.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpcell.00226.2001

Received 18 May 2001; accepted in final form 4 January 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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