Full length rat EPO was amplified from a cDNA library prepared from total RNA extracted from rat lung, cloned into BamH1-EcoR1 cut HCMV-polyA/SASB3–16 and co-transfected with the nonreplicating HSV recombinant UL41E1G6 on 7b cells, a complementing cell line derived from African green monkey kidney (Vero) cells modified to provide the essential ICP4 and ICP27 gene products in trans. Clones were selected by identification of clear plaques, purified by limiting dilution and the EPO insert confirmed by PCR followed by DNA sequencing. The clone which expressed the highest levels of EPO on infection of 293 cells (designated vEPO) was propagated to high titer (4 × 1010
pfu/ml), and used in the experiments. Control vector vC is identical to vEPO except that a reporter gene for green fluorescent protein (GFP) or Lac Z was inserted in the expression cassette in place of the EPO gene (). These backbone of these non-replicating HSV constructs also differ from the non-replicating HSV recombinants used in the previous studies in that vector is defective in expression of 4 immediate early gene products and the transgene expression cassette has been placed into the ICP4 locus; similar to the HSV recombinant expressing preproenkephalin that is currently in clinical trial in patients with intractable pain from cancer (Wolfe et al., 2009
Female Sprague–Dawley rats weighing 225–250 g were housed one to four per cage approximately 7 days prior to the beginning of the study, with free access to food and water and maintained on a 12:12, light:dark schedule at 21° C and 60% humidity. All housing conditions and experimental procedures were carried out in accordance with approved institutional animal care and use protocols. Under isoflurane anesthesia a C6–8 vertebral laminectomy was performed, exposing the C7 spinal cord segment without opening of the dura, and 160 Kdyne blunt force trauma was applied asymmetrically to the right dorsum of C7 spinal cord using a 1.5 mm diameter probe controlled by a computerized impactor device (Infinite Horizons, Precision Systems and Instrumentation, Lexington, KY). One hour after injury 1 μl containing 4×1010 plaque forming units (pfu) of either vEPO or vC was injected above the lesion, and an additional 1 μl injected below the lesion on right side of the spinal cord at a rate of 0.5 μl/min (total injected volume 2 μl). Sham animals underwent the same spinal cord exposure but no injury or injection was performed. For the behavioral analysis, there were 8 animals in each SCI group (vEPO and vC) and 4 sham controls. At the conclusion of the behavioral experiments (8 weeks after injury), animals were perfused through the heart with 100 ml of phosphate-buffered saline (PBS) followed by 500 ml of 4% paraformaldehyde in PBS. The spinal cord was removed, postfixed at 4°C overnight, and cryoprotected with 30% sucrose in PBS. For protein analysis, 2 and 8 weeks after injury animals were perfused through the heart with 100 ml of PBS and spinal cord removed and quickly frozen in dry-ice.
Forelimb motor function was evaluated using the following tests:
The open-field environment consisted of a circular plexiglas enclosure 95 cm in diameter, 40 cm high with an anti-skid floor. Rats were tested in pairs in a shadowy light to encourage locomotor activity. Animals that remained stationary for a period longer than 20 sec were picked up and placed at the center of the open-field arena to reinitiate locomotion. All rats were scored over a time window of a maximum duration of 4min, although the observation period was sometimes extended to make sure that the animals’ abilities were not underestimated.
Limb-use asymmetry test (Cylinder test)
Forelimb use during explorative activity was analyzed by putting rats in a transparent cylinder (20 cm diameter and 30 cm height) for 5 min or 20 touchs during the trial. A mirror was placed behind the cylinder at an angle to enable the rater to record forelimb movements when the animal was turned away from the rater. The cylindrical shape encourages vertical exploration of the walls with the forelimbs as well as landing activity.
Measurement of forelimb placing
Forelimb placing asymmetry was scored using the vibrissae-elicited forelimb placing test (Schallert et al., 2000). Animals were held by their torsos allowing forelimbs to hang free. While holding the animal, the experimenter made gentle up and down movements in space prior to place testing, which facilitated muscle relaxation and eliminated any struggling movements. Independent testing of each forelimb was induced by brushing the respective vibrissae on the edge of a table top once per trial for 10 trials. Intact animals place the forelimb of both sides quickly onto the countertop. Animals with unilateral damage, depending on the lesion site, will show varying degrees of impaired limb placing ability, while still placing the unimpaired limb reliably. A score of one was given each time the rat placed its forelimb on the edge of the tabletop in response to the vibrissae stimulation. Percent unsuccessful placing responses were determined (number incorrect ×10) for impaired forelimb and non-impaired responses. Typically, animals place successfully on 100% of the trials with the unimpaired limb; therefore, only the responses of the impaired limb are shown.
The grid walk test was initially developed to assess the ability of the animal to precisely control hind paw placement (Bresnahan et al. 1987; Kunkel-Bagden et al. 1992). We used a runway with regularly spaced horizontal bars 1.0 cm apart. Errors were coded when the animal misplaced either the forepaw or the hindpaw, such that the paw fell through the space in the grid, up to a maximum of 20 missteps. Animals that did not display at least frequent stepping in the BBB test were unable to cross the walkway and were assigned the maximum score.
Hindlimb motor function was evaluated using the following tests:
Basso–Beattie–Bresnahan (BBB) test
Hindlimb motor behavior was assessed once a week after injury for 5 weeks. Animals were trained to walk in an open area and the hindlimb movement rated on a 21-point scale, with a score of 0 corresponding to no observable hind limb movement and a score of 21 given when plantar stepping toe clearance, trunk stability, coordinated limb movement, and an erect tail was present (Basso et al., 1995
‘CatWalk' automated gait analysis
The animals traverse a walkway (plexiglass walls, spaced 8 cm apart) with a glass floor (100 × 15 × 0.6 cm3) (length × width × thickness) located in a darkened room. Light from an otherwise completely encased white fluorescent tube (length 110 cm, 30 W) enters the distal (from the observer) long edge of this glass floor. Sufficiently far from the edge, it strikes the surface below the critical angle and is entirely internally reflected. Only at those points where a paw touches the glass, light exits the floor and scatters at the paw, illuminating the points of contact only. Via a mirror, the corridor’s floor is monitored by a Pulnix TM-765E CCD camera (Pulnix Inc., UK) equipped with a wide angle objective (Cosimar 8.5 mm).
Primary cell culture
Primary spinal cord neurons from E17 rat pups were plated on 12 well plates coated with poly-D-lysine, at a concentration of 1.5×106 cells per well and cultured in Neurobasal media containing B27, Glutamax I, Albumax II, and penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA). At 14 days in vitro (DIV) vectors vEPO or vC at a multiplicity of infection (MOI) of 2 were added to the well for 2 hrs. Two and three days after infection cell lysates of spinal cord neurons and their culture media were collected for Western blot analysis and ELISA determination of transgene expression. Each experiment was repeated three times.
10 mm in length of spinal cords around epicenter were dissected and homogenized in lysis buffer containing 50 mM Tris, 10 mM NaCl, 1% Nonidet P-40, 0.02% NaN3, protease inhibitor, and phosphatase inhibitor mixtures (Sigma) at pH 7.4. Cultured E17 spinal cord neurons were collected in the same lysis buffer after being dislodged from the culture plates with a cell scraper. Cell lysates and tissue homogenates were sonicated and centrifuged at 15,000 × g for 10 min at 4 at 15,000ere so DRG cell cultures was centrifuged at 1,000 × g for 10 min at 4 °C. Protein concentration in tissue homogenates and cell lysates was determined using the BCA assay (Pierce) and spectrophotometry (AD340; Beckman Coulter). Aliquots containing 20 μg of protein were dissolved in Laemmli buffer and boiled at 95 vC for 5 min.
Proteins were separated on 4–20% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels and then transferred onto a polyvinylidene diflouride membrane (Millipore, Medford, MA, USA). Immunoblots were probed with primary antibody to anti-HA (Sigma, Saint Louis, MO, USA), anti-synaptophysin (Millipore Corporation, Billerica, MA, USA), anti-PSD-95 (Millipore Corporation, Billerica, MA, USA), anti-phosphorylated neurofilament H and tau (SMI31 Covance Corporation, Emeryville, CA, USA), anti-nonphosphorylated neurofilament H (SMI32 Covance Corporation, Emeryville, CA, USA), anti-GAPDH (Millipore Corporation, Billerica, MA, USA), or anti-β-actin (Santa Cruz Biotechnology), then incubated with horseradish peroxidase-conjugated secondary antibody, followed by enhanced chemiluminescence detection (Amersham Biosciences, Arlington Heights, IL, USA). Chemiluminescence detection values were used to quantitate the Western blot results, and the value of the protein of interest normalized to the appropriate internal control. Each in vitro experiment was repeated four times and each animal experiment represented the results of samples from eight different animals. Data are presented as mean ± SEM.
Enzyme-linked Immunosorbent Assay
The amount of EPO released in vitro of produced in vivo was determined using a commercially available ELISA kit (R&D System). Each of the experiments was repeated four times.
Total RNA was isolated from rat spinal cord via Trizol (Invitrogen, Carlsbad, CA, USA). cDNA prepared from mRNA isolated from rat spinal cord was amplified using following primer sets: b-actin-forward (5′-CAG TTC GCC ATG GAT GAC GAT ATC-3′) and b-actin-reverse (5′-CAC GCT CGG TCA GGA TCT TCA TG-3′) for β-actin, EPO-forward (5′-CCG GAA TTC GCC AGG CGC GGA GAT G-3′) and EPO-reverse (5′-CGG GAT CCT CAA GCG TAA TCT GGA ACA TC-3′) for EPO. Amplification was carried out by denaturation at 94° C for 5 min followed by 28 cycles (94° C for 30 sec, 68° C for 3 min, and 1 cycle 68° C for 8 min) using a GeneAmp PCR 2700 (Applied Biosystems, Foster City, CA). Each in vitro experiment was repeated four times and each animal experiment represents the results of samples from five different animals. Data are presented as mean ± SEM.
Measurement of lesion cavity
At 8 weeks post-injury, cervical spinal cord was removed, post-fixed and cryoprotected and sectioned using a cryostat (Leica). Twenty μm serial sections of spinal cord were thaw-mounted onto cold Superfrost glass slides (Fisher Scientific), heated at 37° C and stained with hematoxylin–eosin following standard protocol. Five serial sections were selected every 10 sections at the epicenter of the lesion. The area of the cavity containing tissue damage was determined from images captured using a Nikon Eclipse E1000 microscope equipped with a Plan Apo2X/0.1 lens using Metamorph 7.0 software (Molecular Devices, Palo Alto, CA).
Similar serial cryosections through the epicenter of the lesion, as described above, from 8 week post-injury cervical spinal cords of vEPO and vC treated animals were examined for CD-45 expression using a monoclonal antibody (1:1000; Invitrogen) followed by complementary secondary tagged fluorescent antibody (Alexa Fluor 488; Invitrogen).
The statistical significance of the difference between groups for the in vitro and in vivo western results was determined by one-way ANOVA (SPSS 12.0) using Bonferonni’s correction for the multiple post hoc analyses. Parametric statistics using the general linear model for repeated measures were used to identify significant effects of treatment conditions on the behavioral analysis of motor function (SPSS 18.0). The statistical significance of the difference between groups for the neurite length measurements was determined using the Student’s t-test. All results were expressed as mean ± SEM with p < 0.05 considered significant.