All procedures were carried out in accordance with NIH Guidelines and with the approval of Children’s Hospital Animal Care and Use Committee. Adult male Sprague-Dawley rats (275–300g; Charles River Laboratories, Wilmington, MA) were maintained on a 12 h light/dark cycle (lights on at 7:00 AM). Surgery was carried out between 8:30 AM and 3:00 PM, with animals from different experimental groups intermingled. Strokes were centered in the right caudal forelimb motor area and included varying amounts of adjacent cortical areas (Zai et al., 2009
). Rats were anesthetized with ketamine (75 mg/kg) and Domitor (medetomidine; 0.5 mg/kg), the skin was incised along the midline, muscles and skin were retracted, and a craniotomy was performed to create a rectangular window that extended medio-laterally from the sagittal sinus to the temporal ridge, and rostro-caudally between Bregma +3.5 mm and −2.5 mm. Rose Bengal, a photosensitive dye, was injected into the femoral vein through a small opening in the inner thigh. A fiber-optic cable connected to a xenon light source was positioned over the craniotomy, focusing light directly on the exposed region of the brain to activate Rose Bengal for 30 minutes. A green filter restricted illumination to ca. 525 nm. Under these conditions, Rose Bengal releases free radical species that damage endothelial cells in the exposed portions of the cortical vasculature, resulting in platelet aggregation and focal ischemia. Alzet minimpumps were tucked between the shoulder blades and infusion needles were secured onto the cranium with a silicon-based glue. Incisions were closed with 3-0 silk sutures and cleaned with betadine and ethanol pads. Animals were kept on a warming pad during and after surgery, and were returned to their cages when fully awake, mobile, and able to thermoregulate (as judged by absence of piloe-rection). For 3 days following surgery, animals received twice-daily subcutaneous injections of Buprenex (Buprenorphine; Reckitt Benkiser Pharmaceuticals Inc.) for pain management. Animals in the first study were randomly assigned to receive continuous infusion of saline (0.9%, Baxter Scientific), inosine (50 mM in saline, Sigma-Aldrich), NEP1-40 (500 μM in 2.5% DMSO/97.5% saline), or inosine + NEP1-40 into the lateral ventricle of the undamaged hemisphere via osmotic minipumps (0.25 μl/h, Alzet model 2004, Durect Corporation). In order to achieve rapid drug build-up in the CSF, animals in all studies received a 25 μl intraventricular bolus of the agent to be infused prior to pump placement. Treatments were assigned randomly prior to surgery. Unless noted otherwise, rats were housed in pairs.
The study examining the interactions of inosine and NEP1-40 included two behavioral experiments, one in which rats survived for 4 weeks after stroke and another in which they survived for 8 weeks. In the 4-week study, rats treated with either NEP1-40 alone (N = 11) or NEP1-40 plus inosine (N = 11) were generated concurrently with animals reported in another study (Zai et al., 2009
), but were not themselves described previously. Another group of rats, treated either with inosine alone (N = 7) or saline (N = 7), were generated specifically for this study. Post-hoc analysis revealed that the behavioral and anatomical effects of inosine- and saline were nearly identical between the newly-generated cases and the ones reported on earlier, justifying our combining the newly generated inosine- and saline-treated animals with the previously-generated animals treated with NEP1-40. Animals in the 8-week study treated with NEP1-40, either with or without inosine (N = 11 for each), have not been described earlier but were also generated concurrently with saline- and inosine-treated animals reported in a previous study (Zai et al., 2009
). Behavioral testing was carried out during daylight hours, with no systematic bias towards type of treatment.
Following pre-training and surgery, animals for anatomical and behavioral studies were implanted with minipumps delivering either saline (N = 21) or inosine (N = 20) and housed in pairs in standard cages. Three days later, half the rats in each treatment group were selected at random and transferred to specially designed chambers for environmental enrichment (EE). The effects of brain injury can be exacerbated by physiological activity within the first week (Humm et al., 1999
) although delaying exposure to EE by just 1–3 days enables the beneficial effects of EE to manifest themselves (Ohlsson and Johansson, 1995
; Risedal et al., 2002
; Dahlqvist et al., 2004
The EE chamber was a plexiglas cube 24″ on a side with fixed ladders and a platform to stand on, along with a set of tunneling tubes, toys, and treats that varied daily. At any given time, the chamber contained 4–8 rats, with enough plastic tunnels for each rat to have its own. Rats had free access to water throughout the study and, with the exception of the day prior to behavioral testing, free access to standard rat chow. For the microarray studies, rats were housed individually for 3 days and then either transferred to the EE chambers or left in isolation for 4 more days before preparing tissue for analysis (see below).
Rats were tested for their ability to reach through a narrow slit, retrieve food pellets (Bio-Serv, Inc., Frenchtown, NJ) from a platform, and bring them successfully to their mouth (Allred and Jones, 2004
; Luke et al., 2004
). Variants of this task are widely used in the field because of its established reliance on the integrity of the motor cortex and descending cortical pathways, and because the results are readily quantified. In addition, the task mimics an essential feature of motor strokes in humans by evaluating perfomance on an acquired ability that requires precise motor control. In the acquisition phase, rats were maintained for 3 days prior to and during training on a diet restricted to the banana-flavored pellets used for the retrieval task. Rats were trained daily for 30–60 minutes with each paw for 2 weeks or until they attained a baseline performance of 20–30 successful reaches in a 2-minute period. Immediately after reaching this criterion, each animal was tested for the number of pellets it grasped successfully and consumed in two 2-minute trials. The average of these became the baseline to which subsequent scores were normalized. Rats had free access to food and water for at least three days between baseline testing and surgery. Post-operative testing was carried out in 10–20 minute sessions at 7, 14, 21, and 28 days after surgery by an experimenter blind to the animals’ treatment. Performance was evaluated in two 2-minute blocks using each paw. Scores were recorded only if animals were able to perform at or near their pre-operative level with the unimpaired paw. In instances in which this did not occur immediately, we waited until rats were more active to test them again. In the 8-week studies, rats were tested weekly as above for 4 weeks while receiving the various agents, and then for an additional 4 weeks after minipumps and catheters were removed. Data were analyzed using a two-way ANOVA (repeat measures) with Bonferroni’s post-test to compare data sets.
Anterograde Tracing of Crossing Fibers
At the completion of behavioral testing, rats were re-anesthetized, the infusion needle and pump were removed, and a craniotomy was performed over the uninjured sensorimotor cortex (SMC). Biotinylated dextran amine (BDA: Molecular Probes: 10,000 MW, 10% wt/vol solution in sterile saline) was used as an anterograde tracer and was injected stereotaxically at depths of 0.5, 1.0, and 2.0 mm below the cortical surface at 18 standardized points covering most of the caudal forelimb motor area (70 nl per injection; Nanoject, Drummond Scientific, Broo-mall, PA). Stereotaxic coordinates of the injection sites are shown in Suppl. Table 1
. Two weeks later, animals were anesthetized and perfused transcardially with 0.9% saline followed by 4% paraformaldehyde. The brain and spinal cord were dissected and post-fixed in the same fixative for one hour at room temperature and overnight at 4 °C, then transferred successively to 10% and 30% sucrose solutions (4 °C) over the next few days. Tissue was embedded in OCT Tissue Tek Medium (Sakura Finetek USA Inc.) and frozen on dry ice. Forty-micron free-floating sections were cut in the coronal plane on a Frigo-Jung 8500 cryostat and stained with avidin-biotin complex conjugated to horseradish peroxidase (Vectastain ABC Kit; Vector Laboratories) followed by Vector SG (Vector) as a chromagen. Sections were mounted on slides (Fisher Super Frost PLUS
) and lightly counterstained with eosin to distinguish grey and white matter boundaries. Six to ten equally spaced sections spanning a distance of 1.2 mm were examined and quantified for (a) axons within the dorsal funiculus (BDA-labeled axon profiles ≥ 40 μm in length in the transverse plane within the dorsal funiculus on the denervated side of the spinal cord); (b) axons within the gray matter (as above, but within the gray matter on the denervated side), and (c) lengthy axons in the gray matter (as above, but ≥ 200 μm in length in the transverse plane). Average numbers of axons were calculated and numbers were converted to axons per mm length of spinal cord. Bouton-like structures were identified under a 100X oil objective as regions over 2X the thickness of the axon shaft. Counts were performed in a 0.5 mm2
box aligned with Laminae VI/VII, and were not extrapolated to any volumetric totals. Camera lucida drawings were performed using representative spinal cord sections from control and treatment groups. Statistical significance was determined by one-way ANOVA followed by Tukey’s Multiple Comparisons Test to evaluate significance between treatment groups.
Determination of Lesion Severity
Lesion sizes were calculated from serial sections through the telencephalon as described earlier (Zai et al., 2009
). Lesions were re-drawn onto standard sections from the rat brain atlas (Paxinos and Watson, 1986
) using grey/white matter boundaries and standard structures. Representations of the injury were created from tracings of scanned sections in Adobe Photoshop.
Cortical Regeneration Assay
Primary cortical cultures were established from embryonic day 18 (E18) Sprague-Dawley rat brains. All tissue culture reagents were from Invitrogen unless otherwise stated. Brains were dissected in Hibernate E without calcium (BrainBits LLC) supplemented with B-27, 1 mM sodium pyruvate and 10 μg/ml gentamicin (dissection medium). Reagents and brains were kept on ice except during the initial dissection. Meninges were removed, cortices were dissected and combined in a 10 cm Petri dish in 1 ml of dissection medium. Tissue was diced approximately 100 times into small pieces with a razor blade. Dissection medium (12 ml) containing papain (4.2 mg/ml: Sigma, P4762) and DNase (0.8 mg/ml) were added to the Petri dish, which was then placed on an orbital shaker (Fisher Scientific, 11-671-50Q) in a 37° incubator (without CO2
regulation) and shaken at 30 rpm for 1 hour. The dish was removed from the incubator and the medium was transferred to a 15 ml polystyrene Falcon tube on ice. Chunks of cortex were allowed to settle to the bottom of the tube for 1–2 min. The medium was removed and chunks were resuspended in 1.5 ml of dissection medium plus 10% FBS and triturated 10–15 times with a fire-polished Pasteur pipette. Medium containing dissociated cortices was transferred to another 15 ml tube containing 12.5 ml of dissection medium plus 10% FBS and centrifuged at 600 x g for 6 minutes at 4° C. The supernatant was removed and cells were resuspended in 12 ml of Neurobasal A supplemented with B-27, 1 mM sodium pyruvate, 0.5 mM GlutaMAX-I and 10 g/ml gentamicin (in Neurobasal A plus supplements). Cells were filtered through a 70 m cell strainer into a 50 ml Falcon tube and counted. Cells were removed from ice and diluted to 3.33×105
cells/ml in Neurobasal A plus supplements which had been pre-warmed and pre-equilibrated in a 37° tissue culture incubator (5% CO2
). Cells were plated at a density of 1.2×105
cells/well in 120 l) in 96-well plates coated with poly-D-lysine (Becton Dickinson Labware, BD BioCoat). Door openings over the ensuing 21 days were kept to a minimum. At 7 and 14 days in vitro (DIV), cultures were fed by replacing 50% of the medium with fresh, pre-warmed and pre-equilibrated Neurobasal A plus supplements. At 21 DIV, cultures were injured by scraping with a 96-well floating pin tool with FP1-WP custom parylene-coated pins (0.557 mm tip diameter). To guide the movement of the pin tool, a library copier (V&P Scientific, VP 381NW4.5) with vertical alignment holes was used. Aluminum guide-rails were added on the left and right of the library copier (Electronic and Machine Shop, Yale Univ. School of Medicine), to minimize lateral movement of the pin tool. Immediately after the scrape, 50% of the medium was replaced with fresh medium. In some cases, the fresh medium contained extracted bovine CNS myelin (Cafferty et al., 2010
) and/or agents to be tested. Cultures were returned to the incubator to regenerate neurites for five days.
At 26 DIV, cultures were fixed by adding 100 μl/well of 4% paraformaldehyde + 20% sucrose in PBS for 20 min, then blocked and permeabilized with 10% normal goat serum + 0.1% Triton X-100 in PBS for 30 min. Cultures were immunostained with an anti-βIII- tubulin antibody (Promega, G7121, 1:1000), followed, after rinsing with PBS, by an Alexa Fluor 488 goat anti-mouse IgG secondary antibody (Invitrogen, A11029, 1:1000). Images were acquired with an ImageXpress Micro imaging system (Molecular Devices) using a 10X objective. Neurite regeneration was analyzed using MetaXpress Version 1.7 software. The central 75% of the lesion was analyzed by cropping the image and analyzing neurite growth using an angiotube formation algorithm. The area covered by neurites was measured and all values were normalized to the control. Results were analyzed by 1-way ANOVA and Fisher’s least-significant difference post-hoc test.
We investigated patterns of gene expression in layer 5 pyramidal cells of the forelimb motor area contralateral to the damaged hemisphere in four groups of rats, each containing 3–6 individual animals. These neurons were selected for study because parts of this population, though not necessarily the individual neurons that were isolated, give rise to the novel projections visualized in the anatomical studies. As before, rats underwent stroke surgery and were implanted with minipumps delivering either saline or inosine into the lateral ventricle of the undamaged hemisphere as described above. All rats used in the microarray study were initially housed individually. After 3 days, half the rats in each treatment group were randomly selected and transferred to the EE boxes described above, while the remainder continued to be housed in isolation. To focus on gene changes associated with the early response to EE, rats were anesthetized and killed by decapitation four days later. Brains were rapidly dissected and cut in the coronal plane to prepare tissue blocks that included the forelimb motor cortex. Blocks were rapidly frozen in OCT Tissue Tek Medium (Sakura Finetek USA Inc.) and stored at −80°C until ready for sectioning on a cryostat at 10 μm. Sections were mounted on slides (Gold Seal RITE-ON glass slides), cooled, and stored at − 80° C until ready to use.
Laser-Capture Microdissection (LCM) was carried out as described (Zai et al., 2009
). In brief, sections were thawed, dehydrated under RNAse-free conditions, and ≥ 500 layer 5 pyramidal cells in the forelimb motor area of the undamaged hemisphere were captured individually using the Arcturus VERITAS system and stored in Arcturus extraction buffer (−80° C). We extracted RNA using the Micro-to-Midi TotalRNA Purification System (Invitrogen), then carried out two cycles of RNA amplification (TargetAmp 2-Round Aminoallyl-aRNA Amplification kit 1.0: Epicentre Biotechnologies). Microarray hybridization and analysis was performed essentially as described (Carmichael et al., 2008
; Zai et al., 2009
). Briefly, amplified RNA was checked for average fragment length (RNA 6000 Nano LabChip Kit, Agilent Technologies), biotinylated, and hybridized (1 μg) on RatRef -12 Expression BeadChip arrays (Illumina), querying the expression of >22,000 RefSeq-curated rat transcripts. Slides were processed and scanned (Illumina BeadStation platform), and raw data was analyzed using Bioconductor packages as described (Gentleman et al., 2004
). First-level quality-control analysis was performed using clustering based on variance and by comparing gene expression patterns for individual cases to group means (Pearson correlations: Oldham et al., 2008
). To decrease noise, 3 cases that showed a relatively low correlation to their respective group means (r2
< 0.94) were excluded, increasing the r2
values for the remaining cases to ≥ 0.97. The final analysis included n = 4 samples from rats treated with saline reared in isolation, n = 4 treated with inosine and reared in isolation, n = 4 treated with saline and exposed to EE, and n = 3 treated with inosine and exposed to EE. Data were normalized using quantile normalization, and analysis of differential expression was performed using linear model fitting (LIMMA package, Smyth et al., 2005
). Differentially expressed genes were classified according to gene ontology using Bioconductor packages and online tools (DAVID, http://david.abcc.ncifcrf.gov/
). Pathway analysis was carried out using Ingenuity Pathway Analysis (Ingenuity Systems).