Spinal Astrocyte Glutamate Receptor 1 Overexpression after Ischemic Insult Facilitates Behavioral Signs of Spasticity and Rigidity

Animals.

Male Sprague Dawley rats (300–350 g) were obtained from Harlan (Indianapolis, IN) and were housed in standard cages with corncob bedding. Animals had access to food and water ad libitum and were housed separately after surgery. A 12 h light/dark cycle (lights on at 7:00 A.M.) was used throughout.

Induction of spinal ischemic spasticity and rigidity.

Spinal ischemia was induced using the previously described technique (Taira and Marsala, 1996). Briefly, rats were anesthetized with 4% isoflurane in air and maintained with 1.5–2% isoflurane; body temperature was maintained at 37°C using a homeothermic blanket system. Distal arterial blood pressure and heart rate were monitored by a tail artery catheter. The left carotid artery was cannulated with a 20 gauge polytetrafluoroethylene catheter for blood withdrawal. To induce spinal ischemia, a 2F Fogarty catheter (Am. V. Mueller, CV 1035; Baxter, Irvine, CA) was passed through the left femoral artery to the descending thoracic aorta so that the tip reached the level of the left subclavian artery (10.8–11.4 cm from site of insertion). The intra-aortic balloon catheter was inflated with 0.05 ml of saline, and occlusion of aortic blood flow was confirmed by an immediate and sustained drop in distal arterial blood pressure. Systemic hypotension (40 mmHg) was maintained during occlusion by a blood collecting circuit (37.5°C) connected to the carotid artery and positioned at the height of 54 cm (40 mmHg). After ischemia, the balloon was deflated, and the blood was reinfused during a 60 s period. After blood reinfusion, 4 mg of protamine sulfate was administered subcutaneously. Stabilization of arterial blood pressure was then monitored for an additional 10 min, after which the arterial lines were removed and wounds were closed. Rats were then allowed to recover. Seven to 10 d after ischemia, animals were tested for the presence of spasticity and rigidity. Rigidity was identified by the presence of ongoing tonic electromyographic (EMG) activity measured in gastrocnemius muscle as well as continuous dorsal plantar flexion of paw digits. Spasticity was identified as an increase in muscle resistance during ankle rotation, which correlated with increased EMG activity measured in gastrocnemius muscle during the same time fame (see details below).

Intrathecal catheterization.

At 14–21 d after spinal cord ischemia, intrathecal catheters were implanted as described previously (Yaksh and Rudy, 1976; Hefferan et al., 2003). Under isoflurane anesthesia, an 8.5 cm catheter (polyethylene-5 connected to ∼4 cm of polyehtylene-10 for externalization) was inserted through an incision in the atlanto-occipital membrane of the cisterna magna. The catheter was externalized behind the head and sealed with a piece of stainless steel wire. Rats exhibiting normal forelimb gait, feeding, and grooming behavior were housed separately and allowed to recover for at least 4 d before drug treatment.

Peripheral muscle resistance measurement.

Direct measurement of hindlimb muscle resistance was performed as described previously (Marsala et al., 2005). Briefly, rats were individually placed in a plastic restrainer, and one hindpaw was securely fastened to the paw attachment metal plate, which is interconnected loosely to the “bridging” force transducer (LCL454G, 0–454 g range; or LCL816G, 0–816 g range; Omega, Stamford, CT). After a 20 min acclimation period, rotational force was applied to the paw attachment unit using a computer-controlled stepping motor (MDrive 34 with onboard electronics; microstep resolution to 256 microsteps/full step; Intelligent Motion Systems, Marlborough, CT), causing the ankle to flex. The resistance of the ankle to flexion was measured during 40° of rotation during 3 s (13.3°/s), and data were collected directly to a computer using custom software (Spasticity version 2.01; Ellipse, Kosice, Slovak Republic). Each recorded value was the average of three repetitions.

EMG recordings.

To record EMG activity, a pair of tungsten electrodes were inserted percutaneously into the gastrocnemius muscle 1 cm apart. EMG signals were bandpass filtered (100 Hz to 10 kHz) and recorded before, during, and after ankle flexion. EMG responses were recorded with an alternating current-coupled differential amplifier (model DB4; World Precision Instruments, Sarasota, FL) and stored on a computer for subsequent analysis. EMG was recorded concurrently with peripheral muscle resistance (PMR), and therefore data were recorded as the average of three measurements.

Evaluation of drug treatment on whisker/corneal reflex.

Because of the paraplegic phenotype after spinal ischemia, routine testing for drug-induced motor deficit (e.g., rotorod) is impractical. Therefore, only the supraspinally mediated whisker and corneal reflexes were tested in spastic rats: a cotton-tipped applicator was used to gently displace the whiskers or touch the outer edge of the eye while monitoring reactions. Responses were graded as follows: 0, normal; 1, mildly impaired; 2, consistently impaired (rarely present); 3, absent. Effects on normal motor activity were assessed in naive rats in an open-field paradigm.

Recording of motor evoked potentials.

Motor evoked potentials (MEPs) were recorded using a previously described technique (Kakinohana et al., 2006). Briefly, animals were anesthetized and maintained with ketamine (100 mg · kg⁻¹ · h⁻¹, i.m.). MEPs were elicited by transcranial electrical stimulation (20 V, 200 ms) of the motor cortex using percutaneously placed stainless steel stimulating electrodes on each side of the skull slightly lateral of midline in a rostrocaudal direction. Responses were recorded from the gastrocnemius muscle using silver needle (22 gauge) electrodes (distance between recording electrodes, 1 cm) and a differential amplifier (model DB4; World Precision Instruments) and digitized with custom software (EP 2001; Dr. Jan Galik, Institute of Neurobiology, Kosice, Slovak Republic). For each data point, 10 repetitions were averaged and stored.

Hoffman reflex recording.

Hoffman reflex (H-reflex) was recorded as previously described (Schwarz et al., 1994; Kakinohana et al., 2006). Briefly, under ketamine anesthesia (100 mg · kg⁻¹ · h⁻¹, i.m.) the right hindlimb of the animal was secured, and a pair of stimulating needle electrodes was transcutaneously inserted into the surroundings of the tibial nerve. For recording, a pair of silver needle electrodes was placed into the interosseous muscles between the fourth and the fifth or the first and the second metatarsal right foot muscles. The tibial nerve was stimulated using square pulses with increasing stimulus intensity (0.1–10 mA in 0.5 mA increments, 0.1 Hz, 0.2 ms; Isostim A320; World Precision Instruments), and responses were recorded with an alternating current-coupled differential amplifier (model DB4; World Precision Instruments). The threshold for both the M and H waves was determined, and the H_max/M_max ratio was calculated.

Intrathecal drug delivery and data collection.

For intrathecal drug injection, the externalized portion of the intrathecal catheter was attached to a hand-operated microinjector through an opening on the side of the rat restrainer. NGX424 (tezampanel) (generous gift from TorreyPines Therapeutics, La Jolla, CA), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (Sigma, St. Louis, MO), or dl-2-amino-5-phosphonopentanoic acid (AP-5) (Sigma) were dissolved in 0.9% sterile saline and delivered in a volume of 10 μl, followed by 10 μl of sterile saline flush. All equipment was sterilized with 70% alcohol before injection and thoroughly rinsed with 0.9% sterile saline. After injection, the intrathecal catheter was immediately resealed with the stainless steel plug. Measurements were taken before drug administration, followed by identical measurements taken every 10–20 min for up to 3 h after drug treatment.

GluR1 antisense oligodeoxynucleotide design and treatment.

Oligodeoxynucleotides (19 bases in length) were synthesized (Operon, Huntsville, AL) based on the GluR1 cDNA (GenBank accession number NM031608) listed in www.ncbi.nlm.nih.gov. The specific antisense oligonucleotide (5′-T*A*A*GCATCACGTAAGG*A*T*C-3′; phosphorothiate-DNA chimera) was complementary to positions 1249–1268 of rat GluR1 cDNA. A missense (“scrambled”) antisense, for use as a control, was also prepared in which the bases of the GluR1 oligonucleotide were randomized (5′-A*G*C*GTATCACAGTATA*G*A*C-3′; phosphorothiate-DNA chimera). This control sequence revealed no other rodent sequence homology using the BLAST (basic local alignment search tool) search. In previous in vitro studies, we demonstrated that treatment of cortical or spinal primary cultures with short hairpin RNA targeting the same GluR1 sequence effectively downregulated neuronal and astrocytic GluR1 expression (Kinjoh et al., 2005).

To study the specific effects of GluR1 knockdown, ischemic-spastic rats were implanted with intrathecal catheters. At 4–5 d after implantation, baseline hindlimb muscle resistance and gastrocnemius EMG activity were recorded. Antisense or missense oligonucleotides (50 μg) were then injected intrathecally daily for 5 d. After 5 d of injections, muscle resistance and EMG activity were again recorded. At the end of recording, animals were deeply anesthetized with pentobarbital (100 mg/kg, i.p.) and phenytoin (25 mg/kg, i.p.) and prepared for immunohistochemistry (see below).

Western blotting.

Rats were deeply anesthetized with pentobarbital (100 mg/kg, i.p.) and phenytoin (25 mg/kg, i.p.), and spinal cords were rapidly removed by hydroextrusion and homogenized by sonication in lysis buffer [50 mm Tris-HCl, pH 8.0, with 1 mm EDTA, 150 mm NaCl, 1% NP-40, 0.5% deoxycholic acid, 0.5% saponin, and 0.1% SDS with phosphatase and protease inhibitor cocktails (Sigma)]. After centrifugation of homogenates (14,000 rpm at 4°C, 15 min), supernatants were collected and frozen at −70°C until immunoblotting. Protein concentration for each homogenate was determined using a BCA kit (Pierce, Rockford, IL). Equal amounts of protein (67 μg) were loaded onto an 8–16% Tris-glycine gel for electrophoresis and then transferred to a nitrocellulose membrane. After blocking in 5% nonfat dry milk in 0.1 m Tris-buffered saline (TBS)–0.1% Tween 20 (TBST), membranes were incubated with primary antibody (GluR1/2/3/4; see below) overnight on a shaker at 4°C. Membranes were washed three times for 10 min with TBST and incubated for 1 h on a shaker at room temperature with either goat anti-mouse or goat anti-rabbit horseradish peroxidase-conjugated antibody (1:5000; Pierce). Equal protein loading was adjusted by normalizing all immunoblots against their respective β-actin levels. Briefly, once the GluR protein was detected, the membrane was gently stripped, blocked, and incubated with mouse anti-β-actin (1:5000, 1 h at room temperature; Sigma), and developed as above. Chemiluminescent detection (Pierce) was used to visualize immunoreactive bands, and quantitative analysis of the bands was performed using ImageQuant TL (GE Healthcare, Piscataway, NJ).

Perfusion fixation and fluorescent immunohistochemistry.

For histological analysis of spinal cord tissue, rats were anesthetized with pentobarbital (100 mg/kg, i.p.) and phenytoin (25 mg/kg, i.p.) and transcardially perfused with 200 ml of heparinized saline, followed by 250 ml of 4% paraformaldehyde in 0.1 m PBS, pH 7.4. The spinal cords were dissected and postfixed in the same fixative overnight at 4°C. After postfixation, tissue was cryoprotected in 30% sucrose phosphate buffer. Transverse spinal cord sections (10 μm) were then cut using a Leica (Bannockburn, IL) cryostat and mounted directly on slide glass; sections from naive and ischemic-injured spinal cord were mounted on each slide to ensure identical staining and allow proper comparison. For staining, slides were placed in PBS containing 5% normal goat serum (NGS), 0.2% Triton X-100 (TX) overnight at 4°C to block nonspecific protein activity. This was followed by a 2 d incubation at 4°C on a shaker with primary antibodies: rabbit anti-GluR1 (1:1000; Upstate, Charlottesville, VA), mouse anti-GluR2 (1:500; Chemicon, Temecula, CA), mouse anti-GluR3 (1:500; Chemicon), rabbit anti-GluR4 (1:500; Chemicon), mouse anti-neuronal-specific nuclear protein (NeuN) (1:1000; Chemicon), biotinylated mouse anti-NeuN (1:1000; Chemicon), rabbit anti-glial fibrillary acidic protein (GFAP) (1:1000; Chemicon), and mouse anti-GFAP–cyanine 3 (Cy3) conjugate (1:500; Sigma).

After incubation with primary antibodies, sections were washed three times in PBS and incubated with secondary goat anti-rabbit or -mouse antibodies conjugated to a fluorescent marker (Alexa 488 or 594; 1:250; Invitrogen, Carlsbad, CA) or avidin–Cy3 (1:400; Sigma). All antibody preparations were made in PBS–TX–NGS. For general nuclear staining, 4′,6′-diamidino-2-phenylindole (1:250) was added to the secondary antibody solutions.

Standard control experiments for all antibodies used involved omission of primary antibody. To further test the specificity of the GluR1 antibody, preadsorption studies were performed. For this, GluR1 antibody (1:1000) was incubated with 1 × 10⁻⁵ m GluR1 control peptide (GluR1 primary antibody immunizing peptide; a generous gift from Upstate) in PBS–TX–NGS overnight at 4°C on shaker. This solution was then used the following day as GluR1 primary antibody solution for staining. Identical staining, including double-labeling experiments (e.g., GluR1 plus GluR2 plus GluR1 control peptide), was performed at the same time but without the immunizing peptide to confirm other procedures were correct.

After staining, sections were dried at room temperature and covered with Prolong anti-fade medium (Invitrogen). Slides were analyzed using a Leica fluorescence microscope and captured with a digital camera [Orca-ER (Hamamatsu, Shizuoka, Japan) or microfire S99809 (Olympus Optical, Tokyo, Japan)]. Some slides were selected for confocal imaging using a Leica LCS SP2 confocal microscope (Leica, Bannockburn, IL). All images were processed by Adobe Photoshop CS (Adobe Systems, Mountain View, CA), with equal changes in brightness and contrast when applicable.

Immunohistochemistry with antigen unmasking.

To visualize synaptic AMPA receptor subunits, some sections were pepsin treated (Watanabe et al., 1998; Nagy et al., 2004). For these experiments, 70 μm vibratome sections were cut and stored in 0.3 m PBS (0.1 m PBS with 0.3 m NaCl). Sections were treated with pepsin (1 mg/ml; P-7000; Sigma) in 0.2 m HCl for 10 min at 37°C and washed three times for 10 min with 0.3 m PBS at room temperature. After 30 min in 50% ethanol, sections were again washed three times for 10 min with 0.3 m PBS, placed in primary antibody solution (in 0.3 m PBS–0.3%TX), and incubated 24 h on a shaker at 4°C. Sections were then processed as described above.

Immunoelectron microscopy.

Naive and experimental spinal cord sections, 50 μm thick, were cut on a vibratome and cryoprotected with glycerol–dimethylsulfoxide mixture. After cryoprotection, the sections were frozen and thawed four times and treated with 1% sodium borohydrate. To reduce nonspecific binding, the sections were treated with 0.3% H₂O₂–10% methanol in TBS (100 mm Tris-HCl and 150 mm NaCl, pH 7.6) and 3% NGS–1% bovine serum albumin in TBS. Sections were reacted overnight with rabbit anti-GluR1 (1:500; Chemicon). Bound antibody was detected using biotinylated donkey anti-rabbit IgG (1:500; GE Healthcare, Little Chalfont, UK), the ABC Elite kit (Vector Laboratories, Burlingame, CA), and diaminobenzidine (DAB) as the chromogen. After DAB detection, some sections were processed by an additional antibody labeling cycle using the same method and antibody as above. This staining strategy enhanced the signal-to-background ratio while the background labeling was kept to minimal. Immunoreacted sections were postfixed in buffered 2% OsO₄, rinsed and stained in 1% uranyl acetate, and then dehydrated and embedded in Epon. Ultrathin sections were contrasted with uranyl acetate and analyzed under a Zeiss EM-10 electron microscope operated at 60–80 kV. Electron microscopic negatives were scanned and processed by Adobe Photoshop CS2 (Adobe Systems).

Astrocyte culture and AMPA-stimulated glutamate release.

Spinal cord astrocytes were isolated from lumbar spinal cords of postnatal day 0–1 rat pups using Papain Dissociation System (Worthington, Lakewood, NJ). After isolation astrocytes were cultured with DMEM–10% fetal bovine serum (FBS) on Nunclon six-well plates. To purify astrocytes, mechanical shaking was used on day 7 after isolation. Astrocytes were then fed with fresh DMEM–10% FBS every 3 d until confluent and were then passaged into 24-well plates. After passage, astrocytes were cultured for 3–5 more days before in vitro release experiments were initiated.

On the day of the release experiments, medium was replaced with 300 μl/well artificial CSF (ACSF) (in mm: 151.1 Na⁺, 2.6 K⁺, 0.9 Mg²⁺, 1.3 Ca²⁺, 122.7 Cl⁻, 21.0 HCO₃, 2.5 HPO₄, and 3.5 dextrose; bubbled for 15 min before the experiment with 95% O₂/5% CO₂ to adjust the final pH to 7.3). After 10 min in the incubator, a baseline sample (25 μl) was collected from each well, immediately frozen on dry ice, and stored at −70°C until analysis. AMPA was added to each well at varying concentrations (1, 10, and 30 μm; each well received one concentration and was used only once), and the 24-well plate was placed back in the incubator for 10 min, when another set of samples were collected and frozen. Antagonist studies were performed by adding the antagonist (NGX424, 1 μm) to the wells 10 min before adding AMPA. Each experiment was performed in four independent culture wells. Samples were analyzed for glutamate by HPLC (model HTEC 500; Eicom, Kyoto, Japan) using the E-ENZYMPAK enzymatic derivatization and the GU-GEL column with electrochemical detection.

In vitro immunohistochemistry. After release experiments were completed, cells were washed (three times for 10 min in PBS) and fixed for 30 min with 4% paraformaldehyde/0.1% glutaraldehyde at room temperature. After washing (three times for 10 min in PBS), cells were incubated with primary antibodies recognizing GluR1, GFAP, or NeuN overnight at 4°C. The following day, each well was washed (three times for 10 min in PBS), and appropriate secondary antibodies (Alexa 488 and 594; Invitrogen) were used for visualization. Images were captured with an Olympus Optical inverted microscope equipped with a digital camera (Hamamatsu).

Image analysis.

Effect of GluR1 antisense/missense on spinal cord GluR1-IR was analyzed by University of Texas Health Science Center at San Antonio ImageTool (3.0) using confocal microscope images captured using identical settings for each treatment group. Maximum pixel intensity in each field was normalized to 100%, and the background was set to zero. The total area under the pixel intensity histogram was calculated for each field and two to three fields averaged for each section (using at least three sections per animal and three to four animals per group). To assess changes in individual motoneurons, similar analyses were performed using only outlined motor cells in the images (and normalizing for the analyzed area). Cell counts were done manually.

Data analysis.

Muscle resistance results are presented as either applied resistance (g; grams force) or normalized as percentage of baseline resistance. All dose–response curves were calculated using area-under-the-curve analysis of the time course data. Statistical testing was performed using SigmaStat 2.03 for Windows (SPSS, Chicago, IL). Two-way comparisons were performed with Student's t test. Multiple comparisons were performed using one-way ANOVA, followed by Student–Newman–Keuls test. All results are shown as mean ± SEM. p < 0.05 was considered to be statistically significant. For EMG analysis, the integrated signal was calculated from 20 averaged points and plotted.