Animals and pyramidotomy
Adult female Sprague Dawley rats (250–300gm) were used in this study. All surgical procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Baylor College of Medicine. Unilateral CST lesions were performed at the level of the pyramids above the decussation of the CST as described previously (Zhou, L., Baumgartner, B. J., Hill-Felberg, S. J., McGowen, L. R., and Shine, H. D. 03;Zhou, L and Shine, H. D. 03). Rats were anesthetized with continuous isoflurane using a vaporizing system (Vip 3000; Matrx Medical Inc., Orchard Park, NY). A midline incision was made in the ventral neck region and the basiooccipital portion of the skull was exposed by blunt dissection. A 2 mm craniotomy was performed with a drill burr just lateral to the midline ridge to reveal the left pyramid. Taking the basal artery as the landmark of the midline, a 1.5-mm wide and 0.5-mm deep incision was made into the pyramid followed by aspiration the incision site with a fine-tipped glass suction pipette to ensure that all the CST was completely transected. The exposed brain tissue was covered with gel foam and the skin was closed with wound clips. Buprenorphine (0.1 – 0.5 mg/kg) or ketoprofen (5mg/kg) was administrated by subcutaneous injection for post-operative analgesia.
Anterograde tracing unlesioned CST fibers with biotinylated dextran amine
Biotinylated dextran amine (BDA; lysine fixable, MW 10,000; Molecular Probes, Eugene, OR) was used to label axons of unlesioned CST as described previously (Zhou, L., Baumgartner, B. J., Hill-Felberg, S. J., McGowen, L. R., and Shine, H. D. 03;Zhou, L and Shine, H. D. 03;Chen, Q, Zhou, L, and Shine, H. D. 06). The rats were anesthetized with isoflurane, positioned in a stereotaxic frame, and the sensorimotor cortices were exposed. Using a Nanoliter Injector (World Precision Instruments, Sarasota, FL) fitted with a glass pipette with a 40 μm tip, a solution of BDA (10% in phosphate buffered saline (PBS), pH 7.4) was injected into 12 sites (147 nl per site) at a depth of 1.2 μm in the sensorimotor cortex (Paxinos, G. and Watson, C. 86;Grill, R. J., Murai, K., Blesch, A., Gage, F. H., and Tuszynski, M. H. 97). The skin was closed with wound clips and ketoprofen (5mg/kg) or buprenorphin (0.1–0.5 mg/kg) was administered as an analgesic.
Retrograde delivery of adenoviral vectors to motoneurons of the rat lumbar spinal cord
Replication-defective adenoviral vectors (Adv) carrying the DNA sequences for rat NT-3 (Adv. NT-3) or LacZ gene (Adv. LacZ) under control of the mammalian EF1α promoter(Baumgartner, B. J. and Shine, H. D. 97;Baumgartner, B. J. and Shine, H. D. 98) were delivered into the rat lumbar spinal cord using the same method previously reported (Zhou, L., Baumgartner, B. J., Hill-Felberg, S. J., McGowen, L. R., and Shine, H. D. 03;Zhou, L and Shine, H. D. 03;Chen, Q, Zhou, L, and Shine, H. D. 06). Rats were anesthetized and an incision was made posterior and parallel to the femur on the right leg to expose the sciatic nerve. The sciatic nerve was transected approximately 2 mm proximal to the bifurcation of the common peroneal and tibial branches. The proximal end of the cut nerve was placed into a small polyethylene chamber filled with 1 × 109 infectious units of either Adv. NT-3 or Adv. LacZ, fixed with cyanoacrylate glue, and the incision was closed with sutures and wound clips.
Immunosuppression with anti-leukocytic antibodies and lipopolysaccharide treatment
Purified monoclonal antibodies against the rat CD4 (W3/25) and CD45 (MRC OX-22) was used to transiently suppress the immune system (Romero, M. I. and Smith, G. M. 98;Caballero, F., Pelegri, C., Castell, M., Franch, A., and Castellote, C. 98). One day before CST lesion, rats received an intraperitoneal injection (i.p.) of a combination of 50 μg of each antibody. To reactivate the immune response to WD, rats were given intraperitoneal injections of LPS (250 μg/kg prepared in sterile saline; from Escherichia coli, serotype 055:B5, Sigma, L-2880). This dose was selected based upon discussions with V. H. Perry to reactivated WD in the lesioned CST without causing a significant elevation in fever. Rectal temperatures were measured before and 1, 5, 18, and 24 hours after LPS injection.
Isolation of CNS mononuclear cells from the spinal cord and flow cytometry analysis
A modified method based on methods described by others (Campanella, M., Sciorati, C., Tarozzo, G., and Beltramo, M. 02;Mack, C. L., Vanderlugt-Castaneda, C. L., Neville, K. L., and Miller, S. D. 03) and avoids the alteration of surface antigens caused by enzymatic digestions (Ford, A. L., Foulcher, E., Goodsall, A. L., and Sedgwick, J. D. 96) was used to isolate mononuclear cells from the dorsal columns. Rats were anesthetized with isoflurane and perfused through the left ventricle with cold PBS until the effluent ran clear. Spinal cords were removed and placed in cold Hank’s Basic Salt Solution (HBSS). The dorsal columns were dissected from the spinal cords and forced through nylon mesh (100 μm, Falcon) into cold HBSS with 10% FBS to dissociate the tissue. The homogenate was resuspended in 30% Percoll solution (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and underlaid with 70% Percoll solution. The gradients were centrifuged at 1000 × g at 24°C for 20 minutes. CNS mononuclear cells were collected from the Percoll interface, washed once with HBSS supplemented with 10% FBS and centrifuged at 200 × g at room temperature for 10 minutes. The pellets were resuspended in FACS buffer (PBS with 0.08% NaN3) and incubated with FITC-conjugated anti-rat CD 45 (1:100, clone OX-1, BD Biosciences Pharmigen, San Jose, CA) and PE-conjugated anti-rat CD 11b/c (1:100, clone OX-42, BD Biosciences Pharmigen) antibodies for 40 minutes at 4°C in FACS buffer with 2% FBS, washed twice in FACS buffer, resuspended in FACS buffer, and analyzed with BD FACS Aria (BD Biosciences Pharmigen). Debris was excluded from analysis by using forward and side scatter parameters.
Histochemistry and immunohistochemistry
At the end of observation time points, rats were transcardially perfused with cold 4% paraformaldehyde in PBS. A portion of the lumbar spinal cord (L3 to L6) was removed and postfixed in the same fixation buffer for 6 hours and then infiltrated with 21% sucrose in PBS for cryoprotection. Cross sections of spinal cord 40 μm thick were cut on a cryostat and stored in cryoprotectant solution at −20°C until processed for histochemistry and immunohistochemistry study. Sections representing different levels of the lumbar spinal cord were picked randomly for BDA staining. The cryosections were washed in PBS and incubated with 0.3% H2O2 in PBS for 30 minutes at room temperature to remove endogenous peroxide activity. After 3 washes in PBST (PBS containing 0.1% Triton X-100), sections were incubated with ABC reagents (Vector Laboratories, Burlingame, CA) overnight at 4°C. Following 2 washes in PBST and one wash in PBS, the CST axons were incubated in freshly prepared diaminobenzidine solution (0.7mg/ml) containing 0.06% nickel chloride and 0.015% hydrogen peroxide until the neurites that were labeled with BDA developed a dark-brown reaction product. The sections were subsequently dried on glass slides, dehydrated in graded ethanol, cleared in xylene and coversliped.
Microglia in the lumbar spinal cord were assessed by immunohistochemistry using OX-42 raised against the complement receptor type 3 (CR3) that is expressed on ramified, amoeboid, and reactivated microglia and on macrophages (Aldskogius, H. and Kozlova, E. N. 98). Sections were washed in PBS and incubated in a blocking solution of 3% goat serum in PBS for 1 hour at room temperature followed by 3 washes in PBST. Then they were sections were incubated with OX-42 (monoclonal mouse anti-rat CD11b; MRC OX-42, Serotec, Oxford, UK, 1:200 in PBST) overnight at 4°C. Following 2 washes in PBST, the sections were incubated for 2 hours in 1:200 Cy2 conjugated goat anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) prepared in PBST with 1.5% goat serum. After 2 PBS washes, the sections were incubated 15 minutes with TO-PRO-3 (1:1000 in PBS, Molecular Probes, Eugene, OR) to stain the nuclei, and then washed in PBS for 2 times. The sections were mounted onto slides and coverslipped with Airvol 205 (Air Products Chemical Inc., Allentown, PA).
The W3/25 antibody reacts with CD4 molecule expressed on helper T-cells (Clark, S. J., Jefferies, W. A., Barclay, A. N., Gagnon, J., and Williams, A. F. 87) as well as some microglia and macrophages (Sroga, J. M., Jones, T. B., Kigerl, K. A., McGaughy, V. M., and Popovich, P. G. 03). After incubating with 0.3% H2O2 in PBS for 30 minutes, the cryosections were washed in PBS and incubated in a blocking solution of 5% horse serum in PBS for 1 hour at room temperature and then washed 3 times in PBST. The sections were then incubated with W3/25 (1:200 in PBST) overnight at 4 °C. Following 3 washes in PBST, the sections were incubated with biotin conjugated horse anti-mouse secondly antibody (1:200) and avidin-biotin complex (ABC) reagents (Vector Laboratories) according to the procedures recommended by the manufacturer. The W3/25 positive cells developed the dark brown reaction product after 4 minutes of incubation in freshly prepared diaminobenzidine solution (0.7mg/ml) containing 0.03% H2O2 and 0.06% nickel chloride. The sections were subsequently dried on glass slides, dehydrated in graded ethanol, cleared in xylene and coversliped.
Morphometric analysis and quantification of axonal plasticity
Slides were coded so that axon densities and number of inflammatory cells were determined by an investigator unaware of the treatment of each animal. Quantification of the amount of axonal crossing from the intact CST to the region of motoneurons over-expressing NT-3 was measured from photomicrographs using image analysis software as described previously (Chen, Q, Zhou, L, and Shine, H. D. 06;Grider, M. H. and Shine, H. D. 06). Dark-field photomicrographs of the spinal cord sections were taken with a digital camera (SPOT RT Color-2000, Diagnostic Instruments, USA) at 100X magnification. The photomicrographs were then analyzed using Image J software and the Feature J software plug-in (Sato, Y., Nakajima, S., Shiraga, N., Atsumi, H., Yoshida, S., Koller, T., Gerig, G., and Kikinis, R. 98) to automatically highlight linear figures representing BDA-labeled axons against a white background. The number of axons in the midline region was measured as the number of pixels in a region 26 μm wide and running from the bottom of the dorsal column to the top of the ventral column. To avoid error resulting from variable BDA labeling efficiencies among animals, the number of pixels in a region adjacent to the midline area and ipsilateral to the intact CST was measured. The value of axons crossing the midline was expressed as the ratio of pixels in the midline region to the number of pixels in the lateral region normalized to values of normal animals.
Quantification of microglia and CD4+ T-cells
Quantification of the amount of activation of microglia was measured from digital photomicrographs taken with a Zeiss LSM 510 confocal microscope (Carl Zeiss, Germany) at 20x magnification. A series of photos were compiled using the Z-stack program in the LSM 510 software suite. Morphology of microglia change from ramified morphology to amoeboid and rounded (macrophage) morphology after activation (Hossain-Ibrahim, M. K., Rezajooi, K., MacNally, J. K., Mason, M. R., Lieberman, A. R., and Anderson, P. N. 06). Only microglia containing identifiable nuclei were counted. T-lymphocytes were counted from digtal photomicrographs at 200x magnification. Since CNS T-cells are typically uniform in shape and size and are sparse in CNS tissue they were easily identified in the spinal cord sections. Only cells that had a clearly defined pattern of membrane staining were counted. For both quantitative analyses, the cells were counted in the dorsal columns of the lumbar spinal cords in 3 cross sections cut from each animal at each of the observation time points. To avoid the possibility of counting the same cells in adjacent sections the sections were spaced 600 μm apart.
All data are reported as group means ± standard error of the mean (SEM). A one-way ANOVA test, followed by the Student–Newman–Keul’s or Dunn’s test, was used to assess variances. All values with a p < 0.05 were accepted as significant.