Neuroprotective effects of the agonist of metabotropic glutamate receptors ABHxD-I in two animal models of cerebral ischaemia

Resuscitation (2006) 68, 119—126

EXPERIMENTAL PAPER

Neuroprotective effects of the agonist of metabotropic glutamate receptors ABHxD-I in two animal models of cerebral ischaemia夽 Dorota Makarewicz a, Roman Gadamski b, Apolonia Ziembowicz a, Alan P. Kozikowski c, Jarda T. Wroblewski d, Jerzy W. Lazarewicz a,∗ a

Department of Neurochemistry, Medical Research Centre, Polish Academy of Sciences, Pawinskiego 5, 02-106 Warsaw, Poland b Department of Neuropathology, Medical Research Centre, Polish Academy of Sciences, Pawinskiego 5, 02-106 Warsaw, Poland c Department of Medicinal Chemistry and Pharmacognosy, University of Illinois, Medical Center, 833 S. Wood Street, Chicago, IL 60612, USA d Department of Pharmacology, Georgetown University Medical Center, 3900 Reservoir Road, NW, Washington, DC 20007, USA Received 31 January 2005 ; received in revised form 1 April 2005; accepted 25 May 2005 KEYWORDS Agonist; Brain ischaemia; Glutamate; Hypoxia; Infant; Neurones

Summary The neuroprotective efficacy of 2-aminobicyclo[2.1.1]hexane-2,5dicarboxylic acid-I (ABHxD-I), a rigid agonist of metabotropic glutamate receptors, was studied using a 3-min global cerebral ischaemia model in Mongolian gerbils and the hypoxia/ischaemia model in 7-day-old rats. The effects on brain damage of ABHxD-I (30 mg/kg, intraperitoneally or 7.5 ␮g intracerebroventricularly) administered 30 min before global ischaemia or 30 min after hypoxia/ischaemia was evaluated 14 days after the insults. Treatment of adult gerbils with ABHxD-I injected i.c.v. but not systemically, prevented post-ischaemic hyperthermia and substantially reduced brain damage. These effects may reflect low permeability of the adult blood—brain barrier to ABHxD-I, and the role of reduced body and brain temperature in neuroprotection after its i.c.v. administration. ABHxD-I given either i.p. or i.c.v. to developing rats reduced brain damage by 55 and 37%, respectively, without affecting the body temperature. Due to immaturity and increased post-ischaemic permeability of the blood—brain barrier in developing rats, ABHxD-I may induce neuroprotection by direct interference with brain metabotropic glutamate receptors. © 2005 Elsevier Ireland Ltd. All rights reserved.

夽 A Spanish translated version of the summary of this article appears as Appendix in the online version at 10.1016/j.resuscitation.2005.05.018. ∗ Corresponding author. Tel.: +4822 6086528; fax: +4822 6685423. E-mail address: [email protected] (J.W. Lazarewicz).

0300-9572/$ — see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.resuscitation.2005.05.018

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Introduction Transient occlusion of both common carotid arteries of the Mongolian gerbil results in reversible forebrain ischaemia, leading to delayed death of neurones in sensitive brain areas, particularly the hippocampus.1 This animal model, corresponding to the brain pathology under conditions of cardiac arrest followed by rapid resuscitation,2 has been commonly used to evaluate the therapeutic potential of various drugs acting at ionotropic and metabotropic glutamate receptors, including agonists of group II metabotropic glutamate receptors (mGluRs).3—5 A rodent model of hypoxia/ischaemia, based on that developed by Levine,6 uses infant rats.7—10 This model has been used in numerous studies aimed at the development of new neuroprotective treatments after perinatal asphyxia.11—16 Metabotropic glutamate receptors (mGluRs) are coupled via G proteins to signal transduction pathways,17 and comprise at least eight subtypes, divided into three groups.18 Group I mGluRs contains subtypes mGluR1 and mGluR5, which are coupled to phospholipase C. Another important effector of signal transduction, adenylate cyclase is negatively coupled to mGluRs of group II (mGluR2 and mGluR3) and group III (mGluR4 and mGluR6—8).19 A number of studies have demonstrated that while agonists of group I mGluRs may be neurotoxic, several group II and III mGluR agonists were found to be neuroprotective, not only in vitro, but also in various in vivo models, including brain ischaemia.3,4,16,20,21 2-Aminobicyclo[2.1.1]hexane-2,5-dicarboxylic acid-I (ABHxD-I) is a potent agonist of mGluR of groups I, II and III.22 The results of our unpublished in vitro and in vivo studies have shown that this mGluR agonist provides robust protection of cultured mouse cortical neurones against NMDA toxicity, oxygen and glucose deprivation, plus the toxic effects of the ␤-amyloid peptide, and it was also effective in reducing neuronal damage in an in vivo model of rat spinal cord injury. These neuroprotective effects of ABHxD-I are most likely due to its interaction with group II and III mGluRs. The aim of this study was to determine the neuroprotective potential of ABHxD-I in brain ischaemia. Since the permeability of the blood—brain barrier (BBB) to ABHxD-I is unknown, this agonist was administered not only intraperitoneally (i.p.), but also intracerebroventricularly (i.c.v.). Moreover, to reduce the possible influence of blood brain barrier permeability two models of brain ischaemia were used: reversible forebrain ischaemia in adult Mongolian gerbils, and

D. Makarewicz et al. hypoxia/ischaemia in 7-day-old rats. The model of hypoxia/ischaemia in developing rats was selected, since in rats of that age the BBB is immature.

Materials and methods Experiments were conducted in agreement with the institutional guidelines, in compliance with Polish governmental regulations concerning experiments on animals (Dz.U.97.111.724) and the European Community Council Directive of 24 November 1986 (86/609/EEC). All efforts were made to minimise animal suffering and the number of animals required. The First Local Ethical Committee in Warsaw approved all procedures involving animals. Mongolian gerbils (Meriones unguiculatus) were bred in the Animal Colony of the Medical Research Centre, Polish Academy of Sciences. They were fed ad libitum, and kept at room temperature. Male gerbils weighing 60—80 g, were randomly distributed into control and experimental groups. The animals were anaesthetized with 4% halothane in a gas mixture containing 30% O2 and 70% N2 O. Two minutes before surgery the halothane concentration was reduced to 2%, and was maintained at this level during ischaemia. The carotid arteries were isolated through an anterior midline cervical incision. Cerebral ischaemia was induced by occlusion of both common carotid arteries with miniature aneurismal clips for 3 min. Throughout surgery the animals were kept on a heating pad at 38 ◦ C. After suturing the wounds, animals were held for 3 h at room temperature (20—23 ◦ C), under constant control, and were then placed in their home cages and left to recover for 14 days. Hypoxia/ischaemia in neonatal rats was induced as described by Rice et al.8 Wistar rat pups of both sexes were bred in the Animal Colony of the Medical Research Centre, Polish Academy of Sciences. Animals at postnatal day 7, weighing 12—18 g, were used for experiments. The pups were anaesthetised with halothane (4% for induction, and 1.5—2.0% for maintenance) in a mixture of nitrous oxide and oxygen (0.6:1). The left common carotid artery was dissected and cut between double ligatures of silk sutures (7—0), or just exposed (sham control). The period of anaesthesia was 3.5—7 min. After surgery, the pups were left to recover for 1 h. Then, the litters were placed in a chamber kept at 35 ◦ C and perfused with humidified gases at a rate of 3 L/min. After 10—30 min exposure to flowing air, a hypoxic gas mixture containing 7.3% oxygen in nitrogen was applied for 65 min. The pups were then returned to their dams housed at environmental temperature (20 ◦ C) with a 12:12 h. light—dark

Neuroprotective effects of the agonist of metabotropic glutamate receptors cycle with ample food and water. The rectal temperature of animals was monitored using animal temperature probes (MD 852 ELLAB A/S: Denmark). To analyse the results of temperature measurements in the control and ABHxD-I-treated groups, initially the summary measures of the temperature were compared. For each animal, the area under curve representing changes in temperature at different time points was calculated, and the analysis of variance (ANOVA) with the statistical significance level of P < 0.05 was used to compare the two groups. In addition, the Friedman ANOVA test was used to determine differences of the results of repeated temperature measurements within each group. Since these differences in all experiments were statistically significant, the Mann—Whitney Utest was used to analyse differences between the control and ABHxD-I-treated animals in their temperature at each time point. ABHxD-I was synthesised as described previously.22 For intraperitoneal (i.p.) injections ABDHxD-I was dissolved in saline to produce a stock solution of 20 mM and the pH adjusted to 5.5. Aliquots diluted in saline were injected i.p. to gerbils and 7-day-old rats 30 min before or after ischaemia, respectively, at a total dose of 30 mg/kg b.w. For intracerebroventricular (i.c.v.) infusions, a total volume of 1.7 ␮l of 20 mM ABHxD (7.5 ␮g of ABHxD-I) was infused into the right ventricle of halothane-anaesthetised gerbils 30 min. before ischaemia, using a CMA/100 microinjection pump (CMA Microdialysis, Sweden) set at 0.2 ␮l/min. In experiments with 7-day-old rats the same volume of the drug was injected i.c.v. into the ipsilateral left ventricle 30 min after the end of hypoxia. The stereotaxic atlases of the Mongolian gerbil and developing rat brain were used.23,24 Fourteen days after ischaemia the gerbils were deeply anaesthetised with nembutal and subjected to intracardial perfusion fixation with 4% neutralised formalin solution. The brains were removed, immersed in a 4% formalin solution for 1 week, transferred to absolute ethanol and embedded in paraffin. Cross sections (10 ␮m thick) from the dorsal part of the hippocampus were cut with a microtome and stained with cresyl violet. For each animal at least five sections of the central part of both hippocampi were analysed using a light microscope at ×400 magnification. The density of viable CA1 pyramidal neurones was quantified in ten 0.1 mm portions per section, and an average number of neurones was expressed as a percentage of the mean neuronal density in the CA1 region of the sham-operated animals (300 neurones per 1 mm). The mean percentage of cells lost for each group of animals was computed. Neuronal loss in indi-

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vidual animals (mean ± S.E.M.) was analysed statistically using ANOVA followed by the two-tailed Student’s t-test. In addition the histological findings were quantified utilising a five point grading scale, exactly as described previously,25,26 and statistical significance was tested using the two-tailed Mann—Whitney U-test. In the model of perinatal asphyxia, 14 days after hypoxia/ischaemia (at postnatal day 21) the pups were anaesthetised with chloroform, decapitated and the brains were dissected. The two cerebral hemispheres were separated at the midline and weighed using a precision balance (Sartorius, Germany). Brain damage was assessed by the deficit in weight of the ipsilateral left hemisphere and expressed as a percentage of the weight of the contralateral hemisphere. Data were analysed using ANOVA followed by the twotailed Student’s t-test.

Results Ischaemia in gerbils, induced by a 3-min occlusion of the carotid arteries, produced a severe lesion of CA1 neurones in the gerbil hippocampus with cell loss 14 days after the insult reaching 75% (Figure 1). When ABHxD-I was applied systemically (30 mg/kg, i.p.) 30 min before ischaemia, there were no statistically significant differences in the histological gradings of CA1 damage compared to the ischaemic animals without the drug treatment (Figure 1A). Also, the differences in the percentage loss of CA1 neurones between these two groups of animals were insignificant (Figure 1B). In contrast, direct infusion of ABHxD-I into the cerebral ventricle (7.5 ␮g, i.c.v.) 30 min before the onset of ischaemia resulted in highly significant neuroprotection. The number of neuronal cells lost decreased from 75% to less than 20% (Figure 1). In gerbils subjected to 3-min carotid occlusion a short period of typical post-ischaemic increase in the rectal body temperature was observed (Figure 2). In gerbils treated i.p. with ABHxD-I 30 min before ischaemia, an increase in the temperature immediately after the drug injection was noted. ABHxD-I did not significantly influence the summary measure of rectal temperature during the whole post-ischaemic period. Detailed analysis demonstrated a statistically significant reduction in temperature only 30 min after ischaemia (Figure 2A). After i.c.v. injection with saline 30 min before ischaemia gerbils developed a longer period of hyperthermia, while after i.c.v. injection of ABHxD-I, post-ischaemic hyperthermia did not develop (Figure 2B). Statistically significant differences between both groups were confirmed by the

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Figure 1 Effect of pre-treatment with ABHxD-I applied intraperitoneally (i.p. 30 mg/kg) or intracerebroventricularly (i.c.v. 7.5 ␮g) 30 min before ischaemia on histological damage in the CA1 area of the gerbil hippocampus evoked by 3-min bilateral carotid occlusion. (A) Histological grading of CA1 neuronal damage. Grades were assigned based on the percentage of damaged neurones. Points represent the grades of individual animals. Horizontal bars represent the mean grades of CA1 pyramidal cells loss. (*) Significantly different from the ischaemic control (p < 0.02) by the two-tailed Mann—Whitney U-test. (B) Percentage of CA1 pyramidal cells loss. (**) Significantly different from the ischaemic control (P < 0.005) by ANOVA followed by the two tailed Student’s t-test for grouped data.

comparison of summary measures of the temperature during the whole post-ischaemic period and by a detailed analysis for each time point. In the model in hypoxia/ischaemia of 7-dayold rats, damage to the ipsilateral hemisphere as compared to the contralateral hemisphere was evaluated 14 days after the insult. In untreated animals, massive damage amounting to a greater than 40% weight deficit of the ipsilateral hemisphere compared to the contralateral hemisphere

D. Makarewicz et al.

Figure 2 Effects of ABHxD-I on rectal temperature of gerbils submitted to 3-min bilateral carotid occlusion. ABHxD-I administered intraperitoneally (i.p. 30 mg/kg) (A) or intracerebroventricularly (i.c.v. 7.5 ␮g) (B) 30 min before ischaemia. Control refers to animals subjected to ischaemia but not treated with ABHxD-I. Indicated times relative to ischaemia represent temperature measurements before drug treatment (−30 min), before anaesthesia (−10 min), immediately before ischaemia (−3 min), during ischaemia (0), and at various times after ischaemia. The bars represent mean ± S.E.M. for the number of animals indicated in figures. As described in section ‘Materials and methods’, differences between summary temperature measures and between groups for each time point were analyzed by ANOVA with the statistical significance level of P < 0.05, and by the two-tailed Mann—Whitney U-test, respectively. In the former case the following levels of significance were taken: * P < 0.05, ** P < 0.01, *** P < 0.005. Difference between summary temperatures was detected only for i.c.v. treated animals (B).

Neuroprotective effects of the agonist of metabotropic glutamate receptors

Figure 3 Effect of post-treatment with ABHxD on brain damage in 7-day-old rats submitted to hypoxia/ ischaemia. ABHxD-I was administered intraperitoneally (i.p. 30 mg/kg) or intracerebroventricularly (i.c.v. 7.5 ␮g) 30 min after hypoxia. Brain damage was evaluated by weighing the brain hemispheres 14 days after hypoxia/ischaemia and expressed as the deficit in weight of the ipsilateral hemisphere as a percentage of the weight of the contralateral hemisphere. Bars represent mean ± S.E.M. Differences from the appropriate controls were analyzed by ANOVA followed by the two-tailed Student’s t-test for grouped data; * P < 0.005, ** P < 0.001. Differences between groups treated with ABHxD-I i.p. and i.c.v. were not significant.

was observed in both control groups injected i.p and i.c.v. with saline (Figure 3). Treatment with ABHxDI, administered 30 min after the insult resulted in a significant decrease in the percentage hemisphere weight deficit, to 19.7% after i.p. injection (30 mg/kg) and to 26.9% after i.c.v. injection (Figure 3). The difference in effects between ABHxD-I administered i.p. and i.c.v. did not reach the level of statistical significance. Moreover, i.p. injections of ABHxD-I did not influence the rectal body temperature monitored for up to 3 h after hypoxia (Figure 4).

Discussion The results of this study demonstrate that ABHxDI, an agonist of mGluRs, administered i.c.v. (but not i.p.) to adult gerbils and systemically (i.p.) and i.c.v. to immature rats possesses neuroprotective potential in brain ischaemia. We interpret these results as an indication that ABHxD-I has a direct neuroprotective effect, although in adult animals this may be limited by low permeability of the mature BBB to the drug. Moreover, based on the lack of post-ischaemic hyperthermia in gerbils treated

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Figure 4 Effect of post-treatment with ABHxD-I on rectal body temperature of 7-day-old rats submitted to hypoxia/ischaemia. ABHxD-I was applied intraperitoneally (i.p. 30 mg/kg) 30 min after hypoxia. Indicated times relative to ischaemia represent temperature measurements at various time points after ischaemia. The bars represent mean ± S.E.M. for 11 control ABHxDtreated animals. Statistical tests were used as described for Figure 2. Differences between the two groups were not significant.

i.c.v. with ABHxD-I, we propose that neuroprotection may be potentiated by an ABHxD-I-evoked drop in the animals’ body temperature. Considering the mechanisms of neuroprotection induced by ABHxD-I, one should take into account not only its pharmacological properties like affinity to receptors of putative neuroprotective potential, but also several methodological aspects of this study. These include differences in the animal models, in the routes of drug administration, in drug bioavailability and in other pathophysiological effects. In this study, we used two experimental models of brain ischaemia. The first consisted of a reversible forebrain ischaemia in adult Mongolian gerbils, induced by bilateral carotid occlusion, while the other model of brain hypoxia/ischaemia in the immature rat is of a mixed, global and focal type. These differences in the type of ischaemia induced in the two animal models might to some extent explain the different results of ABHxD-I application we observed. Previous studies using a group II mGluR agonist demonstrated important differences in the neuroprotective efficacy of the drug in the model of global and focal ischaemia induced in adult animals.5 The mechanism of this variation remains unknown. It is also necessary to

124 remember that our ischaemic models use both adult and infant animals. In spite of general similarities between different brain ischaemic models,27 the model of hypoxia/ischaemia in immature rats may have some specific developmentally-related characteristics.28—30 This aspect will be discussed later in relation to possible receptor-related mechanisms of ABHxD-I-induced neuroprotection. Two different models were employed in this study in an attempt to avoid the influence of BBB permeability to ABHxD-I on the results. With this mind, we also administered ABHxD-I both systemically (i.p.), and locally (i.c.v.) into the brain. Indeed, considering the absence of neuroprotective effect of ABHxD-I given i.p., and the lack of any modification of the post-ischaemic rectal body temperature in gerbils under these conditions, we may assume that the mature BBB represents an impermeable barrier to ABHxD-I. In contrast, neuroprotection, albeit without modification of the body rectal temperature, was observed in neonatal rats treated i.p. and i.c.v. with ABHxD-I. This indicates that this drug may more easily penetrate the immature BBB, especially during its reversible disruption in the post-ischaemic period. Morphological features of BBB immaturity have been demonstrated in the central nervous system of rodents during the first weeks of postnatal life,31—34 and ischaemiaor injury-evoked disruption of developing and even mature BBB have previously been observed. 35—37 Tendency for more effective neuroprotection by the i.p. administered ABHxD-I may be explained by the uniform access of the systemically applied drug to the whole hemisphere, while diffusion of ABHxD-I injected i.c.v. may be limited. In the gerbil model of reversible forebrain ischaemia, high doses of ABHxD-I injected directly into the brain were neuroprotective, but also modified post-ischaemic body temperature. Since neuroprotection was observed in both brain hemispheres, while the drug was injected unilaterally into the right ventricle, these results suggest that the reduction in body temperature38 may have contributed to the neuroprotective effect of ABHxD-I in this experimental group. Previously, various antagonists of ionotropic glutamate receptors, such as MK-801 and NBQX, were found to induce post-ischaemic hypothermia and in this way provide indirect neuroprotection in different models of brain ischaemia.25,26,39—42 Possibly, the mGluR agonist ABHxD-I can also interfere with the activity of thermoregulatory centres in the brain after ischaemia. Recent studies have demonstrated the role of mGluR5 in mediating stress-induced hyperthermia.43 Interestingly, in spite of these effects in adult gerbils, we did not observe any

D. Makarewicz et al. influence of ABHxD-I on body temperature in rat pups after ischaemia. These contrary findings might reflect differences between the separate models and protocols. In particular, in the model of hypoxia/ischaemia in immature rats the brain insult is unilateral and the drug is administered to the ipsilateral hemisphere during recovery after ischaemia. On the contrary, in the gerbil model both hemispheres experience ischaemia, while ABHxD-I is injected unilaterally before carotid occlusion. Since postischemic hypothermia was not observed in the immature rats, this suggests that a direct effect of ABHxD-I on brain neurones unrelated to brain temperature is mainly responsible for neuroprotection this rat model of perinatal asphyxia. ABHxD-I is a rigid agonist of mGluRs,22 thus the mechanism of neuroprotection induced by this compound can be ascribed to interference with these receptor groups. The neuroprotective effects of group II mGluR agonists have been reported by various investigators. Bond et al.3 discriminate between direct neuroprotection resulting from activation of neuronal group II mGluR and possible activation of astroglial mGluRs resulting in the production of protective neurotrophic factors. A role for glial cells in neuroprotection is possible, since mGluRs mediate astroglial responses to glucose—oxygen deprivation in vitro.44 In their in vivo studies, Cai et al.16 reported that activation of group II mGluRs by LY379268 provides neuroprotection in the model of hypoxia/ischaemia in 7-day-old rats; the same receptor mechanism of neuroprotection may be produced by ABHxD-I. However, ABHxD-I also shows affinity for group I mGluRs.22 Usually antagonists of group I mGluRs induce neuroprotection while agonists of these receptors are neurotoxic.45 The available data concerning neuroprotection induced by mGluR1 and mGluR5 antagonists from various in vitro studies and in vivo models of brain ischaemia in adult animals are confusing. Rao et al.46 detected a strong neuroprotective effect of MPEP mGluR5 antagonist in the gerbil model of global forebrain ischaemia, whereas AIDA, mGluR1 antagonists showed no neuroprotective potential. Meli et al.47 also using the gerbil brain ischaemia model described a lack of neuroprotective potential for MPEP, whereas two mGluR1 antagonists, AIDA and CBPG were neuroprotective. Again using the gerbil brain ischaemia model Bruno et al.45 found a strong neuroprotective effect of LY367385 a selective antagonist of mGluR1. According to Bao et al.48 several mGluR5 agonists as well as its antagonist MPEP, were neuroprotective in the adult rat model of focal brain ischaemia. The activities of mGluRs appear to be even more complex in the immature brain. Cambonie et al.29,30 demon-

Neuroprotective effects of the agonist of metabotropic glutamate receptors strated that in the neonatal but not the adult rat brain, group I mGluRs contribute in protecting the brain against the excessive activation of NMDA receptors by glutamate. Activation of the mGluR5 receptors was found to prevent glutamate toxicity in cultured cerebellar granule cells.49 Developmental studies have demonstrated that in the immature rat brain the mGluR5 subtype of group I mGluRs predominates, while limited amounts of mGluR1 appear in rat brain at the end of the first postnatal week.50—52 However, it is unlikely that activation of the mGluR5 could be involved in ABHxD-I-evoked neuroprotection, since the affinity of this mGluR isoform for this compound is weak.22 Thus, the mechanism of neuroprotection induced by ABHxD-I in the developing rat brain seems to be connected with activation of the group II mGluRs. Further studies are necessary to confirm this hypothesis. The mGluR agonist ABHxD-I was found to be neuroprotective in two rodent models of brain ischaemia. In the gerbil model of reversible forebrain ischaemia, neuroprotection by ABHxD-I administered i.c.v. may be partially due to a drug-induced reduction of postischaemic hyperthermia. However, experiments using the model of hypoxia/ischaemia in neonatal rats appear to demonstrate a direct neuroprotective effect of ABHxD-I. Our results also suggest that the permeability of the mature BBB to ABHxD-I may be limited, but the drug can more easily penetrate into the brain of immature rats submitted to hypoxia/ischaemia. This model of a progressive, incomplete insult shares important features with brain injury observed in infants suffering an acute episode of severe birth asphyxia.8,53 While systemic post-treatment with ABHxD-I was neuroprotective in the animal model of perinatal asphyxia, further studies are needed to determine whether this substance may be a precursor of new drugs for the treatment of neonatal cerebral ischaemia in humans.

Conflict of interest statement All authors declare that there are no financial and personal relationships with other people or organisations that could inappropriately influence or bias their work, all within 3 years of beginning the work submitted.

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