Nociceptin/orphanin FQ exacerbates excitotoxic white-matter lesions in the murine neonatal brain

Intracerebral administration of the excitotoxin ibotenate to newborn mice induces white-matter lesions, mimicking brain lesions that occur in human preterm infants. Nociceptin (NC), also called orphanin FQ, is the endogenous ligand of the opioid receptor-like 1 (ORL1) receptor and does not bind classical high-affinity opioid receptors. In the present study, administration of NC exacerbated ibotenate-induced white-matter lesions while coadministration of ibotenate with either of two NC antagonists reduced excitotoxic white-matter lesions by up to 64%. Neither ibotenate plus endomorphin I (a selective μ receptor agonist), nor ibotenate plus naloxone (a classical opioid receptor antagonist) modulated the excitotoxic lesion. Pretreatment with antisense oligonucleotides targeting the NC precursor peptide mRNA significantly reduced ibotenate-induced white-matter damage. Finally, high doses of fentanyl, which stimulates both classical μ opioid receptors and ORL1, exacerbated excitotoxic white-matter lesion. This toxic effect was blocked by inhibiting ORL1 but not classical opioid receptors. Together, these findings show that endogenous or exogenous stimulation of the ORL1 receptor can be neurotoxic and that blocking NC signaling protects the white matter against excitotoxic challenge. These data point to potential new avenues for neuroprotection in human preterm infants at high risk of brain lesions.

Periventricular leukomalacia (PVL) is a hemispheric white-matter lesion that is often cystic and occurs most frequently in infants born before 32 weeks of gestation (1). PVL is the major cause of cerebral palsy among premature infants (2). Based on epidemiological and experimental studies, the pathophysiology of PVL appears to be multifactorial. It potentially involves the combined toxicity of hypoxic-ischemic insults, excess free radicals, maternal-fetal infection with increased cytokine production, and growth factor deficiency (1, 3–8). Excess release of glutamate and the subsequent overactivation of the excitotoxic cascade could represent a common molecular pathway for several of these risk factors (6, 9).

Findings from several experimental studies support this hypothesis. Newborn mice injected intracerebrally with ibotenate, a glutamate analog acting mainly on N-methyl-D-aspartate (NMDA) receptors, develop periventricular cystic white-matter lesions mimicking several aspects of human PVL (10). In this well characterized murine model, several drugs that interfere with the excitotoxic cascade have been shown to be neuroprotective (7, 11–14) while proinflammatory cytokines (15) and iron (16) exacerbated ibotenate-induced white-matter lesions. In order to further understand the pathophysiology of the ibotenate-induced cystic lesions, we targeted the NMDA receptors thought to be present on white-matter glial cells. Newborn mice were injected intracerebrally with sense, antisense, or missense oligonucleotides specific for the NMDA R1 receptor subunit (P. Gressens, unpublished work). The sequences of the oligonucleotides were identical to those previously used by Wahlestedt et al. in an in vitro assay (17). In newborn mice, pretreatment with antisense oligonucleotides was neuroprotective while control sense oligonucleotides had no effect on the ibotenate-induced lesion. Unexpectedly, missense oligonucleotides, which were used as another control, induced a threefold reduction in the severity of the excitotoxic white-matter lesions. A search of databases with Basic Local Alignment Search Tool (BLAST) revealed that 12 successive nucleotides of the so-called “NMDA R1 missense oligonucleotide” were actually complementary to a triplicated motif of the murine nociceptin (NC) mRNA, suggesting that the blockade of NC production is neuroprotective in the ibotenate model.

NC, also called orphanin FQ, is the endogenous ligand of the opioid receptor-like 1 (ORL1) receptor (18, 19). The ORL1 receptor (also referred to as the OP₄ receptor) has significant sequence homology with the classical μ (OP₃), κ (OP₂) and δ (OP₁) opioid receptors. However, the ORL1 receptor does not bind with high affinity to traditional opioid ligands. Moreover, NC does not bind with high affinity to the classical opioid receptors, and naloxone, the nonselective opioid receptor antagonist, is inactive against the effects elicited by NC (for a review, see ref.20). In the central nervous system, NC is produced by both neurons and astrocytes (21, 22). In vitro studies suggest that NC production and secretion is induced by cAMP (23, 24). An in situ hybridization study has shown that the ORL1 receptor and NC mRNAs are both expressed in the neopallium of newborn mice (21). At the cellular level, NC inhibits adenylate cyclase (18, 19), activates potassium channels (25), and inhibits calcium channels (26). At a broader level, NC has demonstrated effects on nociception, locomotion, food intake, and memory processes (for a review, see ref.20). NC has also been shown to interact with the glutamatergic system. For example, (a) NC inhibits glutamate- and kainate-induced currents in spinal cord neurons (27); (b) the NMDA receptor blockade reduces the pronociceptive effects of NC in the land snail (28); (c) incubating neuroblastoma cells with NMDA attenuates NC-induced inhibition of adenylate cyclase (29); and (d) NC can inhibit glutamate release from adult rat cerebral cortex slices (30). Finally, fentanyl, a potent agonist of the classical μ opiate receptors as well as a weak agonist of the NC receptor, is commonly administered to premature neonates to reduce pain and discomfort (31). A recent retrospective study on a small cohort of premature infants (32) suggested that fentanyl treatment could be associated with a higher risk for PVL.

Together these data prompted us to investigate the modulating effects of NC on white-matter lesions in the developing mouse brain induced by intrapallial injections of ibotenate. The potential effects of fentanyl were also evaluated in this model.

Clinical manifestations. In this study, the overall mortality was low (less than 5%). Clinical comparisons of different treatment groups with animals injected with ibotenate alone revealed no statistically significant differences in tests of contingency (exact Fisher test) (data not shown). Epileptic manifestations, including clonic or tonic seizures and apneas, were observed during the first 24 hours after ibotenate injection in almost all treated animals. There was no variation detected in the intensity, clinical phenotype, or incidence of epileptic manifestations across treatments.

Pharmacological manipulation of the ORL1 receptor. Control pups treated at P5 with ibotenate and sacrificed 5 days later displayed both a cortical lesion and a periventricular white-matter cyst (Figure 1a and Figure 2, a and b). The cortical plate lesion was characterized by a neuronal depopulation in all neocortical layers.

Figure 1

NC exacerbates ibotenate-induced white-matter lesions and the blockade of endogenous NC neurotransmission is protective. Cresyl violet–stained sections showing brain lesions induced by ibotenate injected at P5 and studied at the age of P10. (a) Brain injected with ibotenate alone, showing typical neuronal loss in layers II–VI (arrow) and white-matter cystic lesion (arrowheads). (b and c) Brains cotreated with ibotenate and 25 μg/kg NC (b) or 250 μg/kg [Nphe¹]NC(1–13)NH₂ (c). (d) Brain pretreated with precursor pro-NC mRNA antisense oligonucleotide at P3 and P4, and injected with ibotenate at P5. Bar, 40 μm.

Cotreatment with ibotenate and i.p. NC of P5 animals induced a dose-dependant increase of the white-matter lesion size without affecting the cortical plate lesion (Figure 1b and Figure 2, a and b). Intracerebral injection of 2.5 μg NC mimicked i.p. injections of NC, with a significant increase of the white-matter lesion size (mean length of the lesion ± SEM: 498 ± 48 μm in controls vs. 734 ± 6 μm in NC-treated pups; P < 0.0001) and no significant effect on the cortical plate lesion (866 ± 50 μm in controls vs. 905 ± 102 μm in NC-treated pups). Similarly, i.p. NC(1–17)NH₂ exacerbated the excitotoxic white-matter lesion while [desPhe¹]NC(1–17)NH₂ had no detectable effect on ibotenate-induced white-matter lesions (Figure 2, a and b). In contrast, cotreatment with ibotenate and i.p. [F/G]NC(1–13)NH₂ or [Nphe¹]NC(1–13)NH₂ protected the white matter against the excitotoxic challenge in a dose-dependant manner but had no detectable effect on the cortical plate lesion (Figure 1c and Figure 2, a and b). High dose i.p. NalBzoH was also neuroprotective for the developing white matter in this model (Figure 2, a and b). In P5 pups, cotreatment with ibotenate, NC and [F/G]NC(1–13)NH₂, or [Nphe¹]NC(1–13)NH₂ abolished the deleterious effects of NC on the white-matter lesion (Figure 2a). In control animals injected with ibotenate, the white-matter lesion increased in size during the first 24 hours and remained stable thereafter (Figure 2c). Treatment with ibotenate plus i.p. [Nphe¹]NC(1–13)NH₂ induced a reduction of the white-matter lesion size at all ages studied with no significant changes over time (Figure 2c).

The coinjection of i.p. endomorphin I, naloxone, NocII, or nocistatin plus ibotenate had no detectable effect on the excitotoxic white-matter (Figure 2d) or cortical plate (data not shown) lesions. Coadministration of ibotenate, NC, and naloxone did not prevent the NC-induced exacerbation of the excitotoxic white-matter lesion (Figure 2d). Intraperitoneal pretreatment of pups with three 10 μg/kg fentanyl injections led to a significant exacerbation of the ibotenate-induced white-matter lesion (Figure 2e). The effect of fentanyl was blocked by cotreatment with [Nphe¹]NC(1–13)NH₂ but not with naloxone (Figure 2e). Interestingly, pups treated with ibotenate + fentanyl + [Nphe¹]NC(1–13)NH₂ had a white-matter lesion size very similar to animals cotreated with ibotenate + [Nphe¹]NC(1–13)NH₂, suggesting that [Nphe¹]NC(1–13)NH₂ was able to antagonize the toxic effects of both the endogenous NC and the exogenous fentanyl (Figure 2e). Intraperitoneal pretreatment of pups with three 2-μg/kg sufentanil injections (a dose considered as equipotent to 10–20 μg/kg fentanyl in terms of pain alleviation) did not exacerbate the ibotenate-induced white-matter lesion (Figure 2e).

In vivo blockade of the NC production. When compared with controls, newborn mice pretreated with antisense oligonucleotides targeting the precursor pro-NC mRNA were significantly protected against the white-matter change related to excitotoxic challenge (Figure 2f). Sense oligonucleotides had no detectable effect on the excitotoxic lesions (Figure 2f).

Cell distribution of injected oligonucleotides. Following intracerebral administration of antisense oligonucleotide labeled with fluorescein, double immunohistochemistry revealed that, in the injected hemisphere, a large proportion of astrocytes in both the white matter and the cortical plate had taken up the oligonucleotide (Figure 3, a and b) while very few neurons were double-labeled (Figure 3c) (in serial sections from five brains, only two neurons had incorporated the labeled oligonucleotide). In contrast, the injection of fluorescein alone induced a very limited labeling of a few cells at the site of injection (data not shown), suggesting that the above described widespread astrocytic distribution of the fluorescent tag reflected the integrity of the link between the oligonucleotide and fluorescein.

Figure 3

Cell distribution in brains injected with fluorescein-labeled NC mRNA antisense oligonucleotides. Pups were intracerebrally injected with labeled antisense oligonucleotides prior to processing the brains for immunohistochemistry. Arrows point to examples of labeled cells. (a) GFAP immunostaining in the P5 neopallium, showing astrocytes labeled in red. (b) Typical example of double labeling for GFAP (red deposit as in a) and fluorescein (blue deposit) in the P5 neopallium, showing numerous double-labeled cells. (c) Double labeling for MAP-2 (red deposit) and fluorescein (blue deposit) in the P5 neopallium, showing the only two double-labeled neurons that were detected in five injected and serially sectioned brains. Bar, 40 μm.

In vitro blockade of the astrocytic NC production. Preincubation of cultured astrocytes with antisense oligonucleotides targeting the precursor pro-NC mRNA significantly reduced the proportion of astrocytes labeled with anti-NC antibody when compared with control cultures or cultures exposed to sense oligonucleotides (Figure 4, a–c). Dot-blot analysis showed that antisense oligonucleotides, but not sense oligonucleotides, reduced the NC content of cultured astrocytes, both in basal conditions and after exposure to forskolin (Figure 4d).

Figure 4

Antisense oligonucleotides targeting the precursor pro-NC mRNA decrease NC production in cultured astrocytes. (a and b) Typical examples of astrocytes labeled with anti-NC antibody following exposure to control conditions (a) or to antisense oligonucleotides (b). Bar, 5 μm. (c) Quantitative analysis of the proportion of astrocytes labeled with anti-NC antibody following exposure to control conditions (ctrl), antisense oligonucleotides (AON) or sense oligonucleotides (SON). Bars represent mean ± SEM. Statistically significant differences between control and experimental conditions are shown (^AP < 0.001 in ANOVA with Dunnett’s multiple comparison test). (d) Dot-blot analysis of NC content of cultured astrocytes exposed to antisense oligonucleotides, sense oligonucleotides, or control conditions. Cells were cultured in the presence (+) or absence (–) of forskolin. One μl containing 0.8 μg total protein was spotted on the membrane for each experimental condition.

Effects of astrocyte depopulation. Compared with control pups injected with PBS, pups pretreated with the VIP antagonist had significant astrocytic depopulation in the superficial cortical layers (layers I to IV) (Figure 5). This finding confirms data reported previously (48). Astrocytic density in the deep cortical layers (layers V and VI) and in the periventricular white matter was not affected by VIP blockade (Figure 5).

As previously shown (48), pretreatment of neonatal mice with the VIP antagonist significantly protected the white matter against excitotoxic lesions (Figure 6). Similarly, inhibition of PKA by H89 was also neuroprotective (Figure 6). The neuroprotection afforded by the VIP blockade and H89 was in the same range (50% and 60%, respectively, reduction of the ibotenate-induced lesion size) as the neuroprotection obtained with NC antagonist (63% reduction of the lesion size) (Figure 6). Furthermore, any combination of two of these three neuroprotective strategies did not result in any more protection than any single drug used alone (Figure 6).

Figure 6

Effects of astrocyte depopulation and PKA inhibition on the excitotoxic lesions. Bars represent mean length of the lesion along the sagittal fronto-occipital axis ± SEM. Numbers in parentheses are the numbers of animals used in each experimental group. Statistically significant differences between controls and experimental animals are shown (^AP < 0.05, ^BP < 0.01 in ANOVA with Dunnett’s multiple comparison test). Ant, antagonist.

Effects of NC on brain cell death. In order to test the potential neurotoxic effects of NC in the absence of ibotenate challenge, TUNEL staining was performed 24 hours after an i.p. injection of NC or PBS. Figure 7 shows the absence of a detectable effect of NC treatment on developmental neopallial cell death.

Figure 7

NC treatment does not affect developmental cell death in P5 mouse pups. Quantitative analysis of TUNEL-positive cell density in the somatosensory cortical layers and underlying white matter of pups treated with PBS (filled bars) and NC (open bars). Values shown are means ± SD.

The most salient finding of the present study is the demonstration that exogenous NC augments neonatal excitotoxic white-matter lesions and that the blockade of NC endogenous signaling with either ORL1-receptor antagonists or with antisense oligonucleotides targeting the NC precursor peptide protects the developing periventricular white matter against ibotenate insult. In contrast, endomorphin I, a classical opioid receptor agonist, and naloxone, an antagonist, had no detectable effect on the excitotoxic brain lesions. Furthermore, naloxone did not prevent the deleterious effect of NC on ibotenate-induced white-matter lesions.

Previous studies have suggested some interactions between nociceptinergic and glutamatergic pathways in different neuronal populations (27–30). However, to our knowledge, the present study is the first demonstration of an exacerbating effect of NC on NMDA-mediated excitotoxicity at the level of developing white matter. Furthermore, the lack of detectable effects of NC agonists and antagonists on ibotenate-induced cortical plate lesions suggest that the role of NC in the periventricular white matter uses specific mechanisms. In the present ibotenate model, cortical plate lesions are secondary to massive calcium influx into neurons bearing NMDA receptors. In contrast, in the white matter, ibotenate does not directly induce axonal lesions but triggers microglial activation that is responsible for secondary astrocytic cell death and axonal breakdown (50). ORL1 receptors have only been described on neurons, but the target of NC in the present study is likely to be neuronal bodies and/or axons. Altogether, the data from this murine model suggest that NC is able to modulate white-matter axonal damage induced by microglia-derived factors but not the NMDA-mediated excitotoxic cascade in cortical plate neurons.

As previously mentioned, the ORL1 receptor does not bind with high affinity to traditional opioid ligands (for a review, see ref.20), and NC does not bind with high affinity to the classical opioid receptors (18, 19). Naloxone and endomorphin I had no detectable effect on ibotenate-induced lesions, suggesting the specific involvement of ORL1 receptors in the present study.

In previous studies (34, 35), NC(1–17)NH₂ displayed a higher potency than NC, probably due to a greater metabolic stability. Such a benefit was not observed in the ibotenate model, suggesting that NC degradation following systemic injection in neonatal mice is not a fully mature phenomenon. In different experimental paradigms, [F/G]NC(1–13)NH₂ has been shown to behave as an antagonist, an agonist, or a partial antagonist of the ORL1 receptor (35, 37, 38). In the present study, [F/G]NC(1–13)NH₂ clearly behaved as a NC receptor antagonist. The data further confirmed the pure antagonist properties of [Nphe¹]NC(1–13)NH₂ (39, 40) and the lower potency of this compound when compared with [Phe¹ψ(CH₂-NH)Gly²]NC(1–13)NH₂) (37, 39).

Astroglial origin of endogenously released NC. In this murine model, the neuroprotection induced by NC antagonists and by antisense oligonucleotides targeting NC mRNA strongly suggests that endogenous NC is playing a deleterious role on the excitotoxic lesion. NC is present in neurons and astrocytes (21, 22), and in vitro studies suggest that NC production and secretion is induced by cAMP in both cell types (23, 24). The findings from this study support the hypothesis that, following ibotenate injection, NC is released predominantly by astrocytes through a cAMP-dependant mechanism. This conclusion is based on several pieces of evidence derived from our ibotenate model: (a) antisense oligonucleotides targeting NC mRNA are taken up readily by astrocytes but not by neurons; (b) the inhibition of neopallial astrocyte production and/or differentiation leads to neuroprotection of the white matter against excitotoxic lesions; (c) the blockade of cAMP-dependent PKA also decreases ibotenate-induced lesions of the white matter; and (d) the reduction in size of white-matter lesions produced by astrocytogenesis inhibition or PKA blockade is similar to that provided by the ORL1-receptor blockade, but combined neuroprotective strategies did not produce further reduction in lesion size. The precise molecular mechanism linking NMDA receptor activation and NC release by astrocytes, which do not have NMDA receptors (51), is still unknown. Similarly, the physiological role of astroglia-derived NC in neocorticogenesis is also unknown.

Potential implications for human preterm neonates. A large body of evidence supports the hypothesis that our murine model of ibotenate-induced white-matter lesion mimics several aspects of human PVL (7, 10–13, 50), and there are some reasons to be hopeful about treating this condition. For example, the present data suggest that compounds such as [Nphe¹]NC(1–13)NH₂ and [F/G]NC(1–13)NH₂ can cross the neonatal blood-brain barrier. Assuming that we can extrapolate from mechanisms operating in the neonatal mouse brain to human preterm infants, it is possible that NC release by astrocytes could be related to the formation of human PVL. If this is true, selective NC receptor antagonists may represent a new class of neuroprotective agents to treat and prevent PLV in human neonates.

In order to reduce pain and discomfort, numerous human preterm infants have received fentanyl for several days in intensive care units. There is no doubt that fentanyl is highly beneficial to alleviate pain. However, the findings from this study and others (32) suggest that the deleterious effects of high doses fentanyl on ibotenate-induced white-matter lesions are mediated through ORL1-receptor activation and suggest an association between fentanyl and a higher risk of PVL in human preterm infants. Nevertheless, important questions remain. Can we extrapolate from the data obtained in our animal model to human preterm infants? Should a prospective study be conducted in human preterm infants to study potential deleterious effects of fentanyl on PVL occurrence? Should we treat human preterm infants with an opioid drug devoid of any ORL1-receptor agonist activity instead of fentanyl? In our model, sufentanil does not have any detectable effect on excitotoxic brain lesions, although a recent study suggested that sufentanil could modulate ORL1 receptor–mediated effects in SK-N-SH cells (52). Buprenorphine, which was widely used as an analgesic molecule in newborns and infants, has also been shown to have ORL1 agonist activity (31). On the other hand, [Nphe¹]NC(1–13)NH₂ produces an antinociceptive action and potentiates morphine-induced analgesia (39) and a recent study described a new class of drugs with opioid agonist/ORL1 antagonist activities (53), thus opening new possibilities in the field of neuroprotection and analgesia in neonates.

Conclusion. The present study demonstrates that, in a murine model of human PVL, NC production blockade and ORL1-receptor antagonists protect the developing white matter against excitotoxic challenge, while NC and fentanyl exacerbate lesions through ORL1-receptor activation. The NC/ORL1 system could represent a new target for the neuroprotection of human premature infants at high risk for PVL.

We are grateful to Heather G. Miller for her critical reading of the manuscript, and to Leslie Schwendimann and Marie-Hélène Quernin for their excellent technical assistance. This work was supported by the INSERM, Foundation Grace de Monaco and, in part, by a cofin99 grant from the Italian Ministry of the Universities.

Vincent Laudenbach’s present address is: Department of Anesthesiology, Hôpitaux Bichat-Claude Bernard & Robert Debré, Paris, France.

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