mGlu2−/− mice on a C57Bl/6 genetic background were kindly provided by Prof. Shigetada Nakanishi (Osaka, Japan) and the colony was generated by homozygous breeding. Han Wistar rats were purchased by Jackson Laboratories (Bar Harbor, ME) and genotyped for the presence of a stop codon mutation in the Grm2 gene (see below). All animals were housed under standard conditions with food and water ad libitum and a 12:12 h light-dark cycle. Studies were performed in agreement with the national and international guidelines and regulations on animal care and use, and were approved by the Neuromed Institutional Animal Care and Use Committee. All efforts were made to minimize animal suffering and to reduce the number of animals used.
Induction of transient MCAO in mice
Surgery, stroke induction, inclusion criteria
For measurements of mGlu2 receptor protein levels in the cerebral cortex at 6 h following ischemia, we used 4 non-ischemic and 5 ischemic C57Bl mice (20–25 g, b.w.). For the study of focal ischemia in mice lacking mGlu2 receptors we used 15 male mGlu2−/− mice and 11 age-matched male mGlu2+/+ mice (20–25 g, b.w.). Transient MCAO occlusion was induced as described by Nygren and Wieloch . Briefly, anesthesia was induced by inhalation of 2.5% isoflurane in N2O/O2 (70:30) and maintained at 2% by a face mask during the initial phase of surgery. Body temperature was maintained at 37 °C using a heating pad and controlled by a device connected with a temperature probe inserted into the rectum. rCBF was monitored by a flexible optical fiber connected to a laser Doppler (PeriFlux System 5000; Perimed, Jarfalla, Sweden) mounted on the skull in correspondence of the MCA of the right hemisphere. A silicon-coated filament (6–0 MCAO suture, Doccol Corporation, Sharon MA) was introduced into the internal carotid artery through an incision in the external carotid artery. The filament was advanced until it blocked the origin of the right MCA. Filament placement was confirmed by a reduction in laser Doppler flow and then, the isoflurane concentration was decreased to 1.5% during the MCA occlusion (45 min). We have included into analysis only mice with adequate occlusion, as determined by (1) rCBF reduction by at least 70% immediately after filament placement; (2) sustained reduction of rCBF for 45 min during occlusion; and, (3) complete recovery of rCBF within 5 min after the filament was removed. Therefore, 9 mGlu2+/+ and 9 mGlu2−/− mice were included in statistical analysis for evaluation of the infarct volume. Nine mGlu2+/+ and 11 mGlu2−/− mice were included in statistical analysis for evaluation of neurological deficits. Two mGlu2−/− mice died before performing the last behavioural test and therefore were not included in the infarct volume analysis. Seven mGlu2+/+ and 8 mGlu2−/− mice were used for statistical analysis of the grip strength test. After surgery, mice were injected with 0.5 ml of 5% glucose subcutaneously, placed into an incubator (Compact incubator, Thermo Scientific, AHSI, Bernareggio (MI), Italy) at 37C° for 2 h, and then returned back to their home cages. Animals were killed 48 h after MCAO and their brains processed for histologic analysis.
Western blot analysis of mGlu2 receptors following transient focal ischemia
Ischemic mice were killed by decapitation 6 h following reperfusion. A cortical area corresponding to the ischemic core (identified by the presence of a white boundary from the surrounding tissue) and the neighboring cortical area (see Fig. 1b) were dissected. The corresponding regions of the contralateral hemispheres and the corresponding regions of the cerebral cortex of sham-operated mice were also dissected and stored frozen at −80 °C. Samples homogenized at 4 °C in in a solution containing Tris-HCl (pH 7.5), NaCl (50 mM), EDTA (5 mM), and an Ultra Cruz Protease Inhibitor cocktail. Ten μg of proteins from the supernatants were separated by 8% SDS polyacrilamide gel. Proteins were transferred on immuno-blot PVDF membranes (Trans-Blot Turbo Transfer Systems, Bio-Rad, Segrate, MI, Italy), which were incubated with a polyclonal anti-mGlu2 receptor antibody (Abcam, Cambridge, UK; 1:1000 in t-TBS) for 1 h at room temperature, and then incubated for 1 more h with an anti-mouse secondary antibody (Calbiochem, San Diego, CA; 1:7000 in t-TBS). For β-actin immunostaining, membranes were incubated with a mouse monoclonal antibody (Sigma-Aldrich, St Louis, MO; 1:50,000) in milk 5%, for 1 h at room temperature, and then incubated for 1 more h with an anti-mouse secondary antibody (Calbiochem; 1:7000 in t-TBS). Immunostaining was revealed by the enhanced ECL western blotting analysis system.
Evaluation of neurological function
We assessed the neurological function 1 d before (T0) and at 2, 24, and 48 h after MCAO. Neurological function was scored by an investigator who was unaware of the genotype. We used the following grading system: 0 = no deficit; 1 = forelimb weakness; 2 = circling toward the affected, contralateral (left) side; 3 = partial paralysis of the affected side; 4 = no spontaneous motor activity .
Grip strength test
We assessed neuromuscular function by using the Grip strength meter (2 Biological Instruments, Besozzo, VA, Italy). The mouse voluntarily gripped a bar with either the healthy (ipsilateral) or the affected (contralateral) forelimb (FL) and pulled it backward. A mean of five trials was used for analysis. We assessed muscular strength one day before ischemia (T0) and then, at 2, 24, and 48 h after MCAO.
Quantitative analysis of the transcripts of mGlu1, −3, −4, −5, −7, and −8 receptors in the cerebral cortex and corpus striatum of non-ischemic mGlu2+/+ and mGlu2−/− mice
Mice were killed by decapitation and the brain was quickly removed; a cortical area corresponding to both dissected regions in Fig. 1b, and the striatum were dissected on ice and immediately frozen on liquid nitrogen, and stored at −80 °C. Total RNA was extracted using Trizol reagent (Thermo Fisher Scientific, Waltham, MA) according to manufacturer’s protocol. The RNA was then treated with DNAse (Qiagen, Hilden, Germany) and single strand cDNA was synthesized from 2 μg of total RNA using superscript III (Thermo Fisher Scientific) and random hexamers. Real-time PCR was performed as described previously . The following primers were used: mGlu1 receptor, forward CATACGGAAAGGGGAAGTGA and reverse AAAAGGCGATGGCTATGATG; mGlu3 receptor, forward CAGCAAGCTCCCTCTTTTGT and Rev. GCTAAAAGAGCCCGTCACTG; mGlu4 receptor, forward CTCCAGCCGCACGCTTGACA and reverse GTAGGCCGAGTCCTGCCCGA; mGlu5 receptor, forward ACGAAGACCAACCGTATTGC and reverse AGACTTCTCGGATGCTTGGA; mGlu7 receptor forward GGTTTTCGTCAAGCCAGAGA and reverse ATCACTGAGTTCAGGAGCCG; mGlu8 receptor forward CGGAATCTGAACTTGCTCGG and reverse GGGGGAAGGCTTTAGGGATTT; and TFRC (transferrin receptor protein-1) forward CCAGTGTGGGAACAGGTCTT and reverse GCACCAACAGCTCCAAAGTC.
DNA extraction, PCR amplification and sanger sequencing method for Han Wistar genotyping
DNA was extracted from the tail using Wizard genomic DNA purification kit (Promega Corporation, Madison, WI) according to manufacturer’s protocol. Fifty μg of DNA was used for PCR amplification with following primers forward: - 5′ GAACAGGAGTCAAAGATCATG 3′ and reverse: - 5′ CAGCACTATTACCGTCAAAC 3′. Thermal cycler conditions were as follows:10 min at 95 °C, 35 cycles of denaturation (30 s at 95 °C), annealing (30 s at 55°) and extension (45 s at 72 °C); the extension was continued at 72 °C for 7 min. Five μm of amplificated DNA was separated in a 2% agarose gel. All positive PCR products were purified by Minielute PCR purification kit (Qiagen) by standard procedures and 5 ng were sequenced with Big Dye Terminator v. 3.3 mix (Thermo Fisher Scientific) with forward primer by using the following thermal cycler conditions program: 30 cycles of denaturation (10 s at 95°) annealing (5 s at 54°) and extension (4 min 60°). Excess dye terminators were removed using 2.0 spin kit (Qiagen); samples were electrophoresed on an ABI Prism 310 genetic analyzer (Applied Biosystems, Foster City, CA) for genotype analysis.
Et-1-induced focal ischemia in mutant Han Wistar rats
Surgery, stroke induction, inclusion criteria
Twenty-six rats (weight 310 ± 40 g) were used: 13 Grm2 mutant Han Wistar and 13 WT. We used the Et-1 model of ischemia for the induction of transient focal ischemia. Rats were anaesthetized with an intramuscular injection of a mix of ketamine (60 mg/kg) and xylazine (12 mg/kg) dissolved into saline. A guide cannula was placed at the following stereotaxic coordinates: AP +0.2; ML +5.2  avoiding to damage the dura mater. A stainless steel wire was insert into the guide cannula to plug it until the day of the Et-1 infusion. The guide cannula was secured to the skull with two supporting screws and dental cement. We infused Et-1 (200 pmol in 3 μl of saline) 15–16 d after the surgery (a 5 days-treatment with Baytril antibiotic, 0.2 ml/kg, s.c., 3 d of handling, 5 d of habituation to the experimental room and experimental cage and training in the AT test, and 3 d for control sessions before the Et-1 infusion). Et-1 was infused in non-anesthetized rats by inserting an injection cannula into the guide cannula. The tip of the injection cannula targeted the piriform cortex approximately 0.2 mm from the MCA origin in the left hemisphere. The same procedure was carried out in sham-operated rats, infused with vehicle. We assessed the ischemia-induced neurological deficits 10–15 min after Et-1 infusion using the following 7-point scale: 0 = normal; 1 = held to face the table edge (to avoid vibrissae and snout contacts with the table, the rat chin was supported upward), the rat failed to place its right FL on the table when we pushed the limb down with a soft bar (failed proprioceptive dorsal FL placing); 2 = held and slightly pushed to face the table edge from inside, the rat failed to hold its FL on the edge of the table and slipped off (failed proprioceptive ventral FL placing); 3 = suspended by the tail, the rat twisted the torso upward; 4 = suspended by the tail, the rat failed to extend the right FL down, and the limb was flexed and/or clenched; 5 = after being placed on the table, the rat turned its body to the right (spasmodic turning); 6 = the rat circled continuously to the right after being placed on the table. Ischemia was considered as severe with scores from 11 to 21, moderate, from 3 to 10; slight from 1 to 2. Twenty-two rats (10 WT and 12 Grm2 Han Wistar mutant) satisfied these inclusion criteria.
Neurological assessment and PHR test
We assessed spontaneous activity, symmetry in limb movement, forepaw outstretching, and resistance to lateral push and circling behavior . In detail, we assessed: (i) flexion and/or clenching of the digits and/or full flexion of the wrist when the rat was suspended by the tail (PHR test); (ii) asymmetry in resistance to applied lateral gentle pressure from behind the shoulders in the left and right directions (the rat was placed on a flat surface). Ischemic rats showed less resistance of the right part of the body (contralateral to the ischemic hemisphere). The right FL became stiff during the push, while the left FL offered resistance; (iii) body twisting when the rat was suspended by the tail (PHR test); and, (iv) circling or inability to walk straight when the rat was placed on a flat surface. The following 4-score grading system was adopted: 0 = impairment in (i), (ii), (iii), and (iv) (severe motor deficit); 1 = impairment in (i), (ii), and (iii) (moderate motor deficit); 2 = impairment in (i) and (ii) (mild motor deficit); 3 = no deficit.
Two adhesive tapes (1 × 1 cm) were placed on the radial aspect of both FLs alternating the order of their application, i.e., right versus left. Both ATs were then pressed slightly and simultaneously and rats were placed immediately within a box and the time in seconds (precision of measuring 1.0 s) needed to remove the ATs (latency) and the order (preference) of FLs (left or right) for removing the first AT was recorded. Rats were pre-trained (five trials for five successive days) to obtain optimal level of performance (latency ≤28 s) and to achieve absence of asymmetry between right and left FL performance before stroke induction. The experimenter was blind to the rat group (Et-1, sham-operated, WT or Grm2 mutant). A trial ended when three min had elapsed without removing of either of the two ATs. For the preference, the ipsilateral (Ipsi) and contralateral (Contra) values referred to mean percentage of trials (out of five for each pre- or post-Et-1 session) in which the rat removed the AT placed on the left or right FL, respectively. For the latency, the Ipsi and Contra values referred to mean time (for five trials in each pre- or post-Et-1 session) needed to remove the AT from the ipsilateral or contralateral FL, respectively.
Asymmetry assessment in behavioural performance (AT and PHR tests)
The asymmetry in behavioural performance was assessed by means of the Laterality Index (LI) = (Ipsi – Contra) / (Ipsi + Contra). For the PHR test, the range of LI was between 0 (normal PHR, equal scores for the right and left sides of the body) and +1.0 (maximum ischemic deficit, PHR score for the right, contralateral side was 0). For the preference in the AT test, having in mind that the range of the preference was between 0% and 100%, the range of LI was from +1.0 (full ipsilateral preference or maximum ischemic deficit) through 0.0 (normal performance) to - 1.0 (full contralateral preference). For the latency in the AT test, having in mind that the range of the latency was between 1.0 s and (equal to the precision of measuring) 180.0 s, the range of LI was from approximately +1.00 (fast contralateral response and lack of ipsilateral response) through 0.00 (equal responses of both FLs, normal), to approximately −1.00 (fast ipsilateral response and lack of contralateral response, maximum ischemic deficit). The time sessions were T0, 1, 24, 72 h, where T0 was mean value of LIs of Preference or Latency before the Et-1 (or Saline) infusion on three consecutive days, with five trials on each day. The values of laterality index were normalized by subtracting the control values (at T0).
Mice and rats were sacrificed respectively at 48 and 72 h post MCAO and brains were fixed in Carnoy’s solution, embedded in paraffin, and sectioned at 10 μm. Sections regularly spaced every 550 μm (through the extension of the ischemic region) were deparaffinized and processed for staining with thionin (Nissl staining for histological assessment of neuronal degeneration). The infarct area was outlined at magnification of X 2.5 and measured with Scion Image software (NIH, Bethesda, MD, USA), then the infarct volume (V) was calculated by integrating the cross-sectional area of damage on each level and the distance between them: V = Σ (Ai x TS_x n), where Ai is the ischemic area measured at i-th section, the TS is the section thickness (10 μm), and n is the number of sections between two adjacent levels. In addition, we measured in rats the infarct area at level of a single section (AP = + 0.2 mm from bregma) in the S1FL, S1DZ, S1ULp, S2, GI/DI, AI and Cl.
For the neurological test in mice and the PHR test in rats, we used Kruskal-Wallis nonparametric ANOVA for multiple unrelated samples (Statistical package Statistica 7.0, 2004, Statsoft, Tulsa, OK) to determine the overall group effect at each time point. Then Mann-Whitney U-test (corrected for the small size of independent samples) was performed for the evaluation of the differences between Et-1 and sham-operated rats, separately for WT and Grm2 mutant rats. Friedman ANOVA by ranks and subsequent Wilcoxon matched-pairs test for related samples were used for evaluation of changes in neurological scores in mice and asymmetry in the PHR test in rats as a function of time.
Statistical analysis of the grip strength test in mice and asymmetry in the AT test in rats was performed by three-way repeated measures Hotelling ANOVA (General Linear Model, Statistica7.0, Statsoft) with factors: Group (mGlu2+/+, mGlu2−/−), Side (Ipsilateral FL, Contralateral FL) and Time (T0, 2, 24 and 48 h) in mice, and two-way repeated measures Hotelling ANOVA for asymmetry in AT preference and latency with factors: - Group (WT, Grm2 mutant) and Time (T0, 1, 24 and 72 h) in rats. For Post-hoc analysis we used Fisher’s LSD or Dunnett’s tests. Student’s’ t test was used for the analysis of infarct volumes in mice and rats (Statistica 7.0, Statsoft).