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Effect and Reporting Bias of RhoA/ROCK-Blockade Intervention on Locomotor Recovery After Spinal Cord Injury:  A Systematic Review and Meta-analysis

Ralf Watzlawick1; Emily S. Sena, PhD2,3; Ulrich Dirnagl, MD1,4,5; Benedikt Brommer, MSc1; Marcel A. Kopp, MD1; Malcolm R. Macleod, PhD, FRCP2,6; David W. Howells, BSc, PhD3; Jan M. Schwab, MD, PhD1,7
[+] Author Affiliations
1Department of Neurology and Experimental Neurology, Charité Campus Mitte, Clinical and Experimental Spinal Cord Injury Research Laboratory (Neuroparaplegiology), Charité–Universitätsmedizin, Berlin, Germany
2Centre for Clinical Brain Sciences, University of Edinburgh, United Kingdom
3Florey Institute of Neuroscience and Mental Health, Heidelberg, Victoria, Australia
4Center for Stroke Research Berlin, Charité–Universitätsmedizin, Berlin, Germany
5German Center for Neurodegenerative Diseases (DZNE), Charité–Universitätsmedizin, Berlin, Germany
6Department of Neurology, National Health Service Forth Valley, Stirling, Scotland, United Kingdom
7Spinal Cord Injury Center, Trauma Hospital Berlin, Berlin, Germany
JAMA Neurol. 2014;71(1):91-99. doi:10.1001/jamaneurol.2013.4684.
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Importance  Blockade of small GTPase-RhoA signaling pathway is considered a candidate translational strategy to improve functional outcome after spinal cord injury (SCI) in humans. Pooling preclinical evidence by orthodox meta-analysis is confounded by missing data (publication bias).

Objective  To conduct a systematic review and meta-analysis of RhoA/Rho-associated coiled-coil containing protein kinase (ROCK) blocking approaches to (1) analyze the impact of bias that may lead to inflated effect sizes and (2) determine the normalized effect size of functional locomotor recovery after experimental thoracic SCI.

Evidence Review  We conducted a systematic search of PubMed, EMBASE, and Web of Science and hand searched related references. Studies were selected if they reported the effect of RhoA/ROCK inhibitors (C3-exoenzmye, fasudil, Y-27632, ibuprofen, siRhoA, and p21) in experimental spinal cord hemisection, contusion, or transection on locomotor recovery measured by the Basso, Beattie, and Bresnahan score or the Basso Mouse Scale for Locomotion. Two investigators independently assessed the identified studies. Details of individual study characteristics from each publication were extracted and effect sizes pooled using a random effects model. We assessed risk for bias using a 9-point-item quality checklist and calculated publication bias with Egger regression and the trim and fill method. A stratified meta-analysis was used to assess the impact of study characteristics on locomotor recovery.

Findings  Thirty studies (725 animals) were identified. RhoA/ROCK inhibition was found to improve locomotor outcome by 21% (95% CI, 16.0-26.6). Assessment of publication bias by the trim and fill method suggested that 30% of experiments remain unpublished. Inclusion of these theoretical missing studies suggested a 27% overestimation of efficacy, reducing the overall efficacy to a 15% improvement in locomotor recovery. Low study quality was associated with larger estimates of neurobehavioral outcome.

Conclusions and Relevance  Taking into account publication bias, RhoA/ROCK inhibition improves functional outcome in experimental SCI by 15%. This is a plausible strategy for the pharmacological augmentation of neurorehabilitation after human SCI. These findings support the necessity of a systematic analysis to identify preclinical bias before embarking on a clinical trial.

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Figure 1.
Multiple Effector Candidate Mechanisms of RhoA/Rho-Associated Coiled-Coil Containing Protein Kinase (ROCK) Blockade Contribute to Propagated Functional Neurological Regeneration/Recovery After Spinal Cord Injury (SCI)

The RhoA/ROCK pathway is activated after SCI by neurons (A) and glial cells (B). A, The RhoA/ROCK pathway is a converging cascade that mediates signaling from myelin and scar-derived growth inhibitory proteins. Activated RhoA–guanosine triphosphate (GTP) binds to Rho-binding domain of ROCK, thereby leading to its activation. ROCK activation induces neuronal growth cone collapse/neurite retraction bulbs by 2 different ways: one acting on microtubuli, the other affecting the actin cytoskeleton. Activation of collapsing response mediator protein 2 (CRMP2) propagates microtubuli destabilization. Microtubuli stabilization has been shown to prevent growth cone collapse and to enable axonal outgrowth in vivo.17,18 ROCK also activates myosin light chain (MLC) and LIM kinase (LIMK), which control actin-myosin interactions, leading to cell contraction, stress fiber formation, and ultimately also growth cone collapse. Finally, one of the recently identified targets of ROCK is phosphatase and tensin homologue (PTEN), being directly upregulated by ROCK.19 In central nervous system lesions, PTEN signaling abrogates axonal outgrowth, and blocking PTEN activity strongly promotes axon regeneration/plasticity, in part through mTOR-dependent upregulation.20 Together, it appears that the Rho/ROCK-pathway is a key target in the injured spinal cord to foster neurite outgrowth/plasticity. This neurite growth–promoting effect was validated in other central nervous system injury paradigms.21,22 Cross-paradigm verification is considered a prerequisite for a robust translational potential.23 B, Furthermore neuroprotective (eg, anti-exctitotoxic, improved perfusion, and reduced edema formation2428), immunomodulatory (reduced leukocyte infiltration2932), antineurodegenerative,3335 proneurorestaurative,3638 together with neuropathic pain–alleviating, properties39,40 are beneficial modular aspects likely to synchronize to attenuate tissue damage and foster repair after SCI. RhoA/ROCK inhibition can be achieved at different levels in several ways. First, through ADP ribosylation, the exoenzyme C3-ADP-ribosyltransferase (Cethrin/BA-210) prevents the conversion to active GTP-bound Rho, which stabilizes the inactive RhoA-guanine nucleotide dissociation inhibitor complex in the cytosol. Second, certain nonsteroidal anti-inflammatory drugs, including ibuprofen, reduce (but do not eliminate) RhoA signaling activation 55, leading to activation of the transcription factor peroxisome proliferator-activated receptor gamma (PPARγ)41 (reviewed in an article by Kopp et al42). Agents that bind and activate the transcription factor PPARγ have been reported to minimize or prevent deleterious cascades after SCI (reviewed in an article by McTigue43). Third, small interfering mRNA strategies directed at RhoA will block de novo synthesis of RhoA, reducing the amount available for activation. Finally, ROCK inhibition is established by 2 different small-molecule ROCK inhibitors (Y-27632 and fasudil [HA-1077, AT877]) and by the peptidic receptor ROCK inhibitor p21CIP1/WAF1.aROCK I/II activates PTEN in mammalian cells, which has to be investigated for neurons.19

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Figure 2.
Study Selection

Interventional preclinical trials applying Rho/Rho-associated coiled-coil containing protein kinase (ROCK) pathway inhibitory strategies to investigate the effect on neurological locomotor outcome.

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Figure 3.
Effect Sizes of Each Individual Study Ranked According to Efficacy

The blue (horizontal) bar represents the 95% CIs of the overall estimate of all eligible studies. The vertical error bars represent the 95% CIs for the individual studies. In contrast to approaches that aim for minimal variability to allow for a most correct reproduction (FORE SCI Initiatve), the presented CAMARADES (Collaborative Approach to Meta Analysis and Review of Animal Data from Experimental Studies) approach deliberates heterogeneity early on, including multiple spinal cord injury models, difference in anesthesia, sex, and application routes to inform robustness of the interventional approach. The aim is to provide a quantifiable overall effect and to identify the relevance of experimental modeling characteristics on locomotor outcome.

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Figure 4.
Effect of Study Modeling on Locomotor Outcome

A, Stratified meta-analysis for the specific drug used revealed the RhoA-inhibiting C3-ADP-ribosyltransferase being more effective than RhoA/Rho-associated coiled-coil containing protein kinase (ROCK) inhibitors acting downstream. B, Despite a possible variability in lesion extent or depth due to different techniques of injury induction, improvements in effect size were greatest in animals with hemisectioned spinal cords. C, The use of fentanyl/fluanisone in combination with diazepam was associated with higher estimates of effects compared with other reported anesthetics. D, The sex of the animal used was identified as another aspect of the study design characteristics that accounted for a significant proportion of between-study heterogeneity. E, Effect size correlates inversely with study quality (ie, studies with a better improvement in functional outcome are associated with low study quality). The shaded bars represent the 95% CI limits of the global estimate. The vertical error bars represent the 95% CIs for the individual estimates. The width of each bar reflects the log of the number of animals contributing to that comparison. Each stratification accounts for a significant proportion of between-study heterogeneity (P < .002).

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Figure 5.
Detection and Correction for Missing Data

A, Funnel plot shows the precision, which is 1 divided by the standard error of the mean, plotted against the standardized effect size. Each filled dot represents 1 of the 30 included experiments. In the absence of publication bias, the points should resemble an inverted funnel. The black line indicates the reported overall effect size of 21.3%. B, The Egger regression line does not intersect the origin, suggesting the presence of a significant publication bias. C, Trim and fill analysis identified 9 theoretical missing experiments (circles), adjusting the overall effect size to take these into account, lead to an absolute reduction in effect size of 6% and to a relative reduction of 27%.

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