All procedures were carried out following the UCSF Committee on Animal Research guidelines. Adult male (25–31 g) C57BL6 mice (n = 72) were anesthetized intraperitoneally with 2.5% Avertin (0.02 ml/g body weight) and prepared for surgery. An incision was made along the dorsal midline, the dorsal extensors were reflected laterally, and a laminectomy was carried out at the T8 level. The vertebral column was stabilized by forceps attached to the adjacent spinous processes, and then the spinal cord was injured by a 3-g weight dropped 5 cm onto an impactor tip (diameter 1.5 mm). The overlying skin was then closed with wound clips. Surgical controls consisted of sham animals (n = 5) that received a laminectomy but were not subjected to injury.
Barrier permeability to luciferase (Promega, Madison, WI) was evaluated in naïve (n = 3) and sham-operated (n = 5) animals, at 25 min (n = 4), 35 min (n = 5), 1.5 hr (n = 5), 4.5 hr (n = 5), 1 day (n = 4), and 3 (n = 4), 7 (n = 5), 14 (n = 4), and 21 days (n = 4) after injury. Luciferase (1 mg/ml in Luciferase Storage Buffer; Promega) was diluted 1:1 with 0.05 M phosphate-buffered saline (PBS) in 0.001% bovine serum albumin (BSA). At 30 min before euthanasia at a given time point, each animal was anesthetized with Avertin, the jugular vein exposed, and injected with luciferase (3.33 µl/g body weight). The luciferase was delivered using a leur-lock syringe attached to a modified 27G Surflo Winged Infusion Set (Terumo Medical Corporation, Somerset, NJ). After injection, bleeding was controlled with sterile cotton swabs and Gelfoam (Upjohn, Kalamazoo, MI) as required. Upon Avertin-induced euthanasia, a blood sample was taken from the right ventricle and diluted immediately 1/50 in PBS, and the spinal cord was removed, rinsed with PBS, and quickly frozen.
The frozen cord was divided into six 3-mm segments, one centered on the injury site, and the two adjacent sections both rostral and caudal. A sixth section prepared from the cervical cord served as an internal control. Each segment was thawed, homogenized, and diluted 1:50 by weight in 1× cell culture lysis reagent (Promega). Homogenates were centrifuged (12,000 rpm) for 8 min to remove cellular debris and 1.5 µl of each supernatant was taken and diluted individually 1:100 in 1× cell culture lysis reagent. A 10-µl aliquot of the final dilution was added to 100 µl of Luciferase Assay System solution (Promega), briefly mixed, and quantitated in a luminometer (0 sec delay, integrated over 10 sec; TD-2020, Turner Designs, Sunnyvale, CA).
Luminosity values for the cervical region of each animal were used as internal controls. Cervical controls were chosen after demonstrating that luciferase values were unchanged from baseline values over the entire 21-day experiment. Cervical control sections were similar to naïve tissue in luminosity. Relative luminosity was determined by taking the raw luminosity values for each segment and dividing them by the cervical value. A log transformation was then carried out on all data points. Values at a given segment and time point are shown as the mean ± SEM. Statistical comparisons were made using analysis of variance followed by the Scheffe and Dunnett tests. Significance was defined at the P < 0.05 level.
The blood-spinal cord barrier to Evans blue albumin (EBA) was evaluated in naïve animals (n = 5) and injured animals at 1 (n = 8), 3 (n = 5), 7 (n = 5), 14 (n = 8), and 21 days (n = 5). The tracer (1.2% in 0.9% NaCl) was given intravenously 30 min before euthanasia. Each animal was perfused transcardially with fixative (4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4). The perfused spinal cord was removed, post-fixed for 4 hr, and cryoprotected in 20% sucrose for 4 days. The cryoprotected cord was then blocked in Tissue-Tek OCT Compound (Sakura Finetek, Torrance, CA), frozen, and sectioned (14–20 µm thick) on a cryostat. All sections were mounted on slides, dried at 37°C for 30 min, and frozen. A nonspecific fluorescence similar to that of EBA fluorescence was apparent in animals that were euthanized at later time points (either 14 or 21 days after injury). We therefore limit our interpretation of EBA localization to earlier time points. We carried out further experiments to determine if there was similar nonspecific luminescence, but found no evidence of intrinsic nonspecific luminescence at the 1- or 21-day points.
Immunolocalization of Glial Fibrillary Acidic Protein, Glut-1, Platelet-Endothelial Cell Adhesion Molecule-1, and CD11b
We examined glial and vascular response during wound healing. The presence of astrocytes and blood vessels was defined by positive staining for glial fibrillary acidic protein (GFAP) and platelet-endothelial cell adhesion molecule-1 (PECAM-1), respectively. Sections were thawed at room temperature, dried at 37°C, rehydrated in 0.05 M PBS (pH 7.4) for 15 min, and incubated in methanol for 15 min. To localize GFAP or PECAM-1, slides were incubated at room temperature as follows: (1) PBS, 5 min; (2) 2% horse serum/0.1% BSA (HS/BSA), 5 min; (3) 10% HS/BSA, 20 min; (4) goat anti-GFAP polyclonal antibody (1:1,000 in 2% HS/BSA; Santa Cruz Biotechnology, Santa Cruz, CA) or goat anti-PECAM-1 polyclonal antibody (1:1,000 in 2% HS/BSA, Santa Cruz Biotechnology), 24 hr; (5) PBS, 3× 5 min; and (6) affinity-purified biotinylated horse anti-goat Ig (1:200 in 2% HS/BSA, Vector Laboratories, Burlingame, CA), 1 hr. Slides were then rinsed in PBS 3× 5 min, incubated in either fluorescein-avidin D (1:100 in PBS; Vector Laboratories), Texas red-avidin D (1:100 in PBS; Vector Laboratories), or Alexa Fluor 350-NeutroAvidin (1:100 in PBS; Molecular Probes, Eugene, OR) for 1 hr, and then rinsed three more times in PBS.
Glut-1 localization began immediately after the last wash of the previous labeling, and was conducted in relative darkness to avoid photo-bleaching. Slides were incubated as follows: (1) 2% goat serum/0.1% BSA (GS/BSA), 5 min; (2) 10% GS/BSA, 20 min; (3) rabbit anti-Glut1 polyclonal antibody (1:2,000 in 2% GS/BSA; Chemicon International, Temecula, CA), 24 hr; (4) PBS 3× 5 min; and (5) affinity-purified biotinylated goat anti-rabbit IgG (1:200 in 2% GS/BSA; Vector Laboratories), 1 hr. Sections were then rinsed in PBS 3× 5 min and incubated in either fluorescein avidin D (1:100 in PBS) or Alexa Fluor 350-NeutroAvidin (1:100 in PBS) for 1 hr. Slides were rinsed in PBS 3× 5 min, treated with Vectashield mounting medium (Vector Laboratories) and coverslipped.
To localize CD11b, slides were incubated at room temperature as follows: (1) PBS, 15 min; (2) 2% BSA, 1 hr; (3) rat CD11b antibody (1:1,000 in 2% BSA; Serotec, Raleigh NC), 24 hr; (4) PBS 3× 5 min; and (5) affinity-purified biotinylated rabbit anti-rat Ig (1:200 in 2% BSA, Vector Laboratories), 1 hr. Slides were then rinsed in PBS 3× 5 min, incubated in fluorescein-avidin D (1:100 in PBS) for 1 hr, and rinsed again three times in PBS.
Quantification of Revascularization
Glut-1-labeled PECAM-1 confirmed vessels were quantified in the uninjured control cord. In the injured cord, blood vessels labeled consistently with PECAM-1; however, there was a population of blood vessels that were PECAM-1 positive and Glut-1 negative. Therefore, to quantify vessels in the injured segment, we relied on PECAM-1 labeling. We used methods similar to those described by Loy et al. (2002)
to quantify revascularization. In each injured cord, serial epicenter sections were examined and the area of maximal damage was selected for density analysis. The central gray matter region of the cord (751 µm × 590 µm) was captured with a CCD camera mounted to a Nikon Optiphot microscope. Images of injured sections were analyzed in random order by a time point-blinded observer. A grid (divided into 20 µm × 20 µm squares) was then placed over the entire cross section using Adobe Photoshop 6.0
. Within these areas, per-area blood vessel density was collected using NIH Image 1.62
. Statistical comparisons of multiple groups were made using analysis of variance (ANOVA) followed by the Scheffe and Dunnett tests. Significance was defined at the P
< 0.05 level.