SYSTEMATIC REVIEW OF RECOVERY OF SPINAL CORD INJURY WITH ANTIOXIDANT THERAPY REVISÃO SISTEMÁTICA DA RECUPERAÇÃO DE TRAUMA RAQUIMEDULAR COM TERAPIA ANTIOXIDANTE REVISIÓN SISTEMÁTICA DE RECUPERACIÓN DE TRAUMA RAQUIMEDULAR CON TERAPIA ANTIOXIDANTE

The objective of the paper is to analyze the frequency and efficacy of experimental studies with antioxidant therapy. A search was conducted in the pubmed.gov database using the keywords “antioxidants” AND “spinal cord injury”, from January 2000 to December 2015, resulting in 686 articles. Studies of non-traumatic injuries, non-antioxidant therapies, absence of neurological and functional evaluation, and non-experimental studies were excluded, leaving a total of 43 articles. The most used therapies were melatonin (16.2%), quercetin (9.3%), epigallocatechin and edaravone (6.9%). The most frequent route of administration was intraperitoneal (72.09%). The dose and mode of administration varied greatly, with a single dose being the most commonly used (39.53%). The time elapsed from trauma to treatment was 0-15 minutes (41.8%), 15-60 minutes (30%) and over 60 minutes (10.6%). Histological analysis was performed in 32 studies (74.41%). The BBB scale was the main functional measure applied (55.8%), followed by the inclined plane test (16.2%) and the Tarlov scale (13.9%). Positive outcomes were observed in 37 studies (86.04%). The heterogeneity of antioxidant therapy, with different types, doses, and measurements observed, limits the comparison of efficacy. Standardized protocols are required to make clinical translation possible.


INTRODUCTION
Spinal cord injury (SCI) can occur by traumatic or ischemic event.Following the primary injury, cellular necrosis and tissue degeneration are the secondary events, caused mainly by hypoxia and ischemia. 1,2 reduction in blood flow and microvascular abnormalities were demonstrated, leading to an increase in intracellular free radical species. 3,47][8][9] Investigations to find a specific therapy to control the formation of free radicals are ongoing. 10The role of antioxidant drugs and hyperbaric oxygenic therapy is also being discussed. 11,12here are many publications that focus on antioxidant treatment after SCI, with different neurological outcomes.The present study analyzes the various types of antioxidant drugs used in experimental SCI, in order to define the most common and effective ones.

MATERIALS AND METHODS
A literature review was carried out in the database pubmed.gov, on December 13, 2015, using the keywords "antioxidants" AND "spinal cord injury", without filters.The selection of keywords was based on MeSH terms structure.The papers included were in vivo SCI studies treated by antioxidant therapy from January 2000 to December 2015.A total of 686 articles were found, which were then reviewed by two independent observers, considering the exclusion criteria reported in Figure 1.Full text PDFs of potentially relevant articles were obtained, in order to better select the articles.
The inclusion criteria were: experimental study in rats with SCI treated with antioxidant drugs and followed up to verify functional recovery.Articles in English, Spanish or Portuguese were included.The exclusion criteria were: lack of abstract, therapies other than antioxidant therapy, studies in animals other than rats, review articles, epidemiological or case-report studies, and lack of neurological and histological assessment.
The variables analyzed in the selected papers were: (I) year of publication, (II) type of the antioxidant drug used, (III) mechanism of action, (IV) posology, (V) administration form, (VI) elapsed time between the trauma and the treatment, and (VII) assessment of motor recovery and histology.  (Tab 1) The year of publication was shown as a frequency graph, and the annual publication rate was determined by Pearson's Correlation and Simple Linear Regression, obtaining the slope ("b" coefficient).For both, statistics, the software program SPSS Statistics v. 24 for Mac (IBM, New York, USA) was used.Significance was defined as p<0.05.
"The study was a review and systematic analysis of scientific articles published on PubMed.In this case, there was no need for prior authorization by an ethics committee, as the works selected for the study were expressly authorized by their respective ethics committees.This study reviewed and analyzed these publications, and there was no contact with or prospective or retrospective data on animals or patients in any phase."

Paper selection and inclusion
Initially, 686 articles were retrieved.After analyzing each paper and applying the inclusion and exclusion criteria, forty-three articles were included in the study.(Figure 1) The studies were reported according to the year of publication on PubMed: 2000-2004 (5 articles, 11.6%), 2005-2009 (13 articles, 30.2%), and 2010-2015 (25 articles, 58.1%).A mean of 3.6 papers were published per year.There was a significant increase in the number of publications in past 15 years (slope= 0.33, r= 0.69, p= 0.012).(Figures 2 and 3)
The elapsed times from SCI to treatment was 0-15 minutes (18 articles, 41.8%), 15 to 60 minutes (13 articles, 30%), and after 1 hour (5 articles, 10.6%).(Table 2) One study was classified separately because the treatment was started 5 minutes before SCI.Six papers (13.9%) did not specify the time of drug administration after SCI.Eighteen studies started treatment immediately after SCI.

Functional recovery
Basso, Bresnahan and Beattie (BBB scale system) was used to perform the functional measurement in 24 articles (55.8%).The following tests were inclined plane test (16.2%) and Modified Tarlov Scale (13.9%).
Histology was performed to analyze the efficacy in 32 articles (74.41%).

Outcomes
Positive outcomes were observed in 37 papers (86%) after antioxidant treatment.
Negative outcomes were observed in 6 studies (13.9%).Two papers with negative outcomes started treatment immediately after trauma, using melatonin and agmatine as antioxidant therapy.

DISCUSSION
After SCI, the inflammatory response occurs by cellular activation in order to reorganize the damaged tissue.This process increases the intensity and the volume of the lesion.2][3][4] The present study showed better outcomes in 37 studies (86%) where antioxidant therapy was used after experimental SCI.The most common therapy was the use of melatonin by the intraperitoneal route immediately after trauma.
Antioxidants are subdivided into two categories based on their hydrophilic or hydrophobic characteristics.Hydrophilic substances interact with intracellular enzymes, reducing reactive species production in the mitochondrial system by chemical reduction.Hydrophobic substances protect the cell membranes from damage.
Hypoxia secondary to SCI leads to free radical formation and lipid peroxidation, whereby lipids from plasmatic and intracellular membranes are converted by reactive species into malondialdehyde, leading to destruction of the membrane structure.This disarrangement of the intracellular membranes leads to activation of apoptosis, culminating in neuronal damage, with loss of motor, sensitive and autonomic functions, or even death. 56,57Antioxidant therapy blocks this cascade by scavenging free radicals and inhibiting different enzymes, such as superoxide-dismutase, glutathione peroxidase and catalase. 12

Number of doses and start of treatment
Eight papers had an incomplete or partial description of the methods used; they did not specify the number of doses given (n= 6) or time treatment was started (n= 2).This information is essential to analyze its efficacy and verify which drug is the best for a clinical trial.
It was difficult to analyze the efficacy of antioxidant therapy because of the different method and types of drugs used.Starting therapy before trauma was not clinically relevant.Five articles started the treatment 1 hour after trauma, making clinical translation possible.

Analysis of functional recovery
The majority of the articles (28 papers, 82.3%) completed the analysis of functional recovery at 3 weeks.It is known that the inflammatory process is reduced slowly and gradually.There is evidence to support the hypothesis that 4 weeks is the minimum time needed to analyze the histological and functional recovery. 58An analysis period of less than 4 weeks is insufficient to correctly determine the response to the treatment.
The limitations of the study were missing information, the different types of antioxidant therapy, the different doses, and different times elapse between the trauma and the start of therapy.The present article is the most complete and up-to-date review of antioxidant therapy in SCI.
As future perspectives, the research group will design an in vivo experimental study to analyze the efficacy of antioxidant therapy after SCI, to provide evidence for clinical translation.

FINAL REMARKS
The literature shows heterogeneity of antioxidant treatment with different types, doses, measurements that limit the comparison of efficacy.Standardized protocols for antioxidant therapy need to be designed to make the clinical translation viable.

Figure 1 .
Figure 1.Flowchart.The search resulted in 686 articles.After applying the exclusion and inclusion criteria, 43 papers remained.

Figure 2 .
Figure 2. Frequency.The articles were divided by year of publication.

Figure 3 .
Figure 3. Statistical analysis.Pearson's Correlation and Linear Regression were used to obtain the slope.year_trend

Table 2 .
Variables observed in the study.
The variables studied and its frequencies are resumed in this table.*Othertype of antioxidant are presented in Table1.† BBB (Basso, Bresnahan and Beattie scale system).