The impact of riluzole on neurobehavioral outcomes in preclinical models of traumatic and nontraumatic spinal cord injury: results from a systematic review of the literature

Study Design:Systematic review.Objective:To evaluate the impact of riluzole on neurobehavioral outcomes in preclinical models of nontraumatic and traumatic spinal cord injury (SCI).Methods:An extensive search of the literature was conducted in Medline, EMBASE, and Medline in Process. Studies were included if they evaluated the impact of riluzole on neurobehavioral outcomes in preclinical models of nontraumatic and traumatic SCI. Extensive data were extracted from relevant studies, including sample characteristics, injury model, outcomes assessed, timing of evaluation, and main results. The SYRCLE checklist was used to assess various sources of bias.Results:The search yielded a total of 3180 unique citations. A total of 16 studies were deemed relevant and were summarized in this review. Sample sizes ranged from 14 to 90, and injury models included traumatic SCI (n = 9), degenerative cervical myelopathy (n = 2), and spinal cord-ischemia (n = 5). The most commonly assessed outcome measures were BBB (Basso, Beattie, Besnahan) locomotor score and von Frey filament testing. In general, rats treated with riluzole exhibited significantly higher BBB locomotor scores than controls. Furthermore, riluzole significantly increased withdrawal thresholds to innocuous stimuli and tail flick latency following application of radiant heat stimuli. Finally, rats treated with riluzole achieved superior results on many components of gait assessment.Conclusion:In preclinical models of traumatic and nontraumatic SCI, riluzole significantly improves locomotor scores, gait function, and neuropathic pain. This review provides the background information necessary to interpret the results of clinical trials on the impact of riluzole in traumatic and nontraumatic SCI

Delayed administration of a bio-engineered zinc-finger VEGF-A gene therapy is neuroprotective and attenuates allodynia following traumatic spinal cord injury.

Following spinal cord injury (SCI) there are drastic changes that occur in the spinal microvasculature, including ischemia, hemorrhage, endothelial cell death and blood-spinal cord barrier disruption. Vascular endothelial growth factor-A (VEGF-A) is a pleiotropic factor recognized for its pro-angiogenic properties; however, VEGF has recently been shown to provide neuroprotection. We hypothesized that delivery of AdV-ZFP-VEGF--an adenovirally delivered bio-engineered zinc-finger transcription factor that promotes endogenous VEGF-A expression--would result in angiogenesis, neuroprotection and functional recovery following SCI. This novel VEGF gene therapy induces the endogenous production of multiple VEGF-A isoforms; a critical factor for proper vascular development and repair. Briefly, female Wistar rats--under cyclosporin immunosuppression--received a 35 g clip-compression injury and were administered AdV-ZFP-VEGF or AdV-eGFP at 24 hours post-SCI. qRT-PCR and Western Blot analysis of VEGF-A mRNA and protein, showed significant increases in VEGF-A expression in AdV-ZFP-VEGF treated animals (p<0.001 and p<0.05, respectively). Analysis of NF200, TUNEL, and RECA-1 indicated that AdV-ZFP-VEGF increased axonal preservation (p<0.05), reduced cell death (p<0.01), and increased blood vessels (p<0.01), respectively. Moreover, AdV-ZFP-VEGF resulted in a 10% increase in blood vessel proliferation (p<0.001). Catwalk™ analysis showed AdV-ZFP-VEGF treatment dramatically improves hindlimb weight support (p<0.05) and increases hindlimb swing speed (p<0.02) when compared to control animals. Finally, AdV-ZFP-VEGF administration provided a significant reduction in allodynia (p<0.01). Overall, the results of this study indicate that AdV-ZFP-VEGF administration can be delivered in a clinically relevant time-window following SCI (24 hours) and provide significant molecular and functional benefits

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

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

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

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