Prostaglandin EP1 Receptor Contributes to ...

TOXICOLOGICAL SCIENCES 89(1), 265–270 (2006) doi:10.1093/toxsci/kfj022 Advance Access publication October 19, 2005

Prostaglandin EP1 Receptor Contributes to Excitotoxicity and Focal Ischemic Brain Damage Abdullah Shafique Ahmad,*,1 Sofiyan Saleem,*,1 Muzamil Ahmad,* and Sylvain Dore´*,†,2 *Department of Anesthesiology & Critical Care Medicine, and †Department of Neuroscience, Johns Hopkins University, Baltimore, Maryland 21205 Received August 9, 2005; accepted October 9, 2005

Cyclooxygenases (COX-1, COX-2), the rate-limiting enzymes involved in the biosynthesis of prostaglandins, are constitutively present in most cells. COX-2, the inducible isoform, is strongly increased as a result of pathologic events 1

These authors contributed equally to this work. To whom correspondence should be addressed at Dept. Anesthesiology & Critical Care Medicine, Dept. Neuroscience, Johns Hopkins University, School of Medicine, 720 Rutland Ave, Ross Research Building 364-365, Baltimore, MD 21205. Fax: (410) 955-7271. E-mail: [email protected]. 2

associated with the excessive activation of NMDA receptors; these events include cerebral ischemia, seizures, excitotoxic brain injury, and other neurologic disorders (Acarin et al., 2002; Adams et al., 1996; Hewett et al., 2000; Iadecola et al., 2001). A role for COX-2 in excitotoxicity has been supported by the findings that COX-2-deficient mice are less susceptible to both ischemic brain injury and NMDA-mediated neurotoxicity in vivo (Iadecola et al., 2001), while neuronal overexpression of COX-2 in mice shows increased infarction volume (Dore´ et al., 2003). Moreover, treatment of cultured mouse cortical neurons with NMDA induces COX-2 expression, whereas the inhibition of this enzyme is protective against NMDA-induced neurotoxicity (Carlson, 2003; Hewett et al., 2000). Additional evidence of the various effects of COX activity suggests that inflammatory reactions contribute to the late stages of ischemic injury, thereby worsening the neurologic outcome. Therefore, intervention in the inflammatory cascade in excitotoxic brain damage is considered to be a potential effective therapeutic strategy. Recent reports of increased stroke accompanying the use of specific COX-2 inhibitors caused the medical community to question their safety. Here, we are moving the focus from COX to prostaglandins and their G-protein-coupled receptors. Prostaglandins are known to affect the nervous system and can modulate synaptic transmission and neurotransmitter release, the sleep/wake cycle, fever, pain, and the immune system (Kaufmann et al., 1997; Narumiya, 2003; Narumiya et al., 1999). PGE2, often called the proinflammatory prostaglandin, is involved in the normal regulation of brain activities, and conflicting observations regarding its action in the brain have been the topic of debate. These inconsistent effects of PGE2 might be the result of its prostanoid receptors: EP1 to EP4. The EP2 and EP4 receptors are involved in intracellular increases in cAMP levels, while the EP3 receptors decrease cAMP levels and the EP1 receptors are linked with Ca2þ and inositol 1,4,5triphosphate (IP3) regulation (Narumiya et al., 1999). We postulate that, in the central nervous system, EP1/EP3 receptors are responsible for some of the negative effects of PGE2. The recent development of new agonists and antagonists with high specificity toward given EP receptors has created a great deal of interest in exploring the physiologic activities of these

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The clinical side effects associated with the inhibition of cyclooxygenase enzymes under pathologic conditions have recently raised concerns. A better understanding of neuroinflammatory mechanisms and neuronal survival requires knowledge of cyclooxygenase downstream pathways, especially PGE2 and its Gprotein-coupled receptors. In this study, we postulate that EP1 receptor is one of the mechanisms that propagate neurotoxicity and could be a therapeutic target in brain injury. This hypothesis was tested by pretreating C57BL/6 wildtype mice with the EP1 receptor selective agonist ONO-DI-004 and the selective antagonist ONO8713, followed by striatal unilateral NMDA injection. Results revealed that ONO-DI-004 increased NMDA-induced lesion volume up to 128.7 ± 12.0%, while ONO-8713 significantly decreased lesion volume to 71.3 ± 10.9%, as compared to the NMDA-control group. Neurotoxic EP1 receptor properties were also studied using C57BL/6 EP1 receptor knockout (EP1/) mice, which revealed a significant decrease to 74.5 ± 8.2%, as compared to wildtype controls. The protective effect of the antagonist ONO-8713 was also tested in the EP1/ mice, revealing no additional protection in these mice. Together, these results support the selectivity of ONO8713 toward EP1 receptor and suggest the neurotoxic role of EP1 receptor. Furthermore, the EP1 receptor role in ischemic brain damage was investigated using a model of middle cerebral artery occlusion (MCAO) and reperfusion. The infarct volume was significantly reduced to 56.9 ± 11.5% in EP1/ mice, as compared to wildtype controls. This is the first study that demonstrates that EP1 receptor aggravates neurotoxicity and that modulation of this receptor can determine the outcomes in both excitotoxic and focal ischemic neuronal damage. Key Words: cerebral ischemia; cyclooxygenase; neurotoxicity; PGE2; stroke.

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receptors. Two of the new compounds tested in this study are ONO-DI-004, a selective EP1 agonist (Yamamoto et al., 1999), and ONO-8713, a selective EP1 antagonist (Watanabe et al., 2000). We hypothesized that activation of EP1 receptor would facilitate neurotoxicity and that its deletion or blockade would ameliorate brain injury. To address this hypothesis, C57BL/6 mice were pretreated with ONO-DI-004 and ONO-8713, followed by NMDA-induced excitotoxicity. Additionally, EP1 receptor knockout (EP1/) mice were utilized to examine the effect of NMDA toxicity and to investigate the effect of EP1 receptor in cerebral ischemia.

MATERIALS AND METHODS

Mice. Following protocols approved by the Institutional Animal Care and Use Committee of Johns Hopkins University, adult male C57BL/6 wildtype (WT) mice and C57BL/6 EP1/ mice, weighing 20–25 g, were used in this study (Watanabe et al., 1999). Mice were provided by Shuh Naruniya, University of Kyoto Japan. Treatment of mice. Weight and rectal temperature of each mouse were recorded before the surgical procedure. Mice were subjected to intracerebroventricular (ICV) injection of vehicle or freshly prepared ONO-DI-004 or ONO-8713, followed by unilateral intrastriatal injection of NMDA (Ayata et al., 1997). In brief, each mouse was anesthetized, maintained at 1.0% halothane, and mounted on a stereotaxic frame (Stoelting Co., Wood Dale, IL). In a volume of 0.2 ll, varying doses of ONO-DI-004 (0.1 nmol, n ¼ 8; 1 nmol, n ¼ 12; 10 nmol, n ¼ 8) or ONO-8713 (0.1 nmol, n ¼ 8; 1 nmol, n ¼ 12; 10 nmol, n ¼ 9) were injected into the right lateral ventricle (stereotaxic coordinates PA– 0.5 mm, lateral–1.0 mm from bregma, and ventral–2.5 mm relative to dura) over 30 s, with the help of a 1-ll Hamilton syringe, and the needle was left in place for an additional 2 min. Twenty min later, 15 nmol NMDA (in 0.3 ll) was injected into the right striatum over 2 min and the needle was left in place for additional 5 min. The NMDA-lesion control group (n ¼ 12) received 15 nmol NMDA, and the vehicle-NMDA control group (n ¼ 11) received 0.2 ll vehicle ICV, followed by 15 nmol NMDA in the striatum. Sham-control and vehiclecontrol groups were treated similarly with saline. To monitor the effect of NMDA toxicity in EP1/ mice, the mice were divided into two groups: the NMDA-lesion group (n ¼ 10) was treated with NMDA alone, and the other group was pretreated with ONO-8713 (1 nmol, n ¼ 7) following the same protocol as described above. After injections, mice were placed in a thermoregulated chamber maintained at 31°C, and returned to their cages after full recovery from anesthesia. Throughout the experimental procedure, rectal temperature of mice was monitored and maintained at 37.0 ± 0.5°C. Assessment of lesion volume. At 48 h post-injection, weight and rectal temperature were recorded. Thereafter, mice were euthanized by transcardiac perfusion with PBS, followed by 4% paraformaldehyde (pH 7.2), under anesthesia. Brains were immediately removed, post-fixed in paraformaldehyde for 4 h, cryoprotected in sucrose (30%), and frozen in 2-methyl butane (precooled over dry ice). Brain sections were cut on a cryostat and stained with cresyl violet to estimate lesion volume. Images of the brain sections were taken and analyzed with the help of SigmaScan Pro 5.0 software (Systat, Inc., Point Richmond, CA). Middle cerebral artery occlusion (MCAO) and reperfusion. Transient focal cerebral ischemia was induced by MCAO, using an intraluminal filament

Quantification of infarct volume. Four days after reperfusion, mice were deeply anesthetized and decapitated to harvest brains, which were sliced coronally into 2-mm-thick sections and incubated with 1% 2,3,5-triphenyltetrazolium chloride (TTC) in saline for 30 min at 37°C. Images of these slices were taken and analyzed with the help of SigmaScan Pro 5.0 software. The area of infarcted brain, identified by the lack of TTC staining, was measured on the rostral and caudal surfaces of each slice and numerically integrated across the thickness of the slice to obtain an estimate of infarct volume. Volumes from all five slices were summed to calculate infarct volume over the entire hemisphere, expressed as a percentage of the volume of the contralateral hemisphere. Infarct volume was corrected for swelling by comparing the volumes in the ipsilateral and contralateral hemispheres. The corrected infarcted hemisphere was calculated as: volume of corrected infarcted hemisphere ¼ volume of contralateral hemisphere (volume of ipsilateral hemisphere volume of infarcted hemisphere). Statistical analysis. Data were processed using SigmaStat 2.0 (Systat), and significance level was set at p < 0.05. Statistical analysis was performed by Student’s t-test. All data are reported as means ± SD.

RESULTS

Physiologic Data The effect of stimulation of EP1 receptors on weight and temperature was recorded before surgery (before injection) and before euthanasia (48 h after injection) in WT and EP1/ mice (data not shown). Average body weight decreased 48 h after surgery, but the percent of decrease was not significant among treatment groups. Rectal temperature did not change appreciably in any of the groups investigated. NMDA-Mediated Brain Lesion Is Augmented by EP1 Receptor Agonist ONO-DI-004 ICV pretreatment of mice with ONO-DI-004 aggravated the brain injury caused by NMDA injection. At 0.1-nmol and 1-nmol doses of ONO-DI-004, no significant change in brain lesion volume was observed, whereas a 10-nmol dose caused


Chemicals. Unless stated otherwise, all chemicals were purchased from Sigma Co. (St. Louis, MO). EP1 receptor agonist ONO-DI-004 and EP1 receptor antagonist ONO-8713 were generous gifts from Ono Pharmaceuticals Co. Ltd., Osaka, Japan.

technique. Mice were anesthetized and maintained at 1.0% halothane, and rectal temperature was monitored and maintained at 37 ± 0.5°C. Relative cerebral blood flow was measured by laser-Doppler flowmetry (Moor Instruments, Devon, England) with a flexible probe affixed to the skull over the parietal cortex, which is supplied by the MCA (2 mm posterior and 6 mm lateral to bregma). Under aseptic conditions, the neck and carotid bifurcation were dissected. The common carotid artery was temporarily ligated, and the external carotid artery was used as a stump. A small 7–0 Ethilon nylon monofilament (Ethicon, Inc., Somerville, NJ), covered with flexible silicone (Cutter Sil light universal hardener, Heraeus Kulzer GmbH, Hanau, Germany), was inserted through the incision in the external carotid artery stump and advanced through the internal carotid artery to the origin of the MCA. The filament tip was left in position for 90 min to occlude the blood supply. After the occlusion, the incision was sutured, anesthesia was discontinued, and the animals were transferred to a thermoregulated chamber maintained at 31°C. At 90 min after occlusion, the mice were briefly anesthetized and reperfusion was achieved by withdrawing the filament, which allowed the circulation of blood through the MCA, and the incision was sutured. The animals were returned to the thermoregulated chamber until full recovery from anesthesia, when they were returned to their cages and survived for 4 days. Throughout the surgical process, rectal temperature was monitored and maintained at 37 ± 0.5°C.

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a significant increase in brain lesion volume: 28.8%, with p < 0.05 (Fig. 1). NMDA-Mediated Brain Lesion Is Attenuated by EP1 Receptor Antagonist ONO-8713 Injection of 15 nmol of NMDA produced lesions in the ipsilateral striatum that were equivalent in NMDA-lesion control group and vehicle-NMDA control group. Pretreatment with 1 nmol and 10 nmol ONO-8713 significantly decreased the NMDA-induced lesion volume by 28.7 and 17.4%, respectively, with p < 0.05 (Fig. 2). No significant protection was observed with a 0.1-nmol dose. The 10-nmol dose showed slightly less protection than the 1-nmol dose. No lesion was observed in the sham-control or vehicle-control groups (data not shown). Genetic Deletion of EP1 Receptor Is Beneficial in Protecting the Brain from Excitotoxicity EP1/ mice were found to be less vulnerable to NMDAinduced toxicity, compared to WT mice (Fig. 3). The lesion volume in NMDA-treated WT mice was 10.05 mm3, which was significantly (p < 0.05) decreased to 7.49 mm3 in EP1/ mice.

FIG. 2. Effect of ONO-8713, a selective antagonist of EP1 receptor, on NMDA-induced brain lesion in C57BL/6 WT mice. (A) A representative photograph of coronal sections of the brains of mice treated with intrastriatal injection of 15 nmol NMDA. (B) Pretreatment with ICV injection of ONO8713 (1 nmol) followed by NMDA significantly attenuates lesion volume. The dotted line demarcates the lesion and non-lesion areas. Scale bar ¼ 1000 lm. (C) Histograms showing decreased NMDA-induced brain injury following pretreatment with ONO-8713. Significant neuroprotection was observed in groups pretreated with 1.0-nmol (n ¼ 12) and 10-nmol doses of ONO-8713 (n ¼ 9). However, no significant change in brain lesion was observed at the lower dose of 0.1 nmol (n ¼ 8), as compared to NMDA alone (15 nmol, n ¼ 12). Values are reported as means ± SD. *p < 0.05, when compared with NMDAlesion group.

The ICV treatment of EP1/ mice with EP1 receptor antagonist ONO-8713 produced no significant difference (data not shown), compared to the EP1/ group, demonstrating the selectivity of the drug toward EP1 receptor and confirming the genetic deletion of EP1 receptor in these mice. Genetic Deletion of EP1 Receptor Is Beneficial in Limiting Infarct Volume following Transient Cerebral Ischemia Transient occlusion of the MCA with a filament maintained for 90 min, followed by reperfusion for 4 days, was chosen as the model for transient ischemia. EP1/ and WT mice were used to test our hypothesis that EP1 receptor is deleterious in brain injury. Quantification of infarct volume after TTC staining showed effective and significant (p < 0.01) decrease in ischemic infarction by 42.03% in EP1/ mice (Fig. 4).

DISCUSSION

The hypothesized neurotoxic effect of EP1 receptor was investigated in C57BL/6 WT mice. Pretreatment with the EP1


FIG. 1. Effect of ONO-DI-004, a selective agonist of EP1 receptor, on NMDA-induced brain lesion in C57BL/6 WT mice. (A) A representative photograph of coronal sections of the brains of mice treated with intrastriatal injection of 15 nmol NMDA. (B) Pretreatment with ICV injection of ONO-DI004 (1 nmol), followed by NMDA significantly augments lesion volume. The dotted line demarcates the lesion and non-lesion areas. Scale bar ¼ 1000 lm. (C) Histograms showing NMDA-induced brain injury aggravated by pretreatment with ONO-DI-004. Significant neurotoxicity was observed in the group pretreated with 10 nmol dose of ONO-DI-004 (n ¼ 8); however, no significant difference in brain lesion was observed at lower dose of 0.1 nmol and 1 nmol as compared to NMDA alone (15 nmol, n ¼ 12). Values are reported as means ± SD. *p < 0.05, when compared with NMDA-lesion group.

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receptor selective agonist ONO-DI-004 revealed exacerbation of the unilateral intrastriatal excitotoxic lesion induced by NMDA, whereas, treatment with the selective EP1 antagonist ONO-8713 significantly protected against NMDA-induced neurotoxicity. To further support the toxic property of the EP1 receptor, EP1/ mice were subjected to the same excitotoxicity paradigm and revealed a significant decrease in NMDA-induced lesion, as compared to WT mice. Furthermore, pretreatment of EP1/ mice with ONO-8713 did not provide any additional neuroprotective effect. Further extending these findings to investigate whether EP1 receptors are also involved in ischemic brain damage revealed that EP1/ mice were protected from brain damage due to MCAO. All together, these data show that EP1 receptor activation promotes neurotoxicity, while its blockade promotes neuroprotection, suggesting that pharmacologic EP1 antagonists could be considered as therapeutic targets against excitotoxic and ischemic brain damage. Under various abnormal physiologic conditions, excessive release of glutamate in the brain causes over-excitation and activation of NMDA receptors, which facilitates the intracellular influx of Ca2þ and consequent cell death (Fogal et al., 2005). During this process of overexcitation, COX-2 is upregulated, catalyzing the generation of prostanoids. PGE2,

FIG. 4. Effect of cerebral ischemia induced by MCAO in the brains of C57BL/6 WT and EP1/ mice. (A) A representative photograph of coronal sections of the brains of WT mice showing infarction due to MCAO. (B) A representative photograph of coronal sections of the brains of EP1/ mice, showing infarction due to MCAO. (C) Histograms depicting that WT mice are more vulnerable to cerebral ischemia than EP1/ mice. Infarct volume of WT (n ¼ 12) and EP1/ (n ¼ 12) mice was measured at 4 days of reperfusion after 90 min of MCAO. Values are reported as means ± SD. *p < 0.01, when compared with WT mice.

a prostanoid that is often measured as a marker of COX activity, is known to regulate numerous cellular and molecular processes that are involved in various disease conditions (Ho et al., 2000; Manabe et al., 2004; Oka, 2004; Tessner et al., 2004; Vassiliou et al., 2004). These processes are regulated by membrane G-protein-coupled receptors on the target cells. Four subtypes of PGE2 receptors have been cloned, namely EP1, EP2, EP3, and EP4 (Narumiya et al., 1999). Studies have shown that EP1 receptors are expressed throughout the brain and in neurons of dorsal root ganglion (Narumiya et al., 1999; Sugimoto et al., 1994). To study the downstream pathways involved in EP1 receptor function, Narumiya’s group reported that EP1 receptor functions through generation of IP3 and increased levels of cellular Ca2þ, and that activation of EP1 receptor by a selective agonist significantly elevates the intracellular level of Ca2þ. It has also been reported that in a model of pain, intrathecal administration of PGE2 caused allodynia, which was mediated by activation of NMDA receptors and generation of nitric oxide in the spinal cord (Minami et al., 1994, 1995). Thus, the authors propose that PGE2 regulates pain in the peripheral nervous system through EP1/EP3 receptors. Further, it has been shown that EP1/ are protected from allodynia caused by the intrathecal injection of PGE2 (Minami et al., 2001). Likewise, in the present study, a significant increase in the NMDA-induced lesion was observed


FIG. 3. Effect of NMDA toxicity in the brain of C57BL/6 WT and EP1/ mice. (A) A representative photograph of coronal sections of the brains of mice treated with intrastriatal injection of 15 nmol NMDA. (B) Coronal section of brains of EP1/ mice treated with 15 nmol NMDA, showing attenuation in lesion volume. The dotted line demarcates the lesion and non-lesion areas. Scale bar ¼ 1000 lm. (C) Histograms showing neurotoxic effect of NMDA in C57BL/6 WT and EP1/ mice. A single dose of NMDA (15 nmol) was injected into the striatum of C57BL/6 WT (n ¼ 12) and EP1/ mice (n ¼ 10). WT mice were found to be more vulnerable to NMDA toxicity than were the EP1/ mice. Values are reported as means ± SD. *p < 0.05, when compared with NMDA-lesion group.

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ACKNOWLEDGMENTS This work was supported in part by grants from National Institutes of Health, NS046400 and AG022971 (S.D.), and a postdoctoral fellowship from the Mid-Atlantic American Heart and Stroke Association (A.S.A.). We thank Tzipora Sofare, MA, for her assistance in preparing this manuscript.

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when the mice were pretreated with EP1 receptor agonist, whereas this lesion was significantly attenuated in EP1/ mice. Moreover, the infarction due to MCAO was attenuated in EP1/ mice. EP1 receptor agonist ONO-DI-004 and antagonist ONO8713, recently manufactured, have had limited usage thus far in targeting the nervous system. They have been used in other organs to study colon cancer (Watanabe et al., 2000), diabetes (Makino et al., 2002), vasoconstriction (Norel et al., 2004), and renal injury (Suganami et al., 2003), and have been used in only a limited number of brain studies to mainly investigate fever and basic neural functions. EP1 receptors are also involved in thermoregulation, and ICV injection of ONO-DI-004 caused hypothermia in rats for a short period (Oka et al., 2003). Regarding the long-term effect of ONO-DI-004 on body temperature, we did not observe significant differences in any group. Altogether, the present study describes for the first time that the EP1 receptor agonist ONO-DI-004 and antagonist ONO8713 can dictate the outcome of excitotoxicity in the central nervous system. In addition, we propose for the first time that genetic deletion of EP1 receptor aids in decreasing NMDAmediated neurotoxicity and in reducing ischemic lesion following stroke. These neurologic observations suggest that, given the side effects associated with COX inhibitors, focus on prostaglandin receptors might prove to be an alternative therapeutic pathway.

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