All procedures were performed in accordance with National Institutes of Health Guidelines with the approval of Children’s Hospital Animal Care and Use Committee. In all, 118 adult Sprague Dawley rats (275–300 g; Charles River) were used in the experiments outlined in . The first part of the study investigated the effect of inosine on brain reorganization and behavioral outcome after unilateral injury centered in the forepaw motor area. We then investigated whether unilateral stroke per se causes neurons in the undamaged hemisphere to sprout corticospinal tract (CST) axon collaterals; whether inosine induces axonal sprouting in the absence of brain injury; whether the functional effects of inosine persist after treatment ends; and whether inosine alters CST reorganization at an earlier time point. The final part of the study examined the effects of inosine at the molecular level.
With the exception of animals in part III and one group of controls in parts VI and VII, all rats sustained unilateral infarcts in the sensorimotor cortex using the photothrombotic model of focal ischemia (Markgraf et al., 1993
). Briefly, rats were anesthetized with a combination of ketamine (75 mg/kg) and Domitor (medetomidine; 0.5 mg/kg), and a 15 mm skin incision was made at the midline rostral to the posterior suture. A craniotomy was performed over the sensorimotor cortex using a hand-held drill to open a window that spanned mediolaterally between the sagittal sinus and temporal ridge, and rostrocaudally between bregma +2.5 mm and bregma −3.5 mm. The photosensitive dye Rose Bengal was injected into the femoral vein, a fiber-optic cable connected to a xenon light source was centered over the craniotomy, and the exposed region was illuminated for 30 min. A green filter fitted over the bulb restricted illumination to ~525 nm, a wavelength that excites Rose Bengal and causes it to release free radical species. The subsequent damage to endothelial cells in exposed portions of the cortical vasculature causes platelet aggregation, resulting in severe focal ischemia. As shown below, infarcts typically had a diameter of 6–7 mm and were restricted to cortical tissue and some underlying white matter. Uninjured control animals in parts III, VI, and VII were generated using the same surgical procedure but without photo-activation of the injected Rose Bengal.
Animals with strokes were randomly assigned to receive a continuous infusion of either saline (0.9%, Baxter Scientific) or inosine (50 mm in saline, Sigma-Aldrich) into the lateral ventricle of the uninjured hemisphere using osmotic minipumps (0.25 μl/h, Alzet model 2004, Durect Corporation). Catheters were placed 1.4 mm lateral and 0.8 mm caudal to bregma on the uninjured hemisphere within 30 min of surgery. Because CSF drug concentrations presumably require several hours to achieve steady-state levels when delivered via slow-releasing osmotic pumps, all animals received a 25 μl intraventricular bolus of the appropriate agent before pump placement at the time of injury. Pumps were tucked between the shoulder blades and infusion needles were secured onto the cranium with a silicon-based glue. The incision was closed with silk sutures and cleaned with betadine and ethanol pads. For 72 h after surgery, animals received twice-daily, subcutaneous injections of Buprenex (buprenorphine; Reckitt Benkiser Pharmaceuticals) for pain management. Animals in the second experiment had strokes but did not receive intraventricular infusions.
Functional recovery was evaluated using a skilled forelimb-reaching task on which animals were trained before surgery. In the first part of the study, animals (N = 12 per group) were tested at 7, 14, 21, and 28 d after surgery before being injected intracranially with the anatomical tracer. In the fourth part of the study (N = 12 per group), minipumps were removed after 4 weeks and testing was continued weekly for another 4 weeks. All testing was done by an experimenter blind to the animals’ treatments.
The behavioral task requires rats to reach with either paw through a narrow slit in a Plexiglas box, grasp a banana-flavored food pellet (Bio-Serv) from a platform, and bring it successfully to the mouth (Allred and Jones, 2004
; Luke et al., 2004
). Three days before and during preoperative training, animals were maintained on a restricted diet of banana-flavored pellets to remove novelty-induced hesitation and to increase motivation. Rats were trained 30–60 min per day with each paw for 2 weeks or until they reached a baseline performance of 20–30 successful reaches in a 2 min period. At the end of the training period but before surgery, animals were tested for the number of pellets successfully grasped and consumed in two 2 min trials, making sure that they were motivated and stress free. The average of these two tests became the “baseline” score, to which subsequent scores were normalized. This enabled us to account for possible individual differences in motivation and competence as a biasing factor for overall performance. Performance was recorded only if rats retrieved a minimum of 25 pellets within the 2 min interval. In postsurgical testing, we likewise scored performance only when animals were fully engaged and performing the task at a relatively consistent level. Within each session, animals spent 10–20 min in the apparatus, during which they were tested twice with each paw (alternating) for 2 min/session. Scores from the unimpaired paw were used to assess animals’ engagement, motivation, and overall behavioral competence. Data were analyzed using a regular two-way ANOVA. Bonferroni’s post-test was used to compare data sets.
Anterograde tracing of crossing fibers
Animals in part I of the study were anesthetized after the final testing period, the infusion needle and pump were removed, and a craniotomy was performed over the uninjured sensorimotor cortex (SMC). The anterograde tracer biotinylated dextran amine (BDA: Invitrogen: 10,000 molecular weight, 10% w/v in sterile saline) was injected stereotaxically at depths of 0.5, 1.0, and 2.0 mm below the cortical surface at 18 standardized points distributed over the sensorimotor cortex, as determined by the Paxinos and Watson (1998)
rat brain atlas (supplemental Fig. 1
, available at www.jneurosci.org
as supplemental material
) (70 nl per injection; Nanoject, Drummond Scientific). Two weeks later, animals were reanesthetized and perfused transcardially with 0.9% saline followed by 4% paraformaldehyde. The brain and spinal cord were dissected and postfixed overnight in 4% paraformaldehyde, followed by 10% and 30% sucrose solutions over the next few days. Tissue was embedded in OCT Tissue Tek Medium (Sakura Finetek) and frozen on dry ice. Forty-micrometer free-floating sections were cut in the coronal plane on a Frigo-Jung 8500 cryostat. Free-floating spinal cord sections were used to visualize the trajectory of CST axons using avidin-biotin complex conjugated to horseradish peroxidase (Vectastain ABC Kit; Vector Laboratories), followed by Vector SG (Vector Laboratories) as a chromagen. Sections were mounted on precoated slides and lightly counterstained with eosin to distinguish gray and white matter boundaries. Six to ten sections spanning a distance of 1.2 mm were examined in each case and quantified for (1) BDA-labeled axon profiles ≥40 μ
m in length within the dorsal funiculus on the denervated side of the spinal cord (i.e., ipsilateral to the BDA injection); (2) BDA-labeled axons ≥40 μ
m in length in the gray matter of the denervated side of the spinal cord, and (3) BDA-labeled axons ≥200 μ
m in length on the denervated side. Axon length was measured in the transverse plane. The number of axons was normalized by the density of CST staining within the intact dorsal funiculus to adjust for possible differences in labeling between animals. The latter values were obtained by densitometric measures of the intact CST and converting density values to a scale of 0–1 (0 = density in the denervated side of the dorsal funiculus, 1 = density in the case with the most intense labeling). Three coronal sections through the rostral cervical cord were used to obtain the normalization values for each animal. Average numbers of axons, normalized for labeling density, were calculated and converted to axons per millimeter of spinal cord. Bouton-like structures were identified under a 100× oil objective as areas ≥2× the thickness of the axon shaft. Counts were performed in a 0.25 mm2
box aligned with laminae VI/VII, and were not extrapolated to any volumetric totals.
Animals in part II were used to investigate the effect of stroke on CST reorganization in the absence of treatment. We created left-sided strokes in six animals, while carrying out sham surgery (Rose Bengal injections, craniotomy, no illumination) in another six animals. As above, BDA was injected into multiple sites in the right forelimb motor area 4 weeks later and animals were prepared for histology after another 2 weeks. Animals in group III were use to investigate whether inosine would induce sprouting in the absence of brain injury. These animals (N = 6 per group) underwent sham surgery, received either inosine or saline for 4 weeks as above, and were then prepared for anatomical tracing of CST fibers that originate on one side of the brain and project to the ipsilateral cervical enlargement. Animals in group IV (N = 12 per group) were used to investigate whether the functional benefits of inosine persist after treatment ends. Animals were prepared as in the first set of experiments, but at the completion of inosine delivery, behavioral testing continued weekly for another 4 weeks. Animals in group V (N = 6 per group) were used to investigate whether inosine-induced changes in CST projections could be seen as early as 2 weeks after injury.
Determination of lesion severity
Sections through the telencephalon of animals in group I were cut at 10 μ
m, mounted onto slides, and stained with Crystal Violet to determine the extent of the lesions. Sections were scanned using a high resolution Epson Perfection 3490 PHOTO scanner. The cross-sectional area of the injured and uninjured hemisphere of each section was determined using NIH ImageJ software. Lesion area was determined by subtracting the area of tissue remaining in the injured hemisphere from that in the uninjured hemisphere in sections spaced 250 μ
m apart spanning the full rostrocaudal extent of the lesion. Lesion volume was extrapolated from these data. Lesions were redrawn onto standard sections from a rat brain atlas (Paxinos and Watson, 1998
). Representations of the injury were created from tracings of scanned sections in Adobe Photoshop.
Effects of inosine on gene expression
The final parts of this study investigated molecular events induced by inosine in the population of neurons that give rise to the undamaged corticospinal tract. To identify these neurons, animals were anesthetized with a combination of ketamine and Domitor (medetomidine) and a laminectomy was performed at the cervical level of the spinal cord (C2–C4). Alexa Fluor 488-conjugated cholera toxin B subunit (CTB; 300U/μ
l in sterile saline) was stereotaxically injected into four sites lateral to the corticospinal tract. Injections (1.3 μ
l) were spaced 1 mm apart on the rostrocaudal axis and were 0.5 mm to the left and right of the midline, just flanking the CST. CTB is avidly taken up by fibers of passage (Chen and Aston-Jones 1995
), and thus these injections are expected to label layer 5 neurons that project to the level of the injection site and below, including the forepaw region. After allowing 2 weeks for transport of the tracer, we performed either sham surgery with no treatment or stroke surgery combined with saline or inosine delivered via minipumps, as described above. Seven days later, animals were decapitated under transient gas anesthesia, brains were removed and rinsed in cold RPMI medium, and a tissue block containing the sensorimotor cortex was dissected (based on stereotaxic coordinates) and placed in OCT Tissue Tek Medium (Sakura Finetek) on dry ice within 5 min of death. Ten-micrometer sections were cut onto precleaned Gold Seal RITE-ON glass slides (Gold Seal Products), placed on dry ice, and rapidly stored at −80°C until ready to use.
Laser-capture microdissection and microarray analysis
Slide-mounted sections were thawed and dehydrated in RNase-free ethanol gradients and xylene. Retrogradely labeled, fluorescent cortical pyramidal cells in the forelimb area of the undamaged hemisphere were individually captured using the Arcturus VERITAS system. Cells (≥500) were collected from each animal and stored in Arcturus extraction buffer at −80°C. Total RNA was extracted from cells using the Micro-to-Midi TotalRNA Purification System (Invitrogen) and double-amplified using the TargetAmp 2-Round Aminoallyl-aRNA Amplification kit 1.0 (Epicenter). Amplified RNA was checked for average fragment length using the Agilent RNA 6000 Nano LabChip kit (Agilent Technologies) and then biotinylated and hybridized (1 μg) on Illumina RatRef-12 Expression Bead-Chip arrays (Illumina), querying the expression of >22,000 RefSeqcurated rat transcripts. Results were obtained from a total of 15 samples representing 6 untreated controls, 4 animals with stroke treated with saline, and 5 animals with stroke treated with inosine. Slides were processed and scanned with Illumina BeadStation platform according to the manufacturer’s protocol.
Raw data were analyzed using Bioconductor packages [www.bioconductor.org
(Gentleman et al., 2004
)]. Low-level quality-control analysis was performed using interarray Pearson correlation and clustering based on variance. To further control for RNA integrity, three indirect measures of comparable RNA integrity were run across samples.
Agilent Bioanlyzer Nanochips were run on the double-amplified RNA samples to insure uniform RNA degradation, as determined by average fragment size. A subset of samples were run on both Illumina and Affymetrix arrays to insure that 5′ to 3′ ratios were comparable.
Finally, the detection scores—a measure of detection ranging between 0 (not detected) and 1 (strongly detected) provided by the Illumina Bead-Studio software—were compared across samples and showed no difference across samples (supplemental Table 2
, available at www.jneurosci
. org as supplemental material
). Two arrays (one saline- and one inosine-treated) were outliers and were excluded from the analysis. Data were normalized using quantile normalization, and analysis of differential expression was performed using a linear model fitting [LIMMA package (Smyth, 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 performed using Ingenuity Pathway Analysis (Ingenuity Systems).
Immunohistochemistry and quantitation of activated caspase3 and other proteins
Animals in the last part of the study were used to visualize the effect of inosine on the expression of several proteins. This set included four normal controls, eight rats with stroke and saline infusions, and eight with stroke and inosine infusions, prepared as described above. One week after surgery, animals were killed, perfused with saline and paraformaldehyde, and their brains were postfixed and prepared for histology. Sixteen-micrometer cortical sections were mounted onto slides and allowed to dry at room temperature for 2 h. To visualize caspase activation, sections were washed in PBS, boiled for 20 min in citrate buffer, blocked in 5% BSA, and incubated overnight in a 2% BSA solution containing an antibody to activated caspase 3 (1:500, Abcam). The following day, slides were rinsed in PBS and incubated with Alexa Fluor 568-conjugated goat anti-rabbit secondary antibody (1:500, Invitrogen) for 2 h at room temperature. Slides were rinsed in PBS and coverslipped with Fluorosave mounting medium (Invitrogen). Immunopositive profiles bordering the lateral aspect of the lesion were quantified in a 1 mm2 field at bregma 1.7 mm in five animals selected randomly from each group. As stereological methods were not used, these profile counts are strictly focal in the information they convey. To insure that between-group differences were not caused by changes in the diameter of these profiles, we measured the cross-sectional areas of stained profiles in 20 consecutive objects in 4 sections/case.
Immunohistochemistry and quantitation of complement proteins and metallothionein
Slide-mounted sections were postfixed in 10% buffered formalin, blocked in 5% BSA, and incubated overnight in goat anit-C1q (1:500, Sigma), goat anti-C3 (1:500, Cappel), or rabbit anti-metallothionein (1:50, Santa Cruz Biotechnology) primary antibodies overnight. After a rinse, sections were incubated in appropriate Alexa Fluor 488-conjugated secondary antibodies, rinsed, and coverslipped using Fluorosave mounting medium (Invitrogen). Complement intensities were quantified by digital thresholding of images using NIH ImageJ. Metallothionein-positive cells were quantified by individual counts in a 1 mm2 field bordering the lateral aspect of the lesion at bregma 1.7 mm. As with caspase counts, these numbers are from profile counts and represent cell densities at this level only, not volumetric or three-dimensional measures.