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Journal of Cerebral Blood Flow & Metabolism logoLink to Journal of Cerebral Blood Flow & Metabolism
. 2010 Dec 15;31(4):1107–1118. doi: 10.1038/jcbfm.2010.201

Protective role for type 4 metabotropic glutamate receptors against ischemic brain damage

Slavianka G Moyanova 1,5, Federica Mastroiacovo 2,5, Lidia V Kortenska 1, Rumiana G Mitreva 1, Erminia Fardone 2, Ines Santolini 2, Mónica Sobrado 3, Giuseppe Battaglia 2, Valeria Bruno 2,4, Ferdinando Nicoletti 2,4, Richard T Ngomba 2,*
PMCID: PMC3070971  PMID: 21157475

Abstract

We examined the influence of type 4 metabotropic glutamate (mGlu4) receptors on ischemic brain damage using the permanent middle cerebral artery occlusion (MCAO) model in mice and the endothelin-1 (Et-1) model of transient focal ischemia in rats. Mice lacking mGlu4 receptors showed a 25% to 30% increase in infarct volume after MCAO as compared with wild-type littermates. In normal mice, systemic injection of the selective mGlu4 receptor enhancer, N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-caboxamide (PHCCC; 10 mg/kg, subcutaneous, administered once 30 minutes before MCAO), reduced the extent of ischemic brain damage by 35% to 45%. The drug was inactive in mGlu4 receptor knockout mice. In the Et-1 model, PHCCC administered only once 20 minutes after ischemia reduced the infarct volume to a larger extent in the caudate/putamen than in the cerebral cortex. Ischemic rats treated with PHCCC showed a faster recovery of neuronal function, as shown by electrocorticographic recording and by a battery of specific tests, which assess sensorimotor deficits. These data indicate that activation of mGlu4 receptors limit the development of brain damage after permanent or transient focal ischemia. These findings are promising because selective mGlu4 receptor enhancers are under clinical development for the treatment of Parkinson's disease and other central nervous system disorders.

Keywords: functional recovery, ischemia, mGlu4 receptor, neuroprotection, PHCCC

Introduction

Research over the past 25 years highlights the role of excitotoxicity in ischemic neuronal death. In ischemic neurons, an excessive and sustained activation of N-methyl--aspartic acid (NMDA) receptors causes calcium overload, which in turn triggers apoptotic or necrotic cell death depending on the intensity of the insult (Choi, 1988). N-methyl--aspartic acid receptor antagonists have been considered as the most promising neuroprotective drugs in stroke for many years, yet all clinical trials with these compounds have failed because of lack of efficacy and serious adverse effects, such as profound sedation, psychotomimetic effects, and intrinsic neurotoxicity (Lee et al, 1999). Extrasynaptic NMDA receptors containing the NR2B subunit appear to be critically involved in ischemic neuronal death (Hardingham et al, 2002; Tu et al, 2010), and have been proposed as targets for neuroprotective agents in stroke. However, the utility of these agents is limited by the presence of synaptic NMDA receptor containing the NR2B subunit (Thomas et al, 2006), which mediates synaptic transmission and likely supports neuronal survival. An alternative approach is to limit glutamate spillover, thus reducing the probability that extrasynaptic NMDA receptors become recruited during ischemia. This might be achieved by enhancing the activity of glial or neuronal glutamate transporters or, alternatively, by limiting the amount of glutamate released from presynaptic terminals using drugs that do not impair fast excitatory synaptic transmission. In this study, we focused on a particular subtype of metabotropic glutamate (mGlu) receptors, namely the mGlu4 receptor, which is preferentially localized in presynaptic terminals and negatively regulates glutamate release (reviewed by Pin and Duvoisin (1995)). The mGlu4 receptor belongs to group III mGlu receptor subtypes, and is coupled to Gi proteins. Activation of mGlu4 receptors inhibits adenylyl cyclase activity and P/Q- and N-type voltage-sensitive calcium channels, and activates K(2P)2.1 potassium channels (Tanabe et al, 1993; Millán et al, 2002; Cain et al, 2008). There are no orthosteric agonists that can differentiate between mGlu4, mGlu6, mGlu7, and mGlu8 receptors. In contrast, the drug N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-caboxamide (PHCCC) behaves as a selective enhancer of mGlu4 receptors with no activity at other group III mGlu receptor subtypes (Maj et al, 2003). By definition, an enhancer requires the presence of an othosteric agonist to amplify receptor function, and, therefore, PHCCC carries the advantage of recruiting only those mGlu4 receptors that are activated by endogenous glutamate, thus acting in an activity-dependent manner. Using PHCCC in cortical cultures, we have shown that activation of mGlu4 receptors protects neurons against excitotoxic death (Maj et al, 2003), and is therefore a potential target for the treatment of brain ischemia or other neurodegenerative disorders.

We now report that mice lacking mGlu4 receptors are more sensitive to ischemic brain damage, and that systemic treatment with PHCCC at doses that are fully tolerated by intact animals is protective in models of permanent and transient focal brain ischemia.

Materials and methods

Drugs

The drug PHCCC (Tocris Cookson Ltd, Bristol, UK) was dissolved immediately before use in 1 mL of Cremophor EL (Sigma-Aldrich, St Louis, MO, USA). It was injected subcutaneously at a fixed dose of 10 mg/kg in a volume of 1 mL/kg.

Approval of In Vivo Experiments by Ethics Committees

All in vivo experiments were approved by the Ethics Committee of the Neuromed Institute (Pozzilli, Italy) and by the Local Ethics Committee of the Institute of Neurobiology (Sofia, Bulgaria).

Assessment of Motor Coordination

Assessment of motor coordination in C57BL/6 mice was carried out using the rotarod test. The rotarod apparatus consists of a rotating horizontal cylinder (30 mm) and a motor driver control unit (Ugo Basile, Varese, Italy). The cylinder is divided into five separate rotating compartments and is fully enclosed to ensure that mice do not jump out of their area. Mice were placed on the rod, which was rotated at an accelerating speed from 5 to 15 r.p.m. Automatic timers recorded the time that the mice remained on the rod. C57BL/6 mice were tested for 10 minutes on the rotarod nearly every day for 2 weeks before the test. Mice were tested on the rotarod apparatus before and 1 hour after the subcutaneous injection of vehicle or PHCCC.

Permanent Focal Ischemia in Mice

Two strains of mice were used for the induction of permanent focal ischemia: (1) male C57BL/6 mice (Charles River, Calco, Italy) and (2) male mGlu4 receptor knockout (mGlu4−/−) mice or their wild-type (mGlu4+/+) littermates with a genetic Sv129/CD1 background. mGlu4−/− mice were purchased from Jackson Lab. (Bar Harbor, ME, USA). Sv129 nd CD1 mice (Charles River) were backcrossed for the generation of mGlu4+/+ mice. Heterozygous (mGlu4+/−) mice obtained from mGlu4+/+ and mGlu4−/− mice were bred for the generation of knockout mice and their wild-type littermates. All mice used for the induction of ischemia were ∼10-weeks old (22 to 24 g, body weight). Animals were anesthetized with chloral hydrate (320 mg/kg, intraperitoneal). A rectal temperature probe connected to a heating pad was used to maintain body temperature at 37°C throughout the surgical period (up to ∼60 minutes after the induction of focal ischemia). We measured cerebral blood flow routinely in all mice undergoing middle cerebral artery occlusion (MCAO) by removing the skin over the right hemisphere and fixing a flexible optical filament by instant glue on the skull in correspondence to a major branch of the MCA. The optical filament was attached to a laser Doppler flow meter (PeriFlux System; Perimed, Cuggiono (MI), Italy) for assessing cerebral blood flow. Cerebral blood flow was measured throughout the surgical procedure when the animal was under deep anesthesia, including 30 minutes before and ∼60 minutes after MCAO. For induction of focal ischemia, the MCA was exposed by means of a burr-hole craniotomy performed using a dental drill. A thin layer of bone was preserved to protect the dura mater and the cortical surface against mechanical damage and thermal injury, and the remaining bone was removed. The MCA was occluded by electrocoagulation (Backhauss et al, 1992). Sham-operated animals were subjected to the same anesthesia and surgical procedure, except for MCAO. After surgery, all mice were placed in an incubator (Compact incubator, Thermo Scientific, AHSI, Bernareggio (MI), Italy) at 37°C for 2 hours, and then returned back to their home cages. Different groups of Sv129/CD1 mice (mGlu4+/+ or mGlu4−/− mice, n=7 to 8) or C57BL/6 (10 mice) were injected subcutaneously with either vehicle (Cremophor EL) or PHCCC (10 mg/kg) 30 minutes before MCAO. Animals were killed 24 hours after ischemia and their brains processed for histologic analysis. Additional groups of C57BL/6 mice (such as naive mice, mice anesthetized with chloral hydrate without temperature control on the heating pad, and mice undergoing MCAO under the standard procedure (see above)) were treated with vehicle or PHCCC (four mice per group) for measurements of rectal temperature at 0.5, 1, 3, 5, and 24 hours after injections.

Evans Blue Perfusion

Wild-type or mGlu4−/− mice were perfused with Evans blue dye for qualitative analysis of the cerebrovascular anatomy. Animals were anesthetized and perfused with 4% formalin, followed by Evans blue (1% in saline) in the left cardiac ventricle. After decapitation, the brains were carefully removed and the vessels with their branches were examined under a microscope.

Surgery Procedures for Induction of Transient Focal Ischemia and Electroencephalography Recording in Rats

Male Wistar rats (320 to 435 g, body weight) were placed in a stereotaxic apparatus (Narishige Sci. Instr. Lab., Tokyo, Japan). For endothelin-1 (Et-1) infusion, a 21-G stainless steel guide cannula was implanted in the left hemisphere at anterio-posterior (AP) +0.2 mm, lateral-medial (LM) 5.2 mm, and dorsal-ventral (DV) −6.0 mm (Paxinos and Watson, 1997) in the vicinity of the MCA. Before the day of surgery, a piece of stainless steel wire (A-M Systems Inc., Carlsborg, WA, USA) was soldered to the upper end of the stainless steel guide cannula, which was insulated manually with two thin layers of epoxylite (Poxidol, Adefal Trading S.A., Montevideo, Uruguay), except at the tip. This electrode served for electroencephalography (EEG) recording of the granular field of the insular cortex (GI). Small stainless steel screw electrodes 1 mm in diameter, 2 mm long (AgnTho's AB, Lidingö, Sweden, cat. Nr MCSM1 × 2) were fixed epidurally in the left hemisphere above the forelimb region of the somatosensory cortex (S1FL) at AP +1.2 mm and LM 4.0 to 4.5 mm, and above the primary visual cortex (V1) at AP −7.0 mm and LM 4.5 mm. Pieces of stainless steel wire (A-M Systems Inc.) coated with Teflon (bare diameter 0.005 inches) were soldered to the caps of two screws after removing the isolation at the end. For recording the EEG from the barrel field of the somatosensory cortex (S1BF), we used a piece of the same stainless steel wire inserted into a small hole made in the lateral convexity of the skull at AP −0.3 mm and LM 6.0 to 6.5 mm. Thereafter, the wire was bent against the bone where it was fixed using a small quantity of tissue glue. Another two miniature stainless steel screws, one fixed on the skull above the frontal bone and the other posterior to the lambda, were used for ground and common reference, respectively. Two wires (stainless steel, 0.008 inches Teflon-coated, A-M Systems Inc.) were inserted into the dorsal neck muscles for recording of muscle activity and rat movements. A mound of Duracryl (Spofa Dental, Prague, Czech Republic) self-curing resin was then constructed around the cannula, screws, and wires, forming a cap to secure them to the skull together with the female connector. During surgery, body temperature was maintained at 37°C using a heating pad.

Induction of Transient Focal Ischemia in Rats

Transient focal ischemia was induced by unilateral infusion of the potent vasoconstrictor, Et-1 (human/porcine Et-1, Sigma-Aldrich, St Louis, MO, USA; 150 pmol/3 μL per 3 min), in the left hemisphere 1 week after implantation of the guide cannula. We could not measure cerebral blood flow in these animals because Et-1 was infused without anesthesia. Endothelin-1 was dissolved in sterile 0.9% NaCl and infused in close proximity to the MCA using a 27-G injection needle inserted into the implanted guide cannula and protruding 2.0 mm from its tip. Animals were placed on a rectal temperature feedback-controlled pad (Digitherm, Yukon-PC, Sofia, Bulgaria), which maintained body temperature at ∼37°C. A total of 40 rats were infused with Et-1; 11 rats were infused with saline alone and considered as nonischemic controls. A maximum of four rats per day were infused with Et-1 to facilitate behavioral assessment and EEG recording. Three of the animals infused with Et-1 died soon after the infusion. Of the remaining 37 rats infused with Et-1, 5 did not show neurologic abnormalities in the first 20 minutes after infusion, and were considered nonischemic. The inclusion/exclusion criteria were determined by examining (1) unequal forelimb flexion in rats held by the tail and suspended above the floor; (2) contralateral shoulder adduction (curling); (3) unequal resistance to left lateral displacement compared with right displacement; and (4) circling contralateral to the infusion side during spontaneous ambulation. These tests were performed at least three times during the 15-minute period after Et-1 infusion. The remaining 32 rats were considered ‘ischemic' on the basis of the neurologic pretest. These animals were randomly divided into 2 groups: 16 rats were treated subcutaneously with 10 mg/kg PHCCC (Et-1/PHCCC) and 16 rats with vehicle (Et-1/vehicle) 20 minutes after Et-1 infusion. Control nonischemic rats that did or did not receive intracerebral infusion of saline were combined into one control group (SHAM) because they did not differ in all pilot tests. SHAM rats were also treated subcutaneously with either PHCCC (6 rats) or vehicle (5 rats) 20 minutes after infusion. Eight animals from the Et-1/vehicle group and eight from the Et-1/PHCCC group were killed after 3 days for histologic analysis. All remaining animals infused with Et-1 underwent behavioral analysis and EEG recording up to 14 days after infusion. Control nonischemic rats were only subjected to behavioral analysis. For each ischemic animal, behavioral analysis always preceded EEG recording by ∼15 minutes.

Assessment of Neurological Function

All behavioral tests were performed three times before Et-1 infusion and then after Et-1 infusion: at 15 minutes, 1 hour, 4 hours, 24 hours, or 1, 3, 7, and 14 days. All rats were coded and randomized to different treatment groups by an experimenter who was not involved in behavioral testing. Behavioral experiments, EEG recording, and histologic analysis were carried out by experimenters who were not aware of treatments. The codes were disclosed after termination of all procedures and processing of data.

Posture/Hang Reflex Test

The posture/hang reflex (PHR) test was carried out as reported previously (Moyanova et al, 2003). In brief, each rat was examined for sensorimotor deficits both contralaterally and ipsilaterally to the hemisphere where Et-1 was infused and consisted of three subtests. First, the rat was examined for degree of abnormal posture when suspended by its tail 50 cm above a platform. It was slowly lowered toward the platform and its posturing was observed. Intact rats extended both forelimbs toward the platform, and this was given a score of 3, with no observable pathologic signs. Rats with consistent flexion of the right forelimb were ranked with score 2. Rats with moderate sensorimotor deficits were scored 1, whereas those with severe neurologic deficits were scored 0.

Forelimb/Hindlimb Placing Test

The forelimb/hindlimb placing (LP) test was carried out as described previously (Moyanova et al, 2003). The test consisted of four tasks assessing the sensorimotor integration of both forelimbs and both hindlimbs in response to visual, tactile, and proprioceptive stimuli. The scores were as follows: 2 (lack of sensorimotor deficit), rats performed immediate and correct placement of forelimbs and hindlimbs on the platform; 1 (moderate sensorimotor deficit), rats performed with a delay (⩾2 seconds) and/or incompletely; and 0 (severe sensorimotor deficit), rats did not perform normally. Summing up the scores of all 8 LP tests yielded a total maximal neurologic score of 16 in a normal rat for each side. A lower total score implicated impairment in the sensorimotor integration for the contralateral (right) side to Et-1 infusion.

Limb-Use Asymmetry Test

The Schallert cylinder test was used to examine asymmetries in forelimb use for postural support behaviors (Schallert et al, 2000). Rats were placed in a 30 cm in height and 20 cm in diameter cylinder. Behavior was quantified by determining the number of ipsilateral and contralateral limb placements on the wall of the cylinder when rearing, and the number of times both forelimbs were used simultaneously for support on the wall. To prevent habituation to the cylinder, the number of movements recorded in any one trial was limited to 20. The following percentages were calculated: (1) percentage use of the right (contralateral) forelimb of total number of limb placements (left+right+both) and (2) percentage use of the left (ipsilateral) forelimb of the total number of placements made. An index of limb-use asymmetry was then obtained by subtracting the percentage use of the contralateral forelimb from the percentage use of the ipsilateral forelimb (a higher score indicates greater asymmetry).

Adhesive Tape Removal Test

The adhesive removal test measures somatosensory neglect. The test was performed as follows: two sticky tapes of equal size 1 cm2 were placed on the radial surface of the wrists of both forepaws as bilateral tactile stimuli. The trial ended when the rat removed both tapes, or after 120 seconds if the tapes had not been removed. The time (in seconds) required to remove the adhesive from each forelimb was measured using a stopwatch. First contact and first removal of the sticky tape are indicative of the more sensitive forelimb. The order (left versus right) in which the animal removed the stimuli was also recorded. Testing sessions consisted of four trials per animal for each time point, with a 1 to 2 minute intertrial interval. Normally, rats take approximately the same amount of time to remove labels from both sides, whereas animals with forelimb somatosensory impairments take significantly longer to remove the tape from the contralateral paw and show a bias toward contacting the stimulus on the ipsilateral (nonimpaired) limb.

Chronic Electroencephalography Recording and Electroencephalography Spectral Analysis

The procedure for EEG recording was described previously (Moyanova et al, 2007). In brief, recording of EEGs started 6 to 7 days after surgery and was performed on freely moving rats during passive wakefulness. Recordings were carried out in a Faraday box equipped with a multichannel mercury swivel commutator. The EEG was recorded using a Nihon Kohden (Tokyo, Japan) EEG machine with a high-pass filter (−3 dB at 0.16 Hz), a low-pass filter (−3 dB at 70 Hz), and a notch filter (50 Hz) to reduce power-line noise. The electromyogram (time constant 0.1 seconds, low-pass filter 500 Hz) and rat movements (time constant 1.0 seconds, low-pass filter 15 Hz) were used to control vigilance and for offline extraction of suitable EEG epochs for further processing. Baseline EEG recordings were made two consecutive days before and immediately before Et-1 infusion. Epochs of 4-second duration were selected offline from EEG trials of 15 minutes and only EEG epochs free from artifact residuals were stored in computer files for subsequent analysis. Calculation of the power spectra was performed by means of fast Fourier transformation (BMDP Statistical Package; BMDP Statistical Package, University of California, Los Angeles, CA, USA). Spectral densities (μV) were computed as square roots of EEG powers (μV2) per 0.25 Hz bin over a spectra of 1 to 32 Hz (125 frequency bins), and spectral densities for all 4-second epochs in a session (mean number 24) were averaged to obtain normalized root mean square (RMS) for each 0.25 Hz frequency bin, each time session, each treatment, and each rat. For EEG recordings, the time sessions following Et-1 were 15 minutes, 1.25 hours, 4.25 hours, and 1, 3, 7, and 14 days. Log10 transformation was then applied for normalization versus before-Et1 values (mean of the three baseline records, see above) for each frequency bin. As a functional measure of brain recovery after the insult, we used a quantification of the postischemic return in EEG amplitude estimated as percentage change in total RMS (1 to 32 Hz) estimated at each time point compared with the RMS at 15 minutes following Et-1 computed according to the formula: (RMS1.25 hours, 4.25 hours, 1 day, 3 days, 7 days, 14 days−RMS0.25 hours/RMS0.25 hours) × 100 (%).

Histologic Analysis and Calculation of the Infarct Volume

The brains were fixed in Carnoy's solution, embedded in paraffin, and sectioned at 10 μm. Sections were deparaffinized and processed for staining with thionin (Nissl staining for histologic assessment of neuronal degeneration). The analysis was performed on sections regularly spaced every 550 μm through the extension of the ischemic region. Infarct volume was calculated by integrating the cross-sectional area of damage on each section and the distance between the various levels. In each stained section, the necrotic area was identified and outlined at a magnification of × 2.5 and measured using Scion Image software (NIH, Bethesda, MD, USA). The infarct volume (V) was calculated by the following formula: V=Σ (Ai × TS × n), where Ai is the ischemic area measured at the ith section, TS the section thickness (10 μm), and n the number of sections between two adjacent levels.

Statistical Analysis

The following nonparametric statistics were used for the analysis of PHR and LP behavioral tests: Friedman's analysis of variance (ANOVA) for evaluation of time-dependent effects and the Wilcoxon-matched pairs test for the effects of Et-1 versus pre-Et-1 scores; and Kruskal–Wallis ANOVA for multiple unrelated samples followed by the Mann–Whitney U-test. Limb-use asymmetry and adhesive tape removal tests were analyzed using the general linear model for two-way ANOVA of the statistical package Statistica 7.0 (Statsoft, Tulsa, OK, USA) for effects of the groups (SHAM, Et-1/vehicle, and Et-1/PHCCC), time (T0, 1 hours, 4 hours, and 1, 3, 7, and 14 days), and group × time interactions. Fisher's least significant difference (LSD) was used as a post hoc test. The EEG data were analyzed using the Wilcoxon-matched pairs test and the Mann–Whiney U-test. Statistical analysis of infarct volume data was performed by Student's t-test or one-way ANOVA plus Fisher's LSD. Differences with a P-value <0.05 were considered significant.

Results

Protective Role of Metabotropic Glutamate-4 Receptors in the Middle Cerebral Artery Occlusion Model of Permanent Focal Ischemia

We first examined whether the lack of mGlu4 receptors could affect the extent of the ischemic lesion in mice subjected to MCAO. Evans blue dye perfusion did not reveal the presence of abnormalities in the cerebrovascular anatomy of mGlu4−/− mice (Figure 1A). However, the extent of infarct volume was 25% to 30% greater in mGlu4−/− mice than in mGlu4+/+ littermates 24 hours after MCAO (Figures 1B and 1C). We then treated both mGlu4+/+ and mGlu4−/− mice with the brain-permeant selective mGlu4 receptor enhancer, PHCCC. The drug was injected subcutaneously 30 minutes before MCAO at a dose of 10 mg/kg, which was known to be fully effective in in vivo studies (Battaglia et al, 2006; Ngomba et al, 2008). Treatment with PHCCC reduced the infarct volume by ∼35% in mGlu4+/+, but was ineffective in mGlu4−/− mice (Figures 1B and 1C), indicating that PHCCC was protective by the activation of mGlu4 receptors. No changes in the reduction of cerebral blood flow in response to MCAO were found among wild-type and mGlu4−/− mice treated with or without PHCCC (blood flow (% of preischemic values)=17+1.5 in wild-type mice treated with vehicle; 15+2.3 in mGlu4−/− mice treated with vehicle; 17+3.4 in wild-type mice treated with PHCCC; and 18+3.6 in mGlu4−/− mice treated with PHCCC (means+s.e.m.; n=7 to 8); PHCCC treatment did not reduce blood flow in the 30-minute interval preceding MCAO, as well as in sham-operated mice at least for 90 minutes after drug injection (not shown)). To exclude that possibility, protection by PHCCC was restricted to hybrid Sv129/CD1 mice, and we extended the study to C57BL/6 mice subjected to MCAO. Treatment with PHCCC in these mice reduced the infarct volume to a slightly greater extent (40% to 45%) than in Sv129/CD1 wild-type mice (Figures 2A and 2B), indicating that pharmacological activation of mGlu4 receptors is protective against ischemic brain damage in different strains of mice. The drug PHCCC did not affect the reduction in cerebral blood flow induced by MCAO in C57BL/6 mice (blood flow (% of preischemic values)=13+3.2 in mice treated with vehicle and 12+2.6 in mice treated with PHCCC (means+s.e.m.; n=10 in each group)). Treatment with PHCCC (10 mg/kg, subcutaneous) had no effect on body temperature (at least at 0.5, 1, 3, 5, and 24 hours after drug injection) in naive mice, in mice undergoing chloral hydrate anesthesia without temperature control by means of the heating pad, and in mice undergoing MCAO 30 minutes after PHCCC according to the standard procedure (not shown).

Figure 1.

Figure 1

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mGlu4-/- mice show an increased infarct volume after permanent focal ischemia and mice are not responsive to PHCCC. Evans blue perfusion showing the absence of anatomic abnormalities of the MCA in mGlu4 receptor knockout (mGlu4−/−) mice with regard to wild-type littermates is shown in A. Brain Nissl staining of male mGlu4 receptor knockout (mGlu4−/−) mice or their wild-type (mGlu4+/+) littermates subjected to permanent MCAO and treated systemically with vehicle or PHCCC (10 mg/kg subcutaneous, administered 30 minutes before the onset of ischemia). Mice were killed 24 hours after MCAO. Representative images are shown in B. In C, values of the infarct volume are means±s.e.m. (n=7 to 8). P<0.05 (one-way ANOVA plus Fisher's PLSD) versus the respective wild-type (mGlu4+/+) mice (*) versus mGlu4+/+ mice treated with vehicle (#). Values were calculated by integrating the cross-sectional area of damage at each bregma level and the distances between the various levels. ANOVA, analysis of variance; MCA, middle cerebral artery; MCAO, middle cerebral artery occlusion; mGlu4, metabotropic glutamate-4; PHCCC, N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-caboxamide.

Figure 2.

Figure 2

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Brain Nissl staining of C57BL/6 mice subjected to permanent MCAO and treated systemically with vehicle or PHCCC (10 mg/kg subcutaneous, administered 30 minutes before the onset of ischemia). Mice were killed 24 hours after MCAO. Representative images are shown in A. In B, values of the infarct volume are means±s.e.m. (n=10). *P<0.05 (Student's t-test) versus mice treated with vehicle. MCAO, middle cerebral artery occlusion; PHCCC, N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-caboxamide.

We also examined the toxicity profile of PHCCC in intact C57BL/6 mice by injecting the drug subcutaneously at doses of 10, 30, 300, and 1,000 mg/kg. None of the mice died within 15 days from the injection, even when administered the highest dose of PHCCC (data not shown). The rotarod test did not reveal any motor impairment in mice 1 hour after treatment with 10 or 30 mg/kg of PHCCC. Treatment with 300 and 1,000 mg/kg of PHCCC reduced the time spent on the rotating rod from 470+115 seconds to 240+95 seconds and 72+42 seconds, respectively (P<0.05 versus mice treated with vehicle; one-way ANOVA+Fisher's PLSD). Thus, ischemic mice were protected against permanent focal ischemia by a subcutaneous dose of PHCCC (10 mg/kg) that did not induce motor incoordination in intact mice and was at least 100-fold lower than the lethal dose.

Pharmacological Activation of Metabotropic Glutamate-4 Receptors is Protective in the Endothelin-1 Rat Model of Transient Focal Ischemia

The drug PHCCC (10 mg/kg) was administered subcutaneously 20 minutes after Et-1 infusion, and ischemic damage was assessed 3 days later (Moyanova et al, 2007; Mastroiacovo et al, 2009). The drug reduced the infarct volume by 35% to 40% as compared with control animals; this reduction was prominent in subcortical regions (Figures 3A and 3B).

Figure 3.

Figure 3

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Brain Nissl staining of rats subjected to Et-1 infusion and treated with vehicle or PHCCC (10 mg/kg subcutaneous, 20 minutes after the onset of transient focal ischemia). Representative images are shown in A. In B, values of the infarct volume are means±s.e.m. (n=8 per group). #,*P<0.05 (Student's t-test) as compared with ischemic rats treated with vehicle. Et-1, endothelin-1; PHCCC, N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-caboxamide.

We also assessed the protective effects of PHCCC using a battery of sensorimotor tests. In the PHR test, all ischemic rats (n=32) showed a moderate-to-severe contralateral postural-motor deficit, which was more severe at 1 hour, 4 hours, and at 1 day after Et-1 infusion, and slowly recovered afterwards. Treatment with PHCCC (see above) increased the rate of recovery, which was already substantial at 1 day after Et-1 infusion (Figure 4A). In the LP test, which examines sensorimotor integration, focal ischemia severely affected contralateral LP placing; no deficit was noted on the ipsilateral side, neither in the forelimb nor in the hindlimb placing tests (not shown). The LP deficit decreased over time in both vehicle- and PHCCC-treated rats. The PHCCC-treated rats showed an even more severe impairment in the LP test within 4 hours after Et-1 infusion. However, these rats showed a faster and more complete recovery (see data at 3, 7, and 14 days after Et-1 infusion) (Figure 4B). In the limb-use asymmetry test, focal ischemia caused an increase in the asymmetry index, indicating preferential use of the unaffected ipsilateral left forelimb for upright postural support during wall exploration. This increase was maintained during the 14 days after Et-1 infusion. Ischemic rats treated with PHCCC rats had less forelimb asymmetry than did those treated with vehicle (particularly at 1 hour to 3 days after Et-1 infusion) (Figure 4C). In the adhesive tape removal test, the latency to remove an adhesive tape from the contralateral forepaw was substantially increased in ischemic rats treated with vehicle at all times after Et-1 infusion. Some of these rats did not remove the contralateral tape until the end (120 seconds) of each trial, thus showing sensory hemineglect on the side contralateral to Et-1 infusion. Ischemic rats treated with PHCCC showed no contralateral hemineglect at any time after Et-1 infusion (Figure 4D), i.e., these rats did not show contralateral hemineglect. In addition, ischemic rats treated with vehicle showed a significant ipsilateral bias, i.e., an increase in the percentage of trials in which the ipsilateral stimulus was contacted before the contralateral stimulus. This deficit was not shared by ischemic rats treated with PHCCC at any time after Et-1 infusion (Figure 4E).

Figure 4.

Figure 4

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Effects of PHCCC treatment (10 mg/kg, subcutaneous) on neurologic function in rats developing transient focal ischemia in response to Et-1 infusion. (A) The PHR test, (B) the LP test, and (panel C) the LUA test were carried out before (T0), and at 1 hour (1h), 4 hours (4h), 1 day (1d), 3 (3d), 7 (7d), and 14 days (14d) after Et-1 infusion. (D, E) The ATR test was not performed at 1 and 4 hours. Values are means±s.e.m. of five to eight determinations. In panel A, the time effect (Friedman's nonparametric ANOVA) in all three groups was significant (χ2=61.5, P<0.001). ANOVA by ranks (Kruskal–Wallis test) revealed a group effect (P<0.05) from day 1 to day 14 because the neurologic score of the Et-1/PHCCC group was higher than the score of the Et-1/vehicle group at 1, 3, and 7 days (Mann–Whitney U-tests, P<0.05). In the Et-1/PHCCC group, recovery was complete at 7 days (no difference between Et-1/PHCCC and SHAM groups, Mann–Whitney U-test, P>0.05). In B, nonparametric Kruskal–Wallis ANOVA on contralateral LP scores in all three groups (namely SHAM, Et-1/vehicle, and Et-1/PHCCC) revealed a significant overall group effect as follows: P<0.001 at 1 and 4 hours; P<0.01 at 3, 7, and 14 days. Ischemic rats treated with vehicle had a severe impairment as compared with the performance of the same rats before Et-1 infusion (Wilcoxon's matched pairs test, P<0.05). The LP deficit decreased over time, as revealed by the nonparametric Friedman's test, showing a time effect in both ischemic groups (P<0.0001). In panel C, two-way repeated-measures ANOVA revealed a significant main effect of group (SHAM, Et-1/vehicle, and Et-1/PHCCC): F2,19=5.6 (P<0.05) and interaction group × time: F12,114=4.2 (P<0.001). Post hoc Fisher's LSD comparisons showed that the asymmetry index increased significantly after Et-1 infusion throughout the experiment, with a tendency for recovery after 7 days. In panels D and E, two-way ANOVA with repeated measures showed significant effects of group (F2,19=22.0, P<0.0001 in panel D; F2,19=4.7, P<0.05 in panel E) and interaction group × time (F8,76=2.4, P<0.05 in both panels D and E). Significant differences among experimental groups in all tests (panels A to E) (P<0.05): #Et-1/vehicle versus determinations at T0; §Et-1/vehicle versus SHAM; *Et-1/vehicle versus Et-1/PHCCC; **Et-1/PHCCC versus SHAM. ANOVA, analysis of variance; ATR, adhesive tape removal; Et-1, endothelin-1; LP, forelimb/hindlimb placing; LUA, limb-use asymmetry; PHCCC, N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-caboxamide; PHR, posture/hang reflex.

Assessment of Electroencephalography Changes

Acute effects of Et-1 infusion on EEG were recorded in four cortical areas (namely GI, S1BF, S1FL, and V1) at 15 minutes, 1 hour, and 4 hours after Et-1 infusion. Chronic effects were then assessed at 1, 3, 7, and 14 days after Et-1 infusion. A control postischemic EEG recording was made 15 minutes after Et-1 infusion, just before vehicle or PHCCC treatment. The time sessions are described in the Materials and Methods section. A significant reduction in EEG amplitude in all four cortical areas was measured at 15 minutes in all ischemic rats. Estimation of this amplitude suppression yielded 32.4%±7.3% (mean±s.e.m.). No EEG suppression was observed in SHAM rats. Representative raw EEG traces in the left somatosensory cortical area, forelimb region (S1FL) in two ischemic rats treated with vehicle, or PHCCC are shown in Figure 5A. Figures 5B to 5E show the time evolution of recovery of EEG power in the four cortical areas versus the corresponding EEG suppression 15 minutes after Et-1 infusion. In the Et-1/vehicle group, EEG was still suppressed at 1 hour, especially in the insular granular cortex GI (Figure 5B) and primary somatosensory area S1BF (Figure 5C) where the suppression was even greater than after 15 minutes: the bars of recovery are below the zero axis at 15 minutes. At 4.25 hours, the EEG in all cortical areas began to recover, but in the majority of rats, the EEG remained suppressed (see the representative EEG in the S1FL, Figure 5A). The EEG amplitude recovered (P<0.05) first in the V1 area (at 4.25 hours, Figure 5E), then in S1FL (at 1 day, Figure 5D), and only much later in S1BF (at 14 days, Figure 5C); it did not recover at all in GI (Figure 5B). In some rats, polymorphic delta activity appeared by 1 day after Et-1, as indicated by large slow-wave activity in the EEG (see the EEG trace at Figure 5A) associated with increase in slow band powers (not shown).

Figure 5.

Figure 5

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Selective activation of mGlu4 receptors induces an earlier recovery of MCA occlusion-induced suppression of the EEG waves in the cortex. Effect of PHCCC compared with the effect of its vehicle on recovery of EEG after Et-1-induced ischemia. The recovery of EEG amplitude is shown for four cortical areas: granular field of the insular cortex (GI) shown in B; barrel field of the somatosensory cortex (S1BF) shown in C; forelimb region of the somatosensory cortex (S1FL) shown in D; and visual cortex (V1) shown in E. The EEG recovery (in %) was estimated by means of power spectral density analysis relative to the EEG amplitude measured in the acute phase of ischemia (15 min after Et-1). Representative raw EEG traces as shown in A from the ipsilateral S1FL from Et-1/vehicle rat and Et-1/PHCCC rat. Vehicle or PHCCC (10 mg/kg) were administered subcutaneously 20 minutes after Et-1 (arrows), i.e., just after the EEG recording at 15 minutes (0.25 hours) after Et-1. All measurements were normalized to corresponding baseline values (before Et-1 infusion) and presented as mean±s.e.m. (n=8 per group). EEG amplitude was recovered after stroke in both groups Et-1/vehicle and Et-1/PHCCC. #Et-1/vehicle and Et-1/PHCCC versus 0.25 hours (zero at the ordinate), i.e., before treatment with vehicle or PHCCC (Wilcoxon's matched pairs test, P<0.05). *Et-1/PHCCC versus Et-1/vehicle (Mann–Whitney U-test, P<0.05). EEG, electroencephalography; Et-1, endothelin-1; PHCCC, N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-caboxamide.

With the administration of PHCCC, the EEG amplitude recovered first in S1FL (P<0.05, 4.25 hours) (Figure 5D). A significant increase in the percentage of EEG recovery was measured later (day 1) in this cortical area in the Et-1/PHCCC group compared with the Et-1/vehicle group. Recovery of the EEG amplitude in Et-1/PHCCC rats began earlier (4 hours) than recovery after administration of vehicle (1 day after MCAO). Most importantly, recovery occurred not only in the S1FL region but also in the SB1F and G1 regions; in vehicle-treated ischemic rats, EEG suppression lasted longer in S1FL. However, the effect of PHCCC on the EEG amplitude in all brain regions was temporary or was masked by spontaneous recovery of the electrical activity. In the majority of Et-1/PHCCC rats, delta polymorphic EEG activity did not appear (see 1 day, Figure 5A).

Discussion

Metabotropic glutamate receptors have a modulatory role in excitatory synaptic transmission, as opposed to AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) or NMDA receptors, which mediate fast excitatory synaptic transmission. For this reason, mGlu receptor ligands are devoid of the typical adverse effects of NMDA or AMPA receptor antagonists and are, therefore, promising drugs for the treatment of brain ischemia or other neurodegenerative disorders (reviewed by Bruno et al (2001)). A large body of evidence indicates that mGlu1 receptor antagonists are neuroprotective in in vivo models of global or focal ischemia and in organotypic hippocampal cultures exposed to oxygen–glucose deprivation (Pellegrini-Giampietro et al, 1999). These drugs act as neuroprotectants by enhancing GABA release and/or by activating the phoshatidylinositol-3-kinase/Akt pathway (Battaglia et al, 2001; Cozzi et al, 2002). Orthosteric agonists of mGlu2/3 receptors show protective activity in models of global ischemia, but not in models of focal ischemia (Bond et al, 1999, 2000; Cai et al, 1999). A potential bias of these drugs is that mGlu2 and mGlu3 receptors have differing influences on excitotoxic neuronal death, with mGlu3 receptors being neuroprotective and mGlu2 receptors being neurotoxic (Corti et al, 2007). Transient global ischemia leads to an early increase in mGlu4 receptor mRNA levels in the hippocampus and parietal cortex, with no changes in the transcript of mGlu1, mGlu2, and mGlu5 receptors, and a decrease in mGlu3 receptor mRNA levels (Rosdahl et al, 1994; Iversen et al, 1994). The selectivity of these changes suggests a critical role for mGlu4 receptors in the mechanisms of neurodegeneration/neuroprotection associated with brain ischemia. However, the study of mGlu4 receptors has been limited by the slow development of selective drugs as compared with other mGlu receptor subtypes. (R,S)-4-Phosphonophenylglycine (PPG), an orthosteric agonist of mGlu4 receptors that also activates mGlu7 and mGlu8 receptors, is protective against excitotoxic neuronal death in culture (Bruno et al, 2000; Henrich-Noack et al, 2000), and improves the recovery of neuronal function in acutely isolated hippocampal slices subjected to oxygen–glucose deprivation (Henrich-Noack et al, 2000). However, PPG has no effect on neuronal damage in models of focal or global ischemia (Henrich-Noack et al, 2000). Perhaps the efficacy of PPG or other orthosteric agonists in vivo is limited by the high levels of ambient glutamate that saturate mGlu4 receptors during ischemia–reperfusion. The use of PHCCC overcomes this limitation because the drug behaves as a positive allosteric modulator of mGlu4 receptors, and, therefore, selectively enhances the function of receptors that are activated by the endogenous glutamate. In cells expressing mGlu4 receptors, PHCCC is inactive on it own, but enhances both the potency and the efficacy of -glutamate or other orthosteric agonists (Maj et al, 2003). In this study, we have used PHCCC in in vivo models of focal ischemia at doses (10 mg/kg, subcutaneous) that were shown to be centrally active (Battaglia et al, 2006; Ngomba et al, 2008) and that did not cause motor impairment in intact mice. The drug PHCCC was protective against permanent or transient focal ischemia, and, at least in mice, its action was entirely mediated by the activation of mGlu4 receptors. Interestingly, mGlu4−/− mice underwent greater damage in response to MCAO, suggesting that endogenous activation of mGlu4 receptors limits the extent of ischemic neuronal death. This evidence is in line with in vitro studies that show greater vulnerability of cortical neurons lacking mGlu4 receptors to excitotoxic neuronal death (Bruno et al, 2000). Thus, PHCCC may act to potentiate an endogenous defensive mechanism against ischemic brain damage. The mGlu4 receptor is expressed presynaptically and negatively modulates glutamate release (Millán et al, 2002; Panatier et al, 2004). The drug PHCCC might limit the amount of synaptically released glutamate in response to ischemia, thus reducing the probability that the potentially harmful extrasynaptic NMDA receptor (see the ‘Introduction' section and references therein) is activated by the ambient glutamate. In the Et-1 model of transient focal ischemia, extracellular glutamate levels are maximally increased ∼30 minutes after Et-1 infusion (Bogaert et al, 2000), and remain high for several hours afterwards both in the striatum and in the cerebral cortex (Herz et al, 1996; Mauler et al, 2001). We injected PHCCC subcutaneously 20 minutes after Et-1 infusion, a time that roughly corresponds to the peak of glutamate release, taking into account the transfer of the drug from the peripheral injection site to the brain. We examined the protective activity of PHCCC against ischemic brain damage in the Et-1 infusion model by assessing the following output measures: (1) volume of brain infarction, (2) brain electrical activity monitored by EEG, and (3) neurologic impairment monitored by four sensorimotor behavioral tests. Treatment with PHCCC reduced total infarct volume by ∼40% in Et-1-infused rats. The reduction was more pronounced in the dorsal striatum (∼50%) than in the cerebral cortex. This is in line with the high expression of mGlu4 receptors in the caudate putamen (Corti et al, 2002). The striatum is generally considered to be the core of the ischemic lesion following MCAO (Tyson et al, 1984). The barrel region of the somatosensory cortex (S1BF) is also part of the ischemic core, as supported by the extensive damage observed in Et-1-injected rats treated with vehicle. In contrast, the forelimb region of the somatosensory cortex (S1FL) is usually considered to be part of the ischemic penumbra in the Et-1 model (Windle et al, 2006, Moyanova et al, 2008). In spite of the accepted view that the ischemic core is nontreatable, some studies have shown reduced striatal damage following various putative neuroprotective compounds (Callaway et al, 2003). Thus, our finding that PHCCC treatment was protective against striatal damage in Et-1-infused rats is remarkable. Interestingly, MK-801, a slow-kinetic NMDA channel blocker that is considered as the gold-standard neuroprotective agent in experimental animal models of brain ischemia, reduces the infarct volume in the cerebral cortex, but not in the dorsolateral striatum in Et-1-infused rats (Moyanova et al, 2007). The broader neuroprotection afforded by PHCCC as compared with MK-801 suggests that drugs that amplify mGlu4 receptor function may be more beneficial than nonsubunit selective NMDA receptor antagonists in models of transient focal ischemia. Once again, it is possible that activation of mGlu4 receptors by PHCCC reduces the amount of synaptically released glutamate, thus limiting the activation of ‘toxic' extrasynaptic NMDA receptors while leaving the activation of ‘trophic' synaptic NMDA receptors intact. As a surrogate of this hypothesis, ischemic rats treated with PHCCC showed a faster and greater recovery of neuronal function both electrophysiologically and behaviorally. Remarkably, EEG activity in PHCCC-treated rats was substantially recovered at 1 to 3 days after ischemia, when control rats showed a dramatic impairment in neuronal function. Behavioral tests were not equally sensitive to PHCCC. Ischemic rats treated with PHCCC did not show sensory hemineglect on the side contralateral to MCAO, and almost no asymmetry in the limb use at all times after drug injection. In contrast, the beneficial effect of PHCCC on postural-motor deficits in the PHR test became visible only at 1 day after MCAO, whereas PHCCC led to a transient impairment in the performance at the LP test from day 3 to day 14 after ischemia. Although these tests show an overall beneficial activity of PHCCC in ischemic rats, one should keep in mind that mGlu4 receptors critically regulate synaptic transmission in the first station of the ‘indirect' pathway of the basal ganglia motor circuitry, and that activation of these receptors is expected to have a strong impact on motor symmetry (reviewed by Conn et al (2005)). Thus, the early effect of the drug on sensory hemineglect and motor asymmetry (<1 day after ischemia) might result from a combination of neuroprotection and functional effects on striatal motor circuitry.

In conclusion, these data provide the first demonstration that activation of mGlu4 receptors limits the development of ischemic brain damage, improves postischemic sensorimotor function, and enhances recovering of the electrical activity of the brain. This finding is interesting from a therapeutical standpoint because mGlu4 receptor enhancers are under clinical development for the treatment of Parkinson's disease and other central nervous system disorders. These drugs are potential candidates for the experimental treatment of stroke, providing that they show a favorable profile of safety and tolerability in humans.

The authors declare no conflict of interest.

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