Neuropathic pain after spinal cord injury and physical exercise in animal models: A systematic review and meta-analysis

Journal Pre-proof Neuropathic pain after spinal cord injury and physical exercise in animal models: a systematic review and meta-analysis Juliete Palandi, Franciane Bobinski, Gabriela Martins de Oliveira, Jocemar Ilha

PII:

S0149-7634(19)30320-3

DOI:

https://doi.org/10.1016/j.neubiorev.2019.12.016

Reference:

NBR 3629

To appear in:

Neuroscience and Biobehavioral Reviews

Received Date:

12 April 2019

Revised Date:

10 December 2019

Accepted Date:

10 December 2019

Please cite this article as: Palandi J, Bobinski F, de Oliveira GM, Ilha J, Neuropathic pain after spinal cord injury and physical exercise in animal models: a systematic review and meta-analysis, Neuroscience and Biobehavioral Reviews (2019), doi: https://doi.org/10.1016/j.neubiorev.2019.12.016

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Neuropathic pain after spinal cord injury and physical exercise in animal models: a systematic review and meta-analysis

Juliete Palandi

a,b*

, Franciane Bobinski

a,c *

, Gabriela Martins de Oliveira b,

Jocemar Ilha a,b

a

Physical Therapy Graduate Program, Department of Physical Therapy, College

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of Health and Sport Science, Santa Catarina State University (UDESC), Florianópolis, 88080-350, SC, Brazil b

Spinal Cord Injury Research Group, Neuromotor System Laboratory,

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Department of Physical Therapy, College of Health and Sport Science, Santa

c

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Catarina State University (UDESC), Florianópolis, 88080-350, SC, Brazil Experimental Neuroscience Laboratory, Graduate Program in Health Sciences,

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University of Southern of Santa Catarina (UNISUL), Palhoça, 88137-272, SC,

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Brazil

Number of figures: 7 Number of tables: 5

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*Authors had contributed equally to this work. Corresponding author: Center for Health and Sports Sciences (CEFID), 158 St. Pascoal Simone, Florianópolis SC 88080-350. Tel: +55 48 3664 8605; E-mail address: [email protected] (J. Ilha)

HIGHLIGHTS 1

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Mechanical and thermal nociception are the most frequently assessed NP components

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Physical exercise shown beneficial effect on mechanical, thermal and cold nociception, and spontaneous pain

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The benefits of physical exercise vary according to its starting and total duration

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There is a large heterogeneity about the type and intensity of exercise to

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alleviating NP after SCI

ABSTRACT

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The aim of this systematic review was to summarize the effects of physical

exercise on neuropathic pain (NP) in animal models of SCI. The search was

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conducted in Medline and Science Direct to identify experimental pre-clinical

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studies involving animal models of SCI, physical exercise as an intervention and the assessment of NP. Fifteen articles met the eligibility criteria. The review shows that in studies of NP involving animal models of SCI, rodents are the most

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common species. Thoracic contusion is the most common injury and mechanical and thermal nociception are the most frequently assessed NP components. The benefits of physical exercise vary according to its starting period and total

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duration. In addition, there is considerable heterogeneity regarding the type and intensity of exercise capable of alleviating NP after SCI. Furthermore, physical exercise has beneficial effects on mechanical, thermal and cold nociception, and spontaneous pain. These results are weakened by the paucity of studies involving these pain outcomes. The review protocol is published for free access on the SyRF platform (http://syrf.org.uk/protocols/).

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KEYWORDS: Neuropathic pain; Spinal cord injury; Physical exercise; Animal model; Preclinical study

Introduction Neuropathic pain (NP) is one of the most common clinical conditions after spinal cord injury (SCI). The presence of NP at or below the SCI level influences

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rehabilitation and quality of life in this population (Finnerup, 2013; Nees et al., 2017). Over 50% of patients experience debilitating NP associated with physical and emotional trauma (Burke et al., 2017; Colloca et al., 2017; Sawatzky et al.,

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2008). Injury to the somatosensory system can affect the transmission of sensory signals and cause NP symptoms (Colloca et al., 2017). Its biological mechanisms

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and treatment are different from those of other chronic pain conditions (Colloca

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et al., 2017). Management of the symptoms of NP, such as allodynia, hyperalgesia, and spontaneous pain, includes pharmacological interventions involving numerous adverse effects, increased visits to healthcare professionals

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and considerable clinical inconsistencies (Colloca et al., 2017; Kramer et al., 2017; Nees et al., 2017).

Treatment largely tends to focus on the symptoms. Physiotherapy for NP in SCI

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patients has been considered a promising alternative. Exercise is able to activate multiple beneficial mechanisms, enabling endogenous treatment (Cobianchi et al., 2017). Moreover, physical inactivity is a risk factor for non-communicable diseases (Hallal et al., 2012). A growing body of evidence shows there is a positive relationship between exercise and decreased pain (Law and Sluka, 2017). The complex nature of development and maintenance of NP after SCI, as

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well as the difficulty in carrying out clinical trials with a significant sample, makes scarce the evidence to show non-pharmacological treatments, such as physical exercise efficacy (Boldt et al., 2014). Previous non-systematic reviews have suggested that therapies involving sensorimotor activity, like physical exercise, may provide beneficial effects by modulating chronic pain states in humans and animals (Cooper et al., 2016; Nees et al., 2017). Animal studies are the first step towards elucidating the mechanisms involved in

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human conditions and developing appropriate interventions (Battistuzzo et al., 2012). Any animal injury model should be similar to those that occur in humans

in terms of cause and function and allow behavioral clinical observation (Sharif-

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Alhoseini et al., 2017). However, preclinical studies mimic reasonably well human

central nervous system injuries and pain evoked-responses but it is difficult to

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completely translate this expressed pain behavioral into the symptoms of the

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individuals in clinical practice since the pain also includes spontaneous and emotional components (Law and Sluka, 2017). Therefore, it is important to systematically collect evidence in preclinical studies to provide researchers with

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the opportunity to identify gaps for advancing knowledge, especially to choose an appropriate animal injury model, physical exercise treatment and behavioral assessment, precisely define the future study goals as well as apply the

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“replacement, reduction and refinement”- 3Rs- in animal research. Then, the aims of this review were to: (1) summarize the effects of physical exercise on NP in animal models of SCI; (2) identify the physical exercise parameters and (3) ascertain the measures used to assess NP. Thus, this review focuses on physical exercise as a therapeutic strategy for NP in animal models of SCI.

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Methods Literature Search This systematic review was reported following the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) (Moher et al., 2009). The review protocol was developed according to the guidelines of the SYstematic

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Review Center for Laboratory Animal Experimentation (SYRCLE) and published for free access in May 2017 on the platform Systematic Reviews Facility (SyRF)

(http://syrf.org.uk/protocols/), as suggested by Leenaars et al. (2012) and De

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Vries et al. (2015). The complete search strategy can be accessed in the supplemental content (Appendix A). The search strategy included: spinal cord

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injury, physical exercise, neuropathic pain, and animal model. The electronic search was performed in the Medline (PubMed) and Science Direct databases,

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without restrictions regarding the year or language of publication. The searches were carried out until May 2017 and an update was performed in October 2018.

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Two reviewers previously trained about the review protocol steps, independently screened the titles and abstracts to ensure the articles met the predefined eligibility criteria. Articles deemed to be unrelated to the research question and

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reviews were excluded at this phase. The remaining full articles were accessed and screened by two reviewers considering the eligibility criteria. Disagreement and discrepancies were resolved by consensus after discussion with a third reviewer. The eligibility criteria were elaborated according to the PICOs strategy for pre-clinical studies with an animal model of SCI, physical exercise as an intervention compared with sedentary or other interventions and any

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assessments of NP. The full articles that did not meet the eligibility criteria were added in the Table of Excluded Studies (Appendix B).

Data extraction and management The data extracted from the included studies comprised the name of first author and year of publication, animal strain and gender, animal age, model and level of SCI, characteristics of physical exercise (type, start period, duration, intensity

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expressed as the speed used and frequency), assessments of motor recovery (for steady injury) and NP. The main outcome measure extracted was the

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assessment of NP.

Risk of bias assessment

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Bias was assessed using SYRCLE’s risk of bias tool (Hooijmans et al., 2014).

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Two reviewers assessed the risk of bias and any disagreement or discrepancies were resolved by consensus with a third reviewer. The symbol “+” was used to indicate the criteria was declared and it implied a low risk of bias, “-” was used to

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indicate the criteria was undeclared and implied a high risk of bias and “?” was used to indicate unclear declaration and implied an unclear risk of bias. For the purposes of this review, we considered studies to be higher quality if they

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reported a low risk of bias in at least half of the rating criteria.

Reporting quality The “Animals in research: reporting in vivo experiments” (ARRIVE) guidelines were used to assess the reporting quality of the studies (Kilkenny et al., 2010). Two reviewers assessed the reporting quality and any disagreements or

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discrepancies were resolved by consensus with a third reviewer. Each ARRIVE item is graduated into three descriptive levels: complete, when all sub-items in the topic have been described; partial, when half or more of the sub-items have been described; and incomplete when less than half or none of the sub-items have been described. A summary was made of the ARRIVE items fully described in the studies and the results are expressed as a relative frequency.

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Meta-analysis The meta-analyses were performed for the different components of NP (mechanical, thermal, cold and spontaneous pain). We considered spontaneous

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pain related to any behavioral test that involved pain processing in supraspinal structures and that there was no previous sensory stimulus, such as freezing,

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anxiety and fear behaviors. Sub-group analysis was performed for the physical

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exercise start period (before or after post-injury day 7). Either random-effect or fixed-effect modeling was used according to the heterogeneity determined by I2. The I2 values were reported in forest plots of each variable. And, I2 values of up

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to 30% were considered low heterogeneity, while values greater than 50% high heterogeneity (Higgins and Thompson, 2002). Only the last assessment after the physical exercise intervention was used. Studies that had more than one physical

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exercise group were included in different comparisons and identified in the forest plot legend. The mean and standard deviation values of the groups of interest were extracted, and in cases where those values were not present in the original article, the corresponding author was contacted. Group values were adjusted for standard mean difference according to the outcome measures presented in the original articles. The results and forest-plots were generated using Review

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Manager (RevMan) Version 5.3 Computer Program (The Nordic Cochrane Centre, The Cochrane Collaboration, Copenhagen, DK). For overall effect p ≤ 0.05 was considered statistically significant.

Results Selection of studies

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The search strategy is shown in Figure 1. A total of 128 hits were identified in the database search. After screening the titles and abstracts, 23 studies were assessed for eligibility, and 8 were excluded because they did not meet our

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Characteristics of the studies

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eligibility criteria, resulting in the inclusion of 15 full-text articles.

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Among the 15 included articles, rats were the most common species (80%) followed by mice (20%), and adult females predominated (73.4%). The most common injury was contusion (80%) at the thoracic spinal cord level (80%). The

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physical exercise interventions involved the use of either treadmill or wheel running. In studies with rats, the most common exercise intervention involved wheel locomotion (33.3%) followed by quadrupedal and bipedal locomotion

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(25%), and quadrupedal or bipedal alone (16.7%). The intervention initiation window varied between 1 and 28 days post-SCI. A session duration of 20 minutes was the most common, with a frequency of 5 times per week (75%). The total extension of the intervention period varied from 1.2 to 12 weeks. Treadmill velocity ranged from 2.5 cm/s to 25 cm/s.

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In the three studies with mice, quadrupedal treadmill training was the most used (66.7%). The intervention initiation window varied between 7 to 52 days post-SCI. Among the 15 studies included in this review, 7 studies started physical exercise before post-injury day 7 (46.7%), 6 studies started physical exercise after postinjury day 7 (40%), and 2 studies started physical exercise before and after postinjury day 7 (13.3%). Two studies were applied with 2 sessions of 15 minutes per day (66.7%), and all studies had a frequency of 5 times per week (100%). The

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total extension of the intervention period varied from 4.5 to 8 weeks. The intensity ranged from 0.5 cm/s to 25 cm/s. The characteristics of the animals, injury-types and physical exercise protocols are shown in Table 1.

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The outcome assessments were classified into the motor function for steady SCI and NP components (see Table 2). Eleven studies (73.3%) had motor recovery

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assessments, studies with rats used the Basso, Beattie and Bresnahan Scale

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(BBB) (66.7%) and studies with mice used the Basso Mouse Scale (BMS) (100%). Fourteen of the fifteen studies assessed mechanical nociception (93.3%). Other assessments were used for thermal nociception, cold nociception,

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and spontaneous pain. The most common test for mechanical nociception was von Frey filaments (85.7%) while for thermal nociception it was the Hargreaves method (70%). In addition to the behavioral tests, fourteen studies performed

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other complementary analyses to verify possible mechanisms associated with NP. The analysis of white and gray matter spared was the most performed (42.8%) followed by neurotrophic factors (35.7%) (a summary of neurobiological assessments was made in Table 3).

Risk of bias

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The quality assessment for all the studies and the individualized scoring of each study can be seen in Figures 2 and 3. Eight studies had a low risk of bias in 50% or more of the rating criteria and were thus considered high-quality studies. Only one study (Ward et al., 2016) presented a low risk of bias in 90% of the rating criteria. Random housing and random outcome assessments presented in only 26.7%. Blinding of outcome assessment and baseline characteristics are the lowest risk criteria declared in the studies (73.3%). Incomplete outcome data and

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selective reporting were found in 53.3% of the studies.

Reporting quality

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The assessment of reporting quality is divided into before and after 2010, the year

of publication of the ARRIVE guidelines (Kilkenny et al., 2010). Reporting quality

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for ARRIVE items and the score for each study can be seen in Tables 4 and 5,

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respectively. The studies published prior to 2010 present some items completely. The title, numbers of animals in the experimental and control groups, procedure details, replication of the experiment, statistical methods, results interpretation

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and funding sources are the most completely reported aspects (100%). However, some key items that would increase reporting and study quality were not cited. Among those unmentioned aspects are objectives, experimental unit, when and

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why the experiment was carried out, experimental animals, housing conditions, sample size calculation, order to treat and assessment, unit of results analysis, baseline data, adverse events, study limitations, and the 3Rs’ implications.

On the other hand, with the publication of the ARRIVE guidelines (2010), 14 items showed an increase in frequency as “complete” in studies. The most significant increase involved details of animals used in experimental procedures (0 to

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83.3%). However, of 17 items not mentioned prior to the publication of the ARRIVE guidelines, 7 remained poorly described. They included, in all articles, relevant information about experimental animals, housing conditions, sample size calculation, adverse effects, study limitations, and the 3Rs’ implications. Only the items of experimental and control groups of the methods were reported in the studies.

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Meta-analysis Seven studies presented the necessary data and were included in the analyzes

(Brown et al., 2011; Detloff et al., 2014; Detloff et al., 2016; Dugan and Sagen,

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2015; Nees et al., 2016; Sliwinski et al., 2018; Tashiro et al., 2015). Six studies

did not provide sufficient data for analysis (Chen et al., 2017; Chhaya et al., 2019;

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Eisen et al., 2014; Endo et al., 2009; Hutchinson et al., 2004; Tashiro et al., 2018).

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In two studies, there was no indication of NP and they were not included in the analysis (Ward et al., 2014; Ward et al., 2016). General analyzes, and subanalyzes (Fig. 4, 5, 6 and 7) considering the time after injury in which the physical

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exercise was started (previous or after post-injury day 7), were performed with the 7 included studies.

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Effect on mechanical nociception of physical exercise intervention vs. sedentary Mechanical nociception was the most commonly assessed outcome for NP. Seven studies were included in this forest plot (Brown et al., 2011; Detloff et al., 2014; Detloff et al., 2016; Dugan and Sagen, 2015; Nees et al., 2016; Sliwinski et al., 2018; Tashiro et al., 2015). Physical exercise presented statistical difference when compared with sedentary (Fig. 4A, n = 411, p = 0.04). When

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those studies that started the physical exercise protocol before post-injury day 7 were separately analyzed, the results were inconclusive (Fig. 4B, n = 186, p = 0.06). Sub-analysis of those studies that started the physical exercise protocol after post-injury day 7 also presented results in favor of physical exercise (Fig. 4C, n = 228, p = 0.01). The analyses presented high heterogeneity.

Effect on thermal nociception of physical exercise intervention vs sedentary

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Thermal nociception was analyzed in six studies (Brown et al., 2011; Detloff et al., 2016; Dugan and Sagen, 2015; Endo et al., 2009; Sliwinski et al., 2018; Tashiro et al., 2015). There was a statistical difference in favor of physical

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exercise compared to sedentary (Fig. 5A, n = 179, p = 0.0009), which suggests

physical exercise is effective regarding thermal nociception. Moreover, sub-

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analysis of those studies that started physical exercise before or after post-injury

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day 7 also showed positive results in favor of physical exercise for thermal nociception (Fig. 5B, n = 113, p = 0.04; and, Fig. 5C, n = 66, p < 0.00001;

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respectively). Heterogeneity of these studies ranged from low to high.

Effect on cold nociception of physical exercise intervention vs sedentary Two studies assessed the effects of physical exercise on cold nociception and

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the meta-analysis found beneficial effects in favor of exercise (Fig. 6A, n = 79, p = 0.04) (Dugan and Sagen, 2015; Nees et al., 2016). When comparing physical exercise initiation before post-injury day 7 the results were inconclusive (Fig. 6B, n = 49, p = 0.25). The positive results are also seen when comparing physical exercise initiation after post-injury day 7 (Fig. 6C, n = 30, p = < 0.00001). Although

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only two studies were included in this analysis, the heterogeneity of those studies was high.

Effect on spontaneous pain of physical exercise intervention vs sedentary Only one study analyzed the effect of physical exercise on spontaneous pain behavior (Nees et al., 2016). The results favored the practice of physical exercise

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(Fig. 7, n = 30, p = 0.05).

Discussion

Systematic reviews of animal studies represent a substantial step towards

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translating the findings from animal studies to human trials (Hooijmans and

Ritskes-Hoitinga, 2013; Ritskes-Hoitinga et al., 2014). To the best of our

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knowledge, this is the first systematic review and meta-analysis to evaluate the

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effects of physical exercise on NP after SCI in animal models as well as to identify the parameters of the physical exercise and measures to evaluate NP. Rodents and thoracic contusion are the most common species and pattern

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chosen for animal models of NP after SCI

Animal models of SCI provide opportunities to elucidate neurobiological mechanisms of the NP and the relationship between painful behavioral and

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possible interventions. These studies also support the use of physical exercise as a treatment and prevention strategy in clinical practice.

Our data search showed that only rodents are employed to study NP after SCI in animal models. Similarly, while several animal species have been reported by SCI studies, rodents are the most widely used model (Sharif-Alhoseini et al., 2017). Different genotypes can mediate nociceptive sensitivity, predispose to NP

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following neural injury and endogenous antinociception (Mogil et al., 1999). Rats were found to be the animals most frequently used to study NP in SCI models. Rat care is low cost and technically easy, they rarely present infectious conditions, and many tissue and behavioral analyses are well established (SharifAlhoseini et al., 2017). In our review, only three studies were found to use mice (Nees et al., 2016; Sliwinski et al., 2018; Tashiro et al., 2018). Models using mice are becoming more widespread, mainly due to their genomic similarity with

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humans, the possibility of knockout models, and like rats, their easy handling (Carpenter et al., 2015). However, different mouse genotypes respond differently

to nociceptive measures (Mogil et al., 1999). Moreover, another difficulty in using

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mice models probably is the small size for the surgical site.

The most common cause of SCI in humans is trauma, when the spinal cord is

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impacted by displaced bone, other tissues or objects after traffic accidents, falls,

compression

SCI

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violence, or sports injury (Jazayeri et al., 2015). Therefore, surgical contusion or models

were

the

best

choices

to

simulate

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pathophysiological mechanism aspects of the human SCI(Sharif-Alhoseini et al.,

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2017). Painful evoked-responses have also been related in contusion or compression models of incomplete SCI (Kilkenny et al., 2010). Otherwise, the main advantage of these models is to allow the selective severity graduation of

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the induced injury and symptoms (Hassannejad et al., 2016). These incomplete SCI models are mainly used to reproduce and treat pain-related behavior that is not present in studies with complete injury models (Nakae et al., 2011; SharifAlhoseini et al., 2017). Tetraplegic, older and chronic injury are the most common conditions associated with NP symptoms in people with SCI (Burke et al., 2017). However, the studies

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included in this review predominantly involve female animals with injury at the thoracic level. That level seems to be chosen because is more difficult to train SCI animals with more rostral and more severe injuries. Furthermore, the thoracic injury models have strength reproducibility and reliability (Sharif-Alhoseini et al., 2017). Furthermore, thoracic models allow for better hind limb evaluation, which is most frequently related to nociception outcomes (Sharif-Alhoseini et al., 2017). Although the traumatic SCI predominantly occurs in men, the choice of females

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may be explained by the fact women are more susceptible to present NP conditions after SCI (Bouhassira et al., 2008; Rosen et al., 2017). In addition,

women and female animals have shorter urethra compared to men and male

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animals, this anatomical sex difference makes easier to empty the urinary bladder

Sharif-Alhoseini et al., 2017).

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in female and reduces the risk of urinary tract infection after SCI (Gao et al., 2017;

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Mechanical and thermal nociception were the most frequently assessed NP components in animal models of NP after SCI Physical exercise was reported to have effects in different components and

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characteristics of NP. Behavioral measures are important in evaluating the effectiveness of the model in mimicking the clinical signs. Approximately half the studies (58.3%) used more than one evaluation to check for signs of NP. In

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humans, NP signs predominantly occur below the injury level (Burke et al., 2017). Of the articles included in this review, just two studies assessed at-level nociception (Ward et al., 2014; Ward et al., 2016). There were considerable differences in the assessment methods for mechanical and thermal nociception between studies. For example, the von Frey method for mechanical nociception was performed as the percentage of response, up-and-down or response

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threshold. For thermal nociception, the Hargreaves method was used where the stimulus is applied to the paw while in the tail flick method, the stimulus is applied to the tail. Different types of assessment also allow different types of outcome measures, where, for example, a higher threshold indicates a decreased nociceptive response, whereas a higher response percentage indicates an increased nociceptive response. Another factor that may influence the results is the different strains, which differ

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significantly in several nociceptive behaviors (Kerr and David, 2007). The great variability of human response to pain is also related to genetic factors. This is

because genetic factors may be related to basal sensitivity to pain, predisposition

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to the development of pain after trauma, sensitivity to pharmacological agents,

among others. These factors also apply to different rodent animal genotypes,

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especially mice strains (Mogil et al., 1999).

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Behavioral assessments for standardizing the severity of the injury and recovery, like BBB for rats or BMS for mice, are widely used to test replicability of the SCI models in scientific research (Basso et al., 2006). In general, the pain behavior

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assessment in experimental models depends on the animal motor capacity to respond to a nociceptive stimulus. This reinforces the difficulty of pain assessment in SCI models, especially because this kind of injury promotes a

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drastic muscle paresis (Nakae et al., 2011). Furthermore, most studies don’t encompass all the NP components observed in patients with SCI as sensory alterations like allodynia or hyperalgesia after a stimulus or spontaneous pain at the level of injury or above (Nees et al., 2017). The affective and emotional components involved with NP were not considered in any of the studies included

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in this systematic review. Therefore, in future studies, it is important to consider the inclusion of its methods, such as the Grimace Scale (Langford et al., 2010).

Methodological and reporting quality can enhance preclinical studies of NP after SCI Methodological quality was considered high in half the studies. Many studies did not report performance or detection bias criteria, such as random housing or

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random outcome assessment. Failure to report methods and results has scientific, ethical and economic implications for preclinical research (Kilkenny et

al., 2010). These failings lead to poor reproducibility and translation of preclinical

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research (Mogil, 2017). Systematic errors can directly influence the magnitude and the direction of the results, and further exacerbate the results of preclinical

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translation studies for clinical practice. In this way, improving and identifying

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different bias risks is one of the main ways to reach high methodological quality in animal studies. Additionally, the ARRIVE guidelines have been developed to promote high-quality, comprehensive and accurate critical review of animal

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research reports. It provides a checklist that can be used to guide authors preparing manuscripts for publication to ensure completeness and transparency of the reported data enhancing the reporting quality (Kilkenny et al., 2010).

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However, due to their relatively recent introduction, they are still underused. Maybe, the recommendation of their use by journals and reviewers might lead to their more widespread application. Higher study quality and more appropriate reporting would strengthen preclinical evidence, which is an urgent requirement in pain models in SCI. Physical exercise had beneficial effects on mechanical nociception

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The meta-analysis results showed beneficial effects of physical exercise on mechanical nociception at the general analysis and when physical exercise program was started after post-injury day 7. On the other hand, the analysis showed no statistical significance in favor of the physical exercise when the training was started before post-injury day 7. The mechanisms by which physical exercise is capable of altering mechanical nociception have been extensively explored; and, the strategy for the prevention

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or treatment of the NP after SCI would combine exercise programs to promote neural plastic changes for neuroprotection or neuroregeneration through multiple mechanisms (Cobianchi et al., 2017; Sandrow-Feinberg and Houlé, 2015). For

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mechanical nociception, positive findings were related to the sensorimotor stimuli applied. This is based on the principle that sensorimotor activity promotes

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repeated and rhythmic neuronal activity that can reinforce the integrity of the

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neuronal circuitry (Cobianchi et al., 2017; Cooper et al., 2016) modulating the maladaptive neuroplasticity and associated peripheral and central sensitization (Nakae et al., 2011).

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Exercise modalities and intensities specific to the pattern of injury can offer greater neuroprotection. This potent protective effect is related to the several processes secondary to SCI that are linked to the development of NP (Liu et al.,

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2012). Physical exercise protects not only the spinal segment where nerve roots corresponding to the active musculature, but also other spinal segments (Côté et al., 2011). This occurs through the release of neurotrophic factors, modulation of afferences or inflammatory mediators, improving neuronal plasticity (Brown et al., 2004; Cooper et al., 2016; Côté et al., 2011). After SCI, low to moderate intensity training may stimulate the sprouting of nociceptive neurons by increasing

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neurotrophins production in CNS while high-intensity training reduces it (Cobianchi et al., 2017; Sawatzky et al., 2008). This information allows us to hypothesize that potential exercise-dependent effects could be related to the dose-response and the time-dependent of sensorimotor stimuli applied. Physical exercise had beneficial effects on thermal and cold nociception The fact the contrasting results were found for the different components of NP may be related to the distinct impacts physical exercise has on the spinal tracts

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(Gruener et al., 2016). The meta-analysis showed that physical exercise has beneficial results on thermal nociception and cold nociception. The finding that physical exercise was effective in thermal heat and cold nociception is in

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accordance with the scientific literature.

Among the common changes characteristic of neuropathic pain, heat and cold

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hyperalgesia seem to be directly influenced by the sensorimotor information

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promoted by physical exercise. Although circuitry for light touch, painful or thermal stimuli is different, the literature suggests, in both human and animal studies, that physical exercise can reverse structural maladaptation and thus

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prevent neuropathic pain (Nees et al., 2017). However, the mechanism by which sensory-motor stimuli in physical exercise affect thermal nociception remains poorly known and not discussed by the studies.

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The exercise-induced analgesia in response to thermal nociception in preclinical studies has important implications for clinical translation and may represent a barrier to exercise-based interventions for the thermal component of NP after SCI in human populations. Post-SCI chronic pain drastically impairs to quality of life and despite a large of therapeutic strategies (Nakae et al., 2011). Thus, an

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analgesic effect of exercise on heat or cold hyperalgesia would positively impact these individuals.

Physical exercise suggests beneficial effects on spontaneous pain Only one study assessed the spontaneous component of NP, in the 5th-week post-SCI (Nees et al., 2016). Long training periods are associated with a better stimulation of neurotrophic factors (Ritskes-Hoitinga et al., 2014). If long-term

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exercise provides better neurotrophic supply to SCI, excessive neuromuscular stimulation can provide increasing axonal regeneration and suitable supraspinal responses (Sandrow-Feinberg and Houlé, 2015). However, the most adequate

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duration of physical exercise cannot yet be determined (Cobianchi et al., 2017).

Sensory pathway remodeling and Inflammation modulation appear to be the

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underlying mechanisms by which physical exercise ameliorates NP after SCI

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Physical exercise attenuates the various NP symptoms (Cooper et al., 2016). This may be related to being a non-specific therapeutic proposal and act in different body systems with several molecular mechanisms associated with pain.

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Maladaptive remodeling of sensory tracts and NP signals were induced by sensory deprivation (Nakae et al., 2011). Structures at the molecular and anatomical levels of the sensory pathways are affected by physical exercise.

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Furthermore, reinforcing existing sensorimotor pathways and promoting neural plasticity may play a role in attenuating pain symptoms and could reverse maladaptive structural changes after physical exercise intervention (Cooper et al., 2016; Nees et al., 2017). Another mechanism explored by the studies included in this review is the sprouting of peptidergic or non-peptidergic fibers after SCI. While peptidergic

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fibers play an important role for thermal nociception, the non-peptidergic fibers correspond to mechanical nociception (Cavanaugh et al., 2009). The aberrant sprouting of peptidergic fibers in regions that would normally process non-nocive mechanical stimuli, as well as increased activity of these fibers may characterize the process of central sensitization and the development of NP (Basbaum et al., 2009). The process by which exercise promotes the amelioration of sprouting still poorly explored. However, it’s suggested to link it to the presence of neurotrophic

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factors, as BDNF and GDNF, in neurons or glial cells (Brown et al., 2004). Neurotrophins, such as BDNF, NGF, and NT-3 analyzed in some of the studies in this review, can modulate NP either directly or indirectly, promoting analgesia

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or hyperalgesia, by regulating neuronal survival, growth, and death (Khan and Smith, 2015). Additional, is suggested a possible relationship with low levels of

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neurotrophins, either in the CNS or PNS, with the occurrence of allodynia due to

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increased receptors expression, increased responsiveness or influence on glial cells activity (Watanabe et al., 2000). Physical exercise is able to increase BDNF and NT-3 levels in the spinal cord, which post-SCI are decreased (Gomez-Pinilla

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et al., 2012; Gómez-Pinilla et al., 2002).

Changes in allodynia and hyperalgesia symptoms are strongly correlated with anti- or pro-inflammatory states. Neuroinflammation promotes hyperactivity in

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neurons and central sensitization, characteristics of the presence of NP (Kramer et al., 2017). The central sensitization after SCI is related to the inflammatory process and the early immune response (Watkins et al., 2001). Exercise can activate anti-inflammatory mechanisms and suppress inflammatory mediators and neurotransmitters in the pain pathway (Chen et al., 2017). However, the full

21

mechanisms by which exercise alleviates NP remain poorly understood and highly complex (Cooper et al., 2016). While a physical exercise intervention following injury can show some beneficial effects, the idea of a preventive intervention has been poorly explored. No study has attempted to assess the effects on the NP components of physical exercise performed prior to SCI. In this way, physical exercise can be a protective strategy for inducing a lesion-like response and then preparing for a similar or worse

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stimulus that will follow (Chang et al., 2014). Only the Gomez-Pinilla; Ying and Zhuang (2012) study evaluated the effects of physical exercise prior to SCI. The

data indicate a large decrease in BDNF in the hippocampus and in the spinal

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cord after SCI, reversed in the animals that performed prior physical exercise. This suggests a strong relationship between previous physical exercise and the

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modulation of physiological CNS responses, especially in molecular adaptations

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that directly interfere in neuronal plasticity. This relationship with pain development has not been explored so far. Limitations

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Our search was conducted in only two databases, thus it may not have detected all the published studies relating to each of the terms (physical exercise, NP, animal model of SCI). A result limitation is that most of the experimental studies

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did not evaluate the entire components of NP and did not report mean and standard deviation values, which meant that not all the studies could be included in the meta-analysis.

Another important limitation point in the data interpretation is the unclear risk of bias, presented by several of the included studies of this review. Moreover, the results of this study can not be directly translated to clinical use in humans,

22

because our results are from preclinical studies, and can only provide subsidies for future clinical trials.

Conclusions Our results suggest that physical exercise, especially when started after the postinjury day 7, can promote an amelioration of mechanical nociception.

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Furthermore, physical exercise seems to offers beneficial effects for thermal, cold nociception and spontaneous pain, suggesting that it can be an effective

therapeutic strategy for some NP symptoms after SCI, especially in contusion

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models. However, these results are weakened by the paucity of studies involving these pain outcomes. More studies are needed in order to establish the real

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effectiveness of the physical exercise. Therefore, researchers must continue to

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investigate how physical exercise training programs can affect different components of NP post-SCI, especially to determine which modality, intensity, frequency and time to start the training after an experimental SCI. In this context,

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future studies will benefit from the data highlighted in this review when attempting to determine goals, formulate research questions and define the methodology. Controlling the risks of bias as well as optimizing reporting in animal studies is

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fundamental for clinical translation. Animal models can be used to explore the molecular mechanisms involved in NP after SCI and shed light on the mechanisms involving physical exercise. While several physical exercise parameters can be adopted, they must be appropriate for the study goals. The understanding of the previous experiences that may reduce the incidence of NP post-SCI is an important point to formulate strategies to prevent this relevant

23

clinical problem. In this context, future studies could explore the use of prior physical exercise as a neuroprotection strategy for NP after SCI.

Acknowledgements This study was a part of the first author’s master degree dissertation and was supported by the Programa de Bolsas de Monitoria de Pós-Graduação

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(PROMOP) of Santa Catarina State University (UDESC), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES; Finance Code 001) and

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (Edital

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Conflict of interest statement

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MCTI/CNPq/Universal 2016 - Proc. 425471/2016-0), Brazil.

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The authors have no conflicts of interest to declare. Authors J. P. and F. B. contributed equally to this study.

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SEARCH STRATEGY

“Neuropathic pain after spinal cord injury and physical exercise in animal models: a systematic review”.

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SEARCH COMPONENT 1 (SC1) Physical Exercise 1.

Exercise

2.

Training

3.

Physical exercise

4.

Treadmill training

5.

Running wheel

6.

Task specific training

7.

Locomotor training 24

SEARCH COMPONENT 2 (SC2) Neuropathic Pain 8.

Neuropathic pain

9.

Nociceptive

10.

Hyperalgesia

11.

Allodynia

12.

Sensory recovery

13.

Spinal Cord Injur*

14.

Spinal Cord Contusion

15.

Spinal Cord Compression

16.

Spinal Cord Hemisection

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SEARCH COMPONENT 3 (SC3) Spinal Cord Injury

SC1 AND SC2 AND SC3

((“Exercise” OR “training” OR “Physical exercise” OR “Treadmill training” OR

-p

“Running wheel” OR “Task specific training”OR “Locomotor training”) AND

(“Neuropathic pain” OR “nociceptive” OR “Hyperalgesia” OR “allodynia” OR

re

“Sensory recovery”) AND (“spinal cord injur*”OR “Spinal Cord contusion” OR

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“Spinal Cord Compression” OR “Spinal Cord Hemisection”))

Excluded Studies

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Identification Code #1

Elegibility criterias

Title

SCI animal

Physical exercise

Neuropa

model

intervention

Yes

Yes

No

Yes

Yes

Ye

asses

Exercise and Peripheral Nerve Grafts as a Strategy To Promote Regeneration

after

Acute

or

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Chronic Spinal Cord Injury

#3

Sequential therapy of anti-Nogo-A antibody treatment and treadmill training

leads

to

cumulative

improvements after spinal cord injury in rats

25

#9

Training-Induced Functional Gains following SCI

#11

Yes

Yes

Y

Yes

Not

N

Yes

Yes

Y

Yes

Yes

Y

Yes

Yes

Y

Yes

Yes

Y

Yes

Yes

Y

BDNF Overexpression Exhibited Bilateral Effect on Neural Behavior in SCT Mice Associated with AKT Signal Pathway

Delayed Exercise Is Ineffective at Reversing

Aberrant

Nociceptive

Afferent Plasticity or Neuropathic

ro of

#17

Pain After Spinal Cord Injury in Rats #30

An Intensive Locomotor Training Paradigm Improves Neuropathic following

Spinal

Cord

Compression Injury in Rats

BDNF Induced by Treadmill

re

#31

-p

Pain

Training Contributes to the Spasticity

lP

Suppression of

and Allodynia After Spinal Cord Injury via Upregulation

#39

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of KCC2

Acute exercise prevents the development of neuropathic pain and the sprouting of

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non-peptidergic (GDNF- and

#41

artemin- responsive) c-fibers after spinal cord injury

Novel

Multi-System

Functional Gains via Task Specific Training in Spinal Cord Injured Male Rats

26

#59

Exercise

therapy

and

recovery after SCI: evidence that shows early intervention improves

recovery

Yes

Yes

Y

Yes

Yes

N

of

function #61

Nogo-A

Antibodies

Training

Reduce

Spasms

in Spinal

and

Muscle Cord-

Injured Rats Differential effects of anti-

ro of

#66

Yes

Yes

Y

-p

Nogo-A antibody treatment and treadmill training in rats

Yes

Y

Not

Not

N

Yes

Yes

Y

No

Yes

Y

with incomplete spinal cord injury Early exercise in spinal cord injured

rats

allodynia

through

signaling

TrkB

Yes

Instrumental Learning Within

lP

#72

induces

re

#67

the Spinal Cord: Underlying Mechanisms

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Implications for

and

Recovery

After Injury

#76

Three exercise paradigms differentially

improve

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sensory recovery after spinal

#50

cord contusion in rats

Altered Gene Expression of RNF34 and PACAP Possibly Involved in Mechanism of Exercise-Induced Analgesia for Neuropathic Pain in Rats

27

#90

Exercise-Induced Changes to

the

Macrophage

Response in the Dorsal Root Ganglia

Prevent

Neuropathic

Pain

Yes

Yes

Y

Yes

Yes

Y

after

Spinal Cord Injury #110

The Amelioration of PainRelated Behavior in Mice

Injury Treated with Neural Stem/Progenitor

ro of

with Chronic Spinal Cord

Cell

Transplantation

Combined

with Treadmill Training #130

Sensorimotor

Activity

-p

Partially Ameliorates Pain

and Reduces Nociceptive Density

in

the

Yes

Yes

Y

No

No

Y

Yes

NO

Y

re

Fiber

Cord #140

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Chronically Injured Spinal

Metaplasticity

within

the

spinal cord: Evidence brain-

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derived neurotrophic factor (BDNF),

tumor

necrosis

factor (TNF), and alterations in

GABA

function

(ionic

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plasticity) modulate pain and

#190

the capacity to learn

Unique Sensory and Motor Behavior

in

Thy1-GFP-M

Mice before and after Spinal Cord Injury

28

#200

Altered

transcription

glutamatergic glycinergic

of and

receptors

spinal

in cord

dorsal horn following spinal

Yes

Yes

N

cord transection is minimally affected by passive exercise

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ur na

lP

re

-p

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of the hindlimbs.

29

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Figure legends

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Fig. 1. Flow diagram displaying the search procedure.

Fig. 2. Risk of bias graph. Review authors’ judgments about each risk of bias item (selection, performance, detection, attrition, reporting or other bias) presented as percentages across all included studies.

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Fig. 3. Risk of bias summary. Review authors’ judgments about each risk of bias item for each included study. (-) High risk of bias, eminent risk of bias for this

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(+) Low risk of bias, free of risk of bias in this item.

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item; (?) Unclear risk of bias, carefully check the article for this item interpretation;

Fig. 4. Forest plot of physical exercise intervention vs. sedentary for mechanical

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nociception. (A) General studies analyses, (B) Studies started physical exercise before post-injury day 7, (C) Studies started physical exercise after post-injury day 7. Standardized (Std.) mean differences (SMD) are demonstrated on the x

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axis. A negative SMD indicates the treatment effect favors physical exercise, while a positive SMD indicates the treatment effect favors sedentary. In the summarized analyses from panels A and B, we use a random effects model, whereas for panel C we use a fixed effects model. Brown et al., 2011 (1) physical exercise intervention started on post-injury day 1; Brown et al., 2011 (2) physical exercise intervention started on post-injury day 8; Detloff et al. 2016 (1) physical

40

exercise intervention started on post-injury day 14, assessment of contralateral forepaw; Detloff et al. 2016 (2) physical exercise intervention started on postinjury day 14, assessment of contralateral hindpaw; Detloff et al. 2016 (3) physical exercise intervention started on post-injury day 28, assessment of contralateral forepaw; Detloff et al. 2016 (4) physical exercise intervention started on postinjury day 28, assessment of contralateral hind paw; Dugan; Sagen, 2015 (1) physical exercise intervention started on post-injury day 5; Dugan; Sagen, 2015

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(2) physical exercise intervention started on post-injury day 21; Nees et al., 2016 (1)* assessment with a 0.16 g von Frey filament; Nees et al., 2016 (2)* assessment with a 0.4 g von Frey filament; Nees et al., 2016 (3)* assessment

-p

with a 0.6 g von Frey filament; Nees et al., 2016 (4)* assessment with a 1.4 g von Frey filament; Sliwinski et al., 2018 (1) assessment with place

re

escape/avoidance paradigm; Sliwinski et al., 2018 (2)* assessment with a 0.16 g

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von Frey filament; Sliwinski et al., 2018 (3)* assessment with a 0.4 g von Frey filament; Sliwinski et al., 2018 (4)* assessment with a 0.6 g von Frey filament; Sliwinski et al., 2018 (5)* assessment with a 1.4 g von Frey filament. SD, standard

ur na

deviation; CI, confidence interval. *, For the meta-analysis, inverted results were used between Physical exercise and Sedentary groups to maintain the greatness

Jo

of the results found in favor of the exercise in the forest plot.

41

ro of -p

re

Fig. 5. Forest plot of physical exercise intervention vs. sedentary for thermal

lP

nociception. (A) General studies analysis, (B) Studies started physical exercise before post-injury day 7, (C) Studies started physical exercise after post-injury

ur na

day 7. Standardized (Std.) mean differences (SMD) are demonstrated on the x axis. A negative SMD indicates the treatment effect favors physical exercise, while a positive SMD indicates the treatment effect favors sedentary. In the summarized analyses from panels A, B and C we use a random effects model.

Jo

Brown et al., 2011 (1) physical exercise intervention started on post-injury day 1; Brown et al., 2011 (2) physical exercise intervention started on post-injury day 8; Dugan; Sagen, 2015 (1) physical exercise intervention started on post-injury day 5; Dugan; Sagen, 2015 (2) physical exercise intervention started on post-injury day 21; SD, standard deviation; CI, confidence interval.

42

ro of -p

re

Fig. 6. Forest plot of physical exercise intervention vs. sedentary for cold

lP

nociception. (A) General studies analysis, (B) Studies started physical exercise before post-injury day 7, (C) Studies started physical exercise after post-injury

ur na

day 7. Standardized (Std.) mean differences (SMD) are demonstrated on the x axis. A negative SMD indicates the treatment effect favors physical exercise, while a positive SMD indicates the treatment effects favor sedentary. For summarized analyses in the panels A and B we use a random effects model,

Jo

whereas for panel C we use a fixed effects model. Dugan; Sagen, 2015 (1) physical exercise intervention started on post-injury day 5; Dugan; Sagen, 2015 (2) physical exercise intervention started on post-injury day 21; SD, standard deviation; CI, confidence interval.

43

ro of -p

Fig. 7. Forest plot of physical exercise intervention vs. sedentary for spontaneous

re

nociception. Standardized (Std.) mean difference (SMD) is demonstrated on the x axis. A negative SMD indicates the treatment effect favors physical exercise,

lP

while a positive SMD indicates the treatment effect favors sedentary. For summarized analyses, we use a fixed effects model. SD, standard deviation; CI,

Jo

ur na

confidence interval.

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45

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Table 1. Summary characteristics of the selected studies.

Reference

Animals

SCI model

Brown et al., 2011

Sprague-

Contusion, T12- SCI non-trained, n=6

Dawley

Experimental groups*

rats, T13

male, adults

E

T

SCI trained 1st day, n=6

S

SCI trained 8th day, n=6

D

I Chhaya et al., 2018

SpragueDawley

Unilateral rats, contusion, C5

SCI non-trained, n=11

T

SCI trainend, n=6

S

Chen et al., 2017

SpragueDawley

D

ro of

famele, age ND

Corticospinal rats, tract

SCI non-trained, n=7

T

SCI trained, n=7

q

transection, T9

Detloff et al., 2014

Sprague-

Unilateral

rats, contusion, C5

lP

Dawley

re

-p

female, age ND

ur na

SpragueDawley

Unilateral rats, contusion, C5

S

D

w

I

SCI non-trained, n=23

T

SCI trained, n=16

S

female, adults

Detloff et al., 2015

I

D

5

I

SCI allodynia non-trained 14th T day, n=7

S

SCI allodynia trained 14th day, D

female, adults

n=9

I

Jo

SCI allodynia non-trained 28th day, n=8 SCI allodynia trained 28th day, n=9

Dugan; Sagen, 2015

Sprague-

Compression,

SCI non-trained, n=15

T

Dawley

rats, T6-T7

SCI trained 5th day, n=12

S

male,

age

SCI trained 21st day, n=15

D

46

based

on

I

weight Endo et al., 2009

SpragueDawley

Contusion, T9 rats,

SCI non-trained, n=11

T

SCI trained, n=12

S

female, adults

D

I Hutchinson

et

al., Sprague-

2004

Dawley

Contusion, T8 rats,

Maier et al., 2009

SpragueDawley

T-shaped, T8 rats,

re

lP

C57BL/6 mice, Contusion, T11

ur na

female, adults

Jo

Sliwinski et al., 2018

Tashiro et al., 2015

SCI treadmill trained, n=7

b

SCI swim trained, n=10

S

SCI standing trained, n=9

D

I

SCI non-trained, n=ND

T

SCI trained, n=ND

q

-p

female, adults

Nees et al., 2016

T

ro of

female, age ND

SCI non-trained, n=6

S

D

w

I SCI non-trained, n=13

T

SCI trained, n=9

S

D

w

I

C57BL/6 mice, Contusion, T9

SCI non-trained, n=10

T

female, adults

SCI trained, n=14

S

D

w

I SpragueDawley

Contusion, T10 rats,

SCI non-trained, n=10

T

SCI trained, n=10

S

female, adults

D

I

47

Tashiro et al., 2018

CG57BL/6J mice,

Contusion, T9

female,

SCI non-trained, n=12

T

SCI trained, n=14

S

adults

D

I Ward et al., 2014

Wistar

rats, Contusion, T10

male, adults

SCI non-trained, n=8

T

SCI trained, n=8

S

D

I Ward et al., 2016

Wistar

rats, Contusion, T8

T

SCI quadrupedal trained, n=13

w

ro of

male, adults

SCI non-trained, n=7

SCI bipedal trained, n=9

-p

*, just interesting groups of studies was cited; T, thoracic level; C, cervical level;

SCI, Spinal Cord Injury; BWS, body-weight support; n, total of animals per group;

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ur na

lP

re

ND, not described; dpi, days post-injury; min, minutes; x/week, times per week.

48

S

D

I

Motor function BBB

Chhaya et al., 2018 Chen et al., 2017

None BBB

Detloff et al., 2014

None

Detloff et al., 2015 Dugan; Sagen, 2015

None BBB

Endo et al., 2009 Hutchinson et al., 2004 Maier et al., 2009 Nees et al., 2016

BBB None BBB BMS

Sliwinski et al., 2018

BMS

Tashiro et al., 2015

BMS

ur na

Tashiro et al., 2018

BBB

Ward et al., 2014

BBB

Ward et al., 2016

BBB

Neuropathic pain Thermal nociception: Tail flick Mechanical nociception: von Frey filaments Mechanical nociception: von Frey filaments, mecha Thermal nociception: Hargreaves method Mechanical nociception: von Frey filaments Thermal nociception: Hargreaves method Mechanical nociception: von Frey filaments Mechanical nociception: von Frey filaments Thermal nociception: Hargreaves method Cold nociception: Acetone droplet Mechanical nociception: von Frey filaments Mechanical nociception: von Frey filaments Mechanical nociception: reflex testing, von Frey filam Thermal nociception: Hargreaves method Thermal nociception: Hargreaves method Cold nociception: Cold Hot plate Mechanical nociception: von Frey filaments Spontaneous pain: Open Field Thermal nociception: Hargreaves method Mechanical nociception: von Frey filaments, place e Thermal nociception: Hargreaves method Mechanical nociception: von Frey filaments Thermal nociception: Hargreaves method Mechanical nociception: von Frey filaments Thermal nociception: Hargreaves method Mechanical nociception: innocuous stimuli scale, ev Mechanical nociception: At-level allodynia score

re

-p

ro of

Reference Brown et al, 2011

lP

Table 2. Specific behavioral assessments.

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BBB, Basso Beattie and Bresnaham Scale; BMS, Basso Mouse Scale.

49

Table 3. Neurobiological assessments.

Analyses White and gray matter spared White and gray matter spared Microglia (Iba1) Macrophages (ED-1/CD68) White and gray matter spared Corticospinal tract sprouting (BDA) Serotonergic synapses (5-HT) Glutamatergic synapses (VGlut1) Neuronal activity (c-Fos) White and gray matter spared Non-peptidergic fibers (IB-4) Neurotrophic factor (GDNF) Neurotrophic factor (Artemin) White and gray matter spared Peptidergic fibers (CGRP) Non-peptidergic fibers (IB-4) None Corticolspinal regenerate fibers (CaMK2a) Peptidergic fibers (CGRP) Neurotrophic factor receptor (TrkB) White and gray matter spared Neutrophic factor (BDNF) Neutrophic factor (NT3) Synaptic plasticity (Synapsin I) Corticospinal tract sprouting (BDA) Peptidergic fibers (CGRP) Serotonergic synapses (5-HT) White and gray matter spared Peptidergic fibers (CGRP) Non-peptidergic fibers (IB-4) Astrocyte (GFAP) Microglia (Iba1) White and gray matter spared Peptidergic fibers (CGRP) Non-peptidergic fibers (IB-4) Synaptic transmission (KCC2) Neurotrophic factor receptor (PLC-y/Shc) Neural fibers (NF-H) Peptidergic fibers (CGRP)

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Local SCI level SCI level SCI level SCI level / DRG Chen et al., 2017 SCI level Thoracic / Lumbar level Lumbar level Lumbar level Lumbar level Detloff et al., 2014 SCI level Cervical level Cervical level Cervical DRG Detloff et al., 2015 SCI level Cervical / Lumbar level Cervical / Lumbar level Dugan; Sagen, 2015 None Endo et al., 2009 Lumber level Lumber level Lumber level Hutchinson et al., SCI level 2004 SCI level / Muscle SCI level / Muscle SCI level / Muscle Maier et al., 2009 SCI level SCI level SCI level Nees et al., 2016 SCI level Lumbar level Lumbar level Lumbar level Lumbar level Sliwinski et al., 2018 SCI level Lumbar level / DRG Lumbar level / DRG Tashiro et al., 2015 Lumbar level Lumbar level Tashiro et al., 2018 SCI level Lumbar level

Jo

ur na

lP

re

-p

Reference Brown et al, 2011 Chhaya et al., 2018

50

Ward et al., 2014

Ward et al., 2016

Lumbar level

GABAergic synapses (GAD65)

SCI level Bladder Bladder Bladder SCI level

White and gray matter spared Neurotrophic factors (NGF) Neurotrophic factors (BDNF) Neurotrophic factors (NT3) White and gray matter spared

BDNF, brain derived neurotrophic factor; CaMK2a, calcium/calmodulindependent protein kinase type II alpha chain; CGRP, calcitonin gene-related peptide; DRG, dorsal root ganglia; GAD65, glutamic acid decarboxylase-65;

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GDNF, glial cell-line derived neurotrophic factor; IB4, isolectin-B4; Iba1, ionized calcium-binding adapter molecule; KCC2, potassium-chloride cotransporter-2;

NF-H, neurofilament-H; NGF, nerve growth factor; NT-3, neurotrophin-3; SCI, spinal cord injury; TrkB, tropomyosin-related kinase B; VGlut1, vesicular

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ur na

lP

re

-p

glutamate transporter 1; 5-HT, 5-hidroxitriptamin.

51

Table 4. Proportion of complete (%) ARRIVE items per year of publication. Introduction Item

1

2

3a

3b

Title

Abstract

100

66.7

33.3

33.3

0

66.7

41.4

66.7

25.0

16.7

Background

4

5

6a

Ethical

Objectives

6b

6c

Study design

statement

7a

7b

Experimen

References published before

66.7

100

66.7

0

100

0

75.0

66.7

25.0

2010

published after

ro of

References 66.7

2010

13c

0

33.3

100

0

100

0

33.3

33.3

66

58.3

41.7

16.0

91.7

33.3

75.0

8.3

58.3

41.7

66

10c

Sample size

11a

before

33.3

0

2010

published after 2010

66.7

ur na

References

100

91.7

0

11b

Allocating

References published

Results

-p

Item

10b

58.3

12

Exp out

13a

13b

Statistical methods

re

10a

50.0

lP

Methods

100

14

15a

15b

Bas

Numbers

data

analyzed

Exp out, experimental outcomes; Bas data, baseline data; Out est, outcomes

Jo

estimation; Gen, Generalizability / translation; Fund, funding.

52

1

Out

Table 5. Proportion of reporting ARRIVE items. % ARRIVE items Partially

Hutchinson et al., 2005

47.4

13.1

Endo et al., 2009

28.9

21.0

Maier et al., 2009

36.8

26.3

Brown et al., 2011

34.2

34.2

Detloff et al., 2014

36.8

34.2

Ward et al., 2014

50.0

26.3

Detloff et al., 2015

39.5

29.0

Dugan; Sagen, 2015

28.9

34.2

published

Tashiro et al., 2015

39.5

18.4

after

Nees et al., 2016

47.3

23.7

58.0

21.0

55.3

15.8

Chhaya et al., 2018

31.6

47.4

Tashiro et al., 2018

31.6

34.2

63.1

31.6

before 2010

References

2010

Ward et al., 2016

ur na

lP

Chen et al., 2017

-p

published

re

References

Jo

Sliwinski et al., 2018

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Complete

53