Tissue sparing quantification at 8 weeks post-SCI.

<p>(A) Residual white matter quantification. (B) Residual grey matter quantification. AdV-ZFP-VEGF improves spinal cord grey matter preservation. (C) Representative sections are shown from each group. Sections shown are taken 2 mm rostral to the epicenter at 8 weeks after SCI. AdV-ZFP-VEGF treated spinal cord exhibited a larger extent of grey matter spared tissue, but not white matter; **p<0.001. Data are mean ± SEM values. n = 8/sham and AdV-ZFP-VEGF groups; n = 10/injured control and AdV-eGFP groups.</p

AdV-ZFP-VEGF improves hindlimb weight support.

<p>Catwalk gait analysis was used to assess hindlimb weight support. A sub-set of animals (with BBB scores >9) were assessed every week between 4–8 weeks, and each animal performed a standardized Catwalk run. A blinded observer analyzed the data. (A) Paw area: the maximal area of the paw print in contact with the detection surface of the CatWalk (expressed in mm²), (B) Paw width: the maximal distance spanning the medial and lateral contact points of the paw (expressed in mm), and (C) Paw length: the maximal distance spanning the cranial and caudal contact points of the paw (expressed in mm). (D) Representative images of CatWalk forelimb (green) and hindlimb (red) prints, which were used to quantify the data presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096137#pone-0096137-g006" target="_blank">Figure 6</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096137#pone-0096137-g007" target="_blank">Figure 7</a>. Data presented is the mean ± SEM, n = 5/group, at 8 weeks following SCI. One-way ANOVA (Holm-Sidak post-hoc). *p<0.05, **p<0.005.</p

Electrophysiological assessment following AdV-ZFP-VEGF administration.

<p>(A) Representative tracings of MEP's recorded from the hindlimb at 8 weeks post-injury. (B) MEP quantification. Recordings were obtained from hindlimb biceps femoris. Stimulation was applied to the midline of the cervical spinal cord (0.13 Hz; 0.1 ms; 2 mA; 200 sweeps). Latency was calculated as the time from the start of the stimulus artifact to the first prominent peak. AdV-ZFP-VEGF did not result in improved MEP's. (C) H-Reflex quantification. Recording electrodes were placed two centimeters apart in the mid-calf region and the posterior tibial nerve was stimulated in the popliteal fossa using a 0.1 ms duration square wave pulse at a frequency of 1 Hz. The rats were tested for maximal plantar H-reflex/maximal plantar M-response (H/M) ratios to determine the excitability of the reflex. AdV-ZFP-VEGF administration did not significantly alter the H/M ratio. n = 6/group.</p

AdV-ZFP-VEGF increases VEGF mRNA and protein.

<p>(A) VEGF mRNA levels encoding for VEGF₁₂₀, VEGF₁₆₄ and VEGF₁₈₈ isoforms were measured by quantitative real-time PCR at 5 days post-SCI. The bar graph illustrates that administration of ZFP-VEGF resulted in an increase of VEGF mRNA compared with AdV-eGFP and SCI injured control groups. Relative mRNA levels are expressed as the mean ± SEM, n = 4/sham and injured control groups, n = 5/AdV-eGFP and AdV-ZFP-VEGF groups. One-way ANOVA (Holm-Sidak post-hoc) was completed individually for each isoform **p<0.001, *p<0.01. (B) Western blot showing administration of AdV-ZFP-VEGF resulted in increased VEGF-A protein levels at 10 days post-SCI, and (C) Quantification shows a significant increase in VEGF-A 42 kD protein in AdV-ZFP-VEGF treated animals compared with control groups. Optical density (OD) of VEGF-A was normalized to actin. Data are presented as mean ± SEM, n = 4/sham, injured control and AdV-eGFP treated groups and n = 5/AdV-ZFP-VEGF treated group. One-way ANOVA (Holm-Sidak post-hoc) **p<0.02, *p<0.05.</p

Transduction of AdV-eGFP/AdV-ZFP-VEGF into the spinal cord.

<p>(A) Photomicrographs showing a transverse section of rat spinal cord obtained adjacent to the injury site 10 days after spinal cord injury and AdV-eGFP injection. eGFP signal was detected in both the gray matter and white matter. (B) High-power (63X) confocal images show that the AdV-eGFP vector (green) transfected neurons (NeuN), astrocytes (GFAP), oligodendrocytes (CC1) and endothelial cells (RECA-1). Cells have been counter-stained with DAPI (blue) as nuclear marker. (C) Bar graph displays quantification of transduced cell types ± SEM, as identified by the cell-specific markers NeuN, GFAP, RECA-1 and CC1. (D) Evaluation of AdV-ZFP-VEGF gene transfer. Western blot showed that the NFκB p65 rabbit polyclonal antibody recognizes the p65 activation domain in the AdV-ZFP-VEGF treated animals. The higher molecular weight bands are endogenous NFκBp65 fragments, which are also recognized by the antibody; however, these bands are present in both the control and treatment groups. The lower band (arrow) corresponds to the AdV-ZFP-VEGF and was only present in the treated animals. Lower panel shows actin expression as a protein control. Scale bar: 1000 µm for A; 100 µm for B.</p

AdV-ZFP-VEGF does not improve open-field walking (BBB) scores following SCI.

<p>Open-field locomotion was assessed using the 21-point BBB scale. Animals were assessed weekly for 8 weeks following injury by blinded observers (n = 8/sham and AdV-ZFP-VEGF groups; n = 10/injured control and AdV-eGFP groups). The left and right limbs were scored individually, but the data presented is the average between left and right hindlimb recovery.</p

AdV-ZFP-VEGF significantly reduces mechanical and thermal allodynia at 8 weeks post-SCI.

<p>Mechanical and thermal allodynia, often used as outcome measures of neuropathic pain, were monitored with von Frey monofilaments and tail-flick tests, respectively. (A) At-level pain. Animals were assessed with 2 g or 4 g von Frey monofilaments around the dorsal incision (above T6–T7 laminectomy and injury). Data are expressed as the average number of adverse reactions out of 10 applications of the monofilament. There was an overall treatment effect with AdV-ZFP-VEGF using the 2 g and 4 g monofilaments at 4 weeks and 8 weeks post-injury; *p<0.05. (B) Below-level pain. Animals were subject to increasing von Frey filaments (2 g–26 g), and the when they elicited a response, this value was taken as the pain threshold value. Data is reported as the average threshold for each group. AdV-ZFP-VEGF increased hindlimb threshold compared to other injured groups; *p<0.05, **p<0.005. (C) Below-level thermal allodynia. A 50°C thermal stimulus was applied to the distal tip of the tail. The data shown is the average time it took for the animals to withdrawl their tail from the stimulus (“tail flick”). Shorter response times indicate a decreased pain threshold. Animals treated with AdV-ZFP-VEGF showed an increased tolerance/threshold to thermal stimuli at 8 weeks post-injury compared to other injured groups; *p<0.05. Data were analyzed by One-way ANOVA. Error bars represent SEM. n = 8/sham and AdV-ZFP-VEGF groups; n = 10/injured control and AdV-eGFP groups.</p

AdV-ZFP-VEGF results in increased vessel counts and angiogenesis.

<p>(A) Left panel: Illustration of the area of spinal cord areas used for RECA-1 counting (2 grey matter areas, 2 white matter areas). (B) Representative sections taken 2 mm rostral to the epicenter from a AdV-ZFP-VEGF treated and AdV-eGFP control animal respectively immunostained with RECA-1 at 10 days after SCI; scale 100 µm. An increased number of vessels were observed in the AdV-ZFP-VEGF treated group. (C) Bar graph illustrating the RECA-1 positive cell counts 10 days after SCI. AdV-ZFP-VEGF administration resulted in a significant increase in vascular counts (2 mm and 4 mm away from the epicenter) as compared with the control group. (D) Representative confocal image from an ADV-ZFP-VEGF treated animal at 5 days post-injury. Image was taken at 2 mm rostral from the epicenter, and shows double-labeled cells. Cells were stained for endothelial cells (RECA-1, green) and proliferation (Ki67, red). Scale bar  = 50 µm (30 µm for magnified panel). (E) Angiogenesis was assessed by quantifying Ki67/RECA-1 co-labeled vessels. Data is presented at the percentage of RECA-1+ vessels that were also Ki67+, with an overall average increase of 10% vascular proliferation observed in the animals receiving AdV-ZFP-VEGF administration. All data are presented as mean ± SEM, and was analyzed by Two-way ANOVA (Holm-Sidak post-hoc). Angiogenesis data were analyzed by performing an arcsine transformation of the values, prior to Two-way ANOVA and post-hoc testing. *p<0.01, **p<0.001. n = 4/sham and injured control groups, n = 5/AdV-eGFP and AdV-ZFP-VEGF groups.</p

Forelimb and Hindlimb locomotion is improved by AdV-ZFP-VEGF administration.

<p>(A) Catwalk gait analysis was used to assess hindlimb swing speed. Animals were assessed every week between 4–8 weeks, and each animal performed a standardized Catwalk run. A blinded observer analyzed the data. Data presented is the mean ± SEM, n = 5/group, at 8 weeks following SCI. One-way ANOVA (Holm-Sidak post-hoc). *p<0.02. (B) Catwalk gait analysis was used to assess forelimb stride length. Animals were assessed every week between 4–8 weeks, and each animal performed a standardized Catwalk run. A blinded observer analyzed the data. Data presented is the mean ± SEM, n = 5/group, at 8 weeks following SCI. One-way ANOVA (Holm-Sidak post-hoc). *p<0.02.</p

AdV-ZFP-VEGF administration attenuated axonal degradation and increased neuron sparing.

<p>(A) Western blot indicates that administration of AdV-ZFP-VEGF resulted in a significant attenuation of NF200 degradation 10 days after injury. Lower panel shows actin protein control. (B) Relative OD value of controls versus AdV-ZFP-VEGF treated animals. Significant NF200 sparing was observed in AdV-ZFP-VEGF-treated animals compared to control groups at 10 days after injury, although all injured groups showed significant NF200 loss following SCI. Optical density of NF200 was normalized to actin. One-way ANOVA (Holm-Sidak post-hoc), *p<0.05. (C) Representative sections taken 2 mm rostral to the epicenter from AdV-ZFP-VEGF treated and AdV-eGFP treated animals immunostained with NeuN at 5 days after SCI; scale 200 µm. A greater number of NeuN-positive cells were observed in animals treated with AdV-ZFP-VEGF. (D) Bar graph shows quantification of the NeuN-positive cell counts at 5 days after SCI. There was a significant preservation of neurons overall in the AdV-ZFP-VEGF group compared to all the other injured groups (two-way ANOVA comparing distance from the epicenter and treatment group). Bar graph shows mean OD values ± SEM. Two-way ANOVA (Holm-Sidak post-hoc), *p<0.02. n = 5/sham, n = 4/injured control, AdV-eGFP and AdV-ZFP-VEGF groups.</p