All animal manipulation procedures complied with NIH regulations and were approved by the Institutional Animal Care and Use Committee guidelines at both the University of Louisville and Case Western Reserve University School of Medicine. Adult male (275–350 g) Sprague Dawley rats were used for these experiments. Each rat was anesthetized with an intramuscular injection of a mixture of ketamine hydrochloride (50 mg/kg), xylazine (6.5 mg/kg) and acepromazine (2.5 mg/kg) for each surgical procedure.
Cholera toxin B subunit (CTB) injection
To anterogradely trace primary afferent axon terminals in the gracile and cuneate nuclei, 5–10 μl of 1% CTB (Sigma-Aldrich, St. Louis, MO), dissolved in 0.25 M Tris HCl, pH 7.4, was injected into the right forelimb median and ulnar nerves and left hindlimb sciatic nerves using a Hamilton syringe with a 30 gauge needle (Webster and Kemplay, 1987
; Maslany et al., 1991
; Onifer et al., 2005
). A nerve crush procedure was not used for the CTB injections, to avoid increased CTB uptake by nociceptive dorsal root neurons (Tong et al., 1999
). CTB tracing prior to cervical SCI allowed us to both visualize the degenerating primary afferent terminals (Sayer et al., 2002
) and to verify the completeness of the SCI (Onifer et al., 2005
Rats subjected to SCI were selected at random one week following CTB injections. The dorsal surfaces of the C3-4 spinal cord segments were exposed by laminectomy of the C3 and C4 vertebrae and an incision was made in the underlying dura. A 1.5 mm deep transverse lesion between the C3 and C4 dorsal roots was made with a Vibraknife™
as previously described (Onifer et al., 2005
), severing most primary afferents in the dorsal column-DCN pathway.
A 15 mm incision was made over the T2 spinous process in the mid-dorsal line between the scapulae. The dorsal surface of the T1 spinal cord segment was exposed by laminectomy. A pair of ultrafine microforceps was then inserted through the dura into the spinal cord parenchyma to a depth of 1 mm between the median dorsal raphe and the right dorsal root entry zone. The forceps were closed tightly twice for a total of 10 seconds. Great care was taken to avoid additional trauma to the cord or roots and the tips of the forceps were not inserted deeper than 1 mm. The animal recovered for 2 weeks before receiving DRG microtransplants (described below).
Tissue preparation for mRNA and protein analyses
Tissues were isolated from randomly chosen normal rats (n=6) and rats 2 days (n=3), 1 week (n=3) or 3 weeks (n=6) following cervical SCI. Each rat was transcardially perfused with 100 ml of cold 0.1 M phosphate buffer, pH 7.4, (PB). The right and left dorsal column nuclei, the right and left visual cortices and the right and left sides of 2mm of the C3-4 spinal cord segments, spanning the SCI site, were carefully dissected, placed immediately in liquid nitrogen and stored at −80°C. The tissue from one side of each rat was used for mRNA analysis and tissue from the opposite side was used for Western blot analysis as described below.
DCN, visual cortices, and spinal cord tissues from one side of each rat were quickly thawed and then homogenized in 1 ml of QIAzol Lysis Reagent (Qiagen, Valencia, CA) using a PolyTron homogenizer. Total RNA was prepared with the RNeasy Lipid Tissue Mini Kit (Qiagen) according to the recommended protocol. The quality and quantity of total RNA was determined by spectrophotometry and agarose gel electrophoresis. There was no detectable difference in 28S and 18S rRNA between normal and injured rats.
Real time polymerase chain reaction (rtPCR)
One microgram of total RNA was reverse transcribed using the high-capacity cDNA archive kit (Applied Biosystem, Foster City, CA) according to the recommended protocol. Probes used for TaqMan quantitative rtPCR were selected for NG2 (Rn00578849), neurocan (Rn01642355), brevican (Rn01495414), aggrecan (Rn00672903) and glial fibrillary acidic protein (GFAP) (Rn00566603) from the pre-validated Assays-on-Demand (Applied Biosystems, Foster City, CA) library. Individual probes were used in a 40 μl reaction utilizing TaqMan PCR Master Mix and subjected to 40 cycles of real time-PCR (ABI 7300, Applied Biosystem, Foster City, CA). The 18S ribosomal RNA was used as an internal control in the reaction. Tissue samples used for comparing each individual gene were run in the same reaction with a common reaction mixture. Each sample was run in triplicate for both the gene of interest and the 18s control gene. The Comparative CT Method (DDCT Method) was used to calculate changes in gene expression. The mean critical threshold (Ct) was calculated for the gene of interest and 18S genes to determine a relative expression value. Individual rat’s Ct values expressed as fold change from normal were then compared by 1-way ANOVA followed by post-hoc Tukey’s HSD comparison tests using the statistical software SPSS v.13.0 (SPSS, Chicago, IL) and Sigma Plot v.8.0 (Systat, Point Richmond, CA).
Electrophoresis and Western blot analysis
The following antibodies were used in this study.: anti-NG2 (clone 7.1, Chemicon), anti-neurocan C-terminal epitope (clone 650.24, which detects both the C-terminal and full-length neurocan isoforms, Chemicon, Temecula CA); anti-neurocan N-terminal epitope (clone 1F6, which labels the N-terminal and full-length neurocan isoforms, Developmental Studies Hybridoma Bank, Iowa City, IA), anti-brevican core protein (rabbit polyclonal antibody B6, which recognizes both the full length and C-terminal brevican isoforms), anti-brevican N-terminal ADAMTS4/5 cleavage product (rabbit polyclonal antibody B50, Viapiano et al., 2003
), anti-aggrecan core protein (clone Cat-301, Matthews et al. 2002
), anti-GFAP (clone GA5, Sigma-Aldrich) and anti-beta tubulin (Invitrogen, Carlsbad CA). Frozen tissue samples from spinal cord, DCN and visual cortex were thawed on ice and homogenized in 10 volumes of 40 mM TrisHCl, pH 7.6, containing 40 mM sodium acetate and a protease inhibitor cocktail (Complete, Roche Applied Science, Indianapolis, IN). Homogenization was performed on ice by three 5sec bursts of tissue sonication, using a 6.5 mm microprobe at a power of 35 W. Tissue homogenates were subsequently passed through a 30G needle to retain and disrupt unbroken debris. Aliquots of the homogenates at a final protein concentration of 2–3 mg/ml were treated with 0.3 U/ml of protease-free chondroitinase ABC (chABC, Seikagaku, Tokyo, Japan) for 8h at 37°C. Chondroitinase activity was stopped by boiling the samples in the presence of 1X gel-loading buffer. Samples containing 10 μg total protein were electrophoresed on reducing 3–10% SDS-polyacrylamide gels and analyzed by Western blotting. Immunoblots were developed by chemiluminescence and the integrated optical density (O.D.) of each target protein was imaged and quantified using the Gel-Pro Analyzer software (v3.1, Media Cybernetics, Silver Spring MD). Blots were stripped and re-probed for beta tubulin as loading control. O.D. ratios (O.D. target protein/O.D. beta tubulin) for each protein and tissue region were compared by 1-way ANOVA followed by Tukey’s multiple comparison test using SPSS v.13.0 and Sigma Plot v.8.0 statistical software.
Two weeks after thoracic SCI, DRG neurons from adult C57Bl/6N mice that constitutively express green fluorescent protein (GFP) were microtransplanted as previously described (Davies et al., 1999
; Grimpe and Silver, 2004
). Briefly, on the day of microtransplantation, mice were terminally anesthetized with isoflurane and decapitated. Approximately 35–40 cervical, thoracic, and lumbar DRG were dissected. They were dissociated for 60 minutes in a solution containing dispase (Roche; Indianapolis, IN) and collagenase (Worthington. Lakewood, NJ). Ganglia were triturated in the presence of 25 μL of 5 mg/mL deoxyribonuclease (DNase, Sigma-Aldrich). The resulting single cell suspension was spun at 3000g for three minutes and the cells resuspended in L-15 media (Gibco, Invitrogen), at an approximate density of 1000 neurons/μL. Five μL of the DNase solution was added to the cells, which were kept on ice until transplantation.
Each injured recipient rat was anesthetized and placed into a stereotaxic frame. The dorsal surfaces of the rostral cervical spinal cord and the caudal medulla were exposed. The cell suspension was transplanted through a pipette inserted into the right fasciculus gracilis, 0.2–0.3 mm below the pial surface at about 1 mm caudal to the gracile nucleus, using a Nanoject II delivery system. All transplants used a total volume of 690nl, delivered over ten minutes as previously described (Davies et al., 1997
; Grimpe and Silver, 2004
). The pipette was withdrawn five minutes after the final injection. Following transplantation, rats received either no further treatment (n=6), a single injection of chABC (n=4), a single injection of NT-3 lentivirus (n=5) or combined injections of chABC and NT-3 lentivirus (n=6). All animals were sacrificed 10 days following microtransplantation.
A pipette was inserted to a depth of 0.3mm at the lateral junction of the right fasciculus gracilus with the right gracile nucleus gracilis and 690nl of protease-free chABC (20U/mL; Seikagaku America, Falmouth, MA) using a Nanojet II delivery system (Drummond Scientific, Broomall, PA; Massey et al., 2006
). A piece of Duragen™
(Integra, Plainsboro, New Jersey) covered the dural defect.
Development of NT-3-expressing lentiviral vector
The vector backbone for the self inactivating lentiviral vector was a gift from S. Gerson at Case Western Reserve University (Zufferey et al., 1998
). Sequences for cloning of the gene encoding full length murine NT3 (777bp) into this vector were provided by G. Landreth and S. O’Gorman at Case Western Reserve University. All vectors were sequenced after cloning to ensure sequence fidelity.
Virions were prepared by triple transfection of packaging, envelope, and transducing vectors into cultured human 293T cells. Virus-enriched media was collected 48 hours post- transfection. Viral particles were concentrated by passing the media through a Microkos ultrafiltration unit (Spectrum Labs, Rancho Dominguez, CA).
Viral titers of the NT-3 containing vectors were determined indirectly using a reverse transcriptase (RT) assay due to the absence of reporter genes. The titer of the GFP-expressing vector was calculated by FACS sorting and quantification of infected cells. Since both vectors had the same sequence, it was assumed that the level of RT activity would be equivalent and would simply reflect the concentration of virus present. The amount of radionucleotide incorporation present in 10 μl aliquots of the GFP-expressing virus with a known titer was compared to the amount of incorporation present in 10 μl aliquots of the NT-3-expressing vectors with unknown titers. Five separate measurements with different dilutions were taken from each stock solution for the assay. Quantification was provided via scintillation counts over three minutes.
DRG survival assay
Adult cortical astrocytes were plated onto poly-lysine coated 6-well culture plates. Cells were transduced with lentiviral constructs containing NT3-lentivirus or GFP-lentivirus, both adjusted to 2 × 108
infectious units (IU) per ml. Mock transducions were performed with medium containing 1× polybrine (6ug/ml). Twenty four hours post transduction, culture medium was changed to fresh Eagle medium containing fetal bovine serum (FBS). Four days post transduction, DRG from E15 rats were prepared as described by Davies et al (1994)
. Four thousand DRG cells were plated onto poly-lysine-coated glass coverslips in 24-well plates. A total of 280ul of conditioned medium from either the transduced or mock transduced astrocytes and 20ul of fresh Eagle medium with serum were added to each well. Each condition was performed at least in triplicate.
Two days after plating, DRG cells were washed and fixed in buffered 4% paraformaldehyde (PFA) for 30 minutes. Fixed cells were blocked at room temperature in PBS containing 5% normal goat serum, 0.1% v/v Triton X-100 and 0.1% w/v bovine serum albumin for 2 hours, before incubation with a mouse monoclonal anti-beta tubulin antibody (Sigma-Aldrich), at a 1:1000 dilution. Slides were incubated with primary antibody overnight, washed three times with PBS and stained with a goat-anti mouse antibody conjugated with the fluorochrome Alexa-594 (Invitrogen, dilution 1:500). The secondary antibody was also incubated overnight, followed by washing and mounting of the coverslips using Citifluor. The surviving number of cells per a well was counted using an Olympus microscope equipped with fluorescence. Results from these experiments were then analyzed by 1-way ANOVA followed by Tukey’s multiple comparison test using SPSS v.13.0 and Sigma Plot v.8.0 statistical software.
NT-3 lentivirus delivery
Injured rats were anesthetized 12 days after the initial injury and placed in a stereotaxic frame. The dorsal surface of the caudal medulla was exposed and a pipette inserted to a depth of 0.3 mm into the medial zone of the right gracile nucleus. Six hundred and ninety nl of viral supernatant (2 × 108 IU/ml) were injected using a Nanoject II delivery system. A second 690 nl injection was made into the lateral zone of the right gracile nucleus. After each injection, the pipette was allowed to remain in place for 5 minutes. Following surgery, animals were isolated for 48 hours before receiving DRG microtransplants with or without concomitant chABC delivery.
ELISA assessment of NT-3 expression
Tissue samples from rats receiving either saline injections (n=3) or injections of NT-3 expressing lentivirus (n=3) ten days earlier were prepared by sonication in lysis buffer (137 mM NaCl, 20 mM Tris-HCl, 1% v/v Nonidet-P40, 10% v/v glycerol and a cocktail of protease inhibitors, pH 7.4), followed by centrifugation and collection of the resulting supernatant. Protein concentration was determined by the Bradford method and equal amounts of each sample (0.2 mg/ml) were analyzed in triplicate by ELISA 96-well plates (Nunc, Rochester, NY) coated with anti-human NT-3 polyclonal antibody (Chemicon) diluted 1:5000 in 0.025 M sodium bicarbonate/0.025 M sodium carbonate, pH = 9.7, overnight at 4° C. Coated plates were blocked for one hour at room temperature, followed by incubation with experimental and control samples overnight at 4° C. NT-3 standards ranging from 4.68 to 300 pg/ml were used to generate a standard curve and run simultaneously with the samples. Following incubation with tissue, the plates were extensively washed with 20 mM Tris-HCl, 150 mM NaCl, 0.05% v/v Tween-20 (TBST). A mouse anti-NT-3 antibody (Chemicon), diluted 1:4000 in blocking buffer, was added to the wells and incubated overnight at 4° C. After washing with TBST, an anti-mouse secondary antibody conjugated to horseradish peroxidase (HRP) diluted 1:1000 in blocking buffer was added to each well and incubated for 2.5 hrs. Following extensive washing, 100 μl of the HRP substrate tetramethylbenzidine was added to each well and allowed to develop. Color development was stopped by adding 100 μl of 1 N HCl to each well, and absorbances were recorded at 450 nm using a microplate reader.
Control, injured and injured/microtransplanted animals were terminally anesthetized by pentobarbital overdose and perfused with oxygenated, heparinized, and calcium-free Tyrodes solution or 0.1M PBS, followed by 4% PFA in 0.1M PB. Cervical spinal cord and brainstem were removed from each rat and cryoprotected at 4°C in PB containing 30% sucrose for 3 to 4 days. Tissues were cryosectioned at 20 μm in the transverse plane and stored at −80°C. Brainstems from rats that received microtransplants were sectioned in the sagittal plane at 50 μm using a Vibratome and transferred to multiwell plates as free floating sections into 0.1M PBS. These sections were mounted onto microscope slides, coverslipped and inspected for fluorescent DRG microtransplants.
The following antibodies and probes were used for histological detection of CSPGs: anti-NG2 (clone 7.1, 1:200 dilution; Chemicon), anti-neurocan (clones 650.24, 1:500 and 1F6, 1:200), and anti-brevican core protein (rabbit polyclonal antibody B756, directed against aminoacids 420–433 of rat brevican, 1:500), and anti-aggrecan (clone Cat-301, 1:200). Additionally, CS chains were detected with Wisteria floribunda (WFA) lectin (1:100, Sigma-Aldrich) as described (Bruckner et. al., 1998
; Massey et al., 2006
To identify the location of the DCN, sections were processed for cytochrome oxidase activity (Wong-Riley, 1979
; Crockett et al., 1993
). Sections were incubated in a solution of 10% sucrose, 0.1M PBS and dimethyl sulfoxide (Sigma-Aldrich) for 10 minutes. These sections were then reacted with ?% 3,3′-diaminobenzidine tetrahydrochloride (Sigma-Aldrich) and 0.03% cytochrome C (Sigma- Aldrich) at 37°C for 1 to 2 hours before being rinsed two times in 0.1M PB and coverslipped.
Antibodies to beta-tubulin III (BTIII) (1:1000; Covance Research Products, Inc., Berkeley, CA), parvalbumin (1:400; Chemicon), neuronal-specific nuclear protein (NeuN) (1:500; Chemicon) or microtubule-associated protein 2 (MAP2) (1:500; Sigma-Aldrich) were used to observe neurons and their processes (Massey et al., 2006
). Antibodies to GFAP (1:2000; Chemicon) and to the iC3b (CD11b) complement receptor (OX42) (1:500; Serotec, Raleigh, NC) were used to visualize astrocytes and microglia, respectively. Goat anti-CTB (1:2000; List Biological Laboratories, Hornby, Ontario, Canada) was used to see CTB-traced primary afferent axon terminals (Massey et al., 2006
). Visualization of GFP expressed by microtransplanted mouse DRG neurons was enhanced by reacting sections containing the right DCN with an anti-GFP antibody (1:1000; Chemicon; Davies et al, 1999
The brainstem and spinal cord sections were incubated with TBS (pH 7.4) containing 10% normal donkey serum (Jackson ImmunoScientific, Inc.) or 5% normal goat serum with 0.1% bovine serum albumin and combinations of the primary antibodies. Sections were then reacted with species-specific donkey IgG (Jackson ImmunoScientific, Inc.) and goat IgG (Invitrogen) secondary antibodies conjugated with either fluorescein isothiocyanate (FITC), Alexa-fluor 488, cyanine 3 (Cy3), Alexa-fluor 594 or 7-amino-4-methyl-3-coumarinylacetic acid (AMCA). Confocal or epifluorecent images of immunoreactivity (IR) from coverslipped sections were obtained using an Olympus Laser Confocal microscope and digitized with an Olympus Optical (Melville, NY) 3 Laser Fluoview 500 or obtained using a Nikon Eclipse E400 (Tokyo, Japan) microscope connected to a SPOT Insight 3.2 color camera.
Quantification of axonal regeneration
Separate images of the brainstems from injured rats that received microtransplants were obtained for each fluorochrome and digitally merged using SPOT Advanced software (Nikon). The NeuN immunoreactivity was used to identify neurons in the gracile nuclei in the caudal medulla along with the rest of the neuronal groupings in this area. The ability of GFP-immunoreactive neuritic processes from the transplanted DRG neurons to enter the nucleus gracilis was quantified by measuring the total length of transplant axons within the host gracile nucleus using Metamorph® software (Molecular Devices, Sunnyvale, CA). Statistical comparisons between treatments were performed using SPSS v.13.0 and Sigma Plot v.8.0 statistical software.