Supplemental information
A systematic review and meta-analysis of Ginsenoside-Rg1 (G-Rg1) in experimental ischemic stroke
Cheng-long Xie1, Wen-Wen Wang2, Xiao-Dong Xue3, Su-fang Zhang1, Jing Gan1Zhen-Guo Liu1,*
1 Department of Neurology, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine 200092, 1665 Kongjiang Road, Shanghai, China.
2 The center of Traditional Chinese Medicine, The Second Affiliated Hospitalof Wenzhou Medical College, Wenzhou 325027, China.
3 Department of Neurology, Cang Nan county hiospital, Wenzhou 325027, China.
* Correspondence author: Zhen-Guo Liu
Tel/fax +86 21 25077501; Email .
Protocolfor systematic review and meta-analysis of Ginsenoside-Rg1 (G-Rg1)in experimental ischemicstroke
Abstract
Background:The neuroprotective actions of Ginsenoside-Rg1 (G-Rg1) have been documented for experimental stroke therapy. We used a systematicreview and meta-analysis to assess the efficacy of G-Rg1 in experimental ischemic stroke.
Methods:We plan to searchPubMed,EMBASE, Google scholar, Chinese National Knowledge Infrastructure (CNKI), VIP informationdatabase, and Wanfang data Information Sitefor potentially eligibletrials thatinvestigate G-Rg1 for acute ischemic stroke compared with control groups. We identified studies describing the efficacy of G-Rg1 in animal models of focal cerebral ischemia. Primary outcomes were infarct volume and neurological function score (NFS). NFS and infarct volumewere considered as continuous data.Two reviewers will independentlyscreen and identify eligible studies using predefined eligibility criteria. Data abstraction will be done independentlyand in duplicate.Allanalyses were performed with RevMan version 5.1.
Discussion:The aim of this systematic review and meta-analysis is to summarize the available evidence on the efficacyof G-Rg1 in experimental ischemic stroke.
Systematic review registration: Without registration
Keywords:Ginsenoside-Rg1; G-Rg1; ischemic stroke; animal model; systematic review; meta-analysis
Background
Acute ischemic stroke (AIS) is defined as a clinical syndrome of sudden break out of focal or global disturbance of central nervous system function result froman interruption of the cerebral blood flow [1]. The estimated annual incidence of AIS is0.25% and has become a leading cause of morbidityand mortality [2]. In addition, AIS is a costly condition incurring treatment, care and indirectcosts. Over the past decades, over 1000 drugs have been tested in experiment studies, and exceed 400 have demonstrated efficacy in animal modelsof stroke[3]. However, it is disappointing that most of these treatments have been confirmed tobe no effective in the acute phase of stroke[4,5], only recombinant-tissueplasminogen activator(rt-PA), aspirin and stroke unitcare have convincingly displayed efficacy in clinicaltrials of AIS [6].Therefore, it is necessary to examine other potential neuroprotectantsfor ischemic stroke.Moreover, various failures of candidate drugs for cerebral ischemiahave generated a series of suggestions that aim to improvethe likelihood of successful translation. Among them, systematic review and meta-analysis of preclinical studies have been advocated to further informcandidate drug development and supply pool information for jumping into stroke clinical trials [7].
For thousands of years, Ginsenghas been used in Traditional Chinese Medicine as a tonic to improvestamina and vitality[8].It also has supposed adaptogenicproperties, making it useful for many health conditions. The main pharmacologicallyactiveingredientsofginseng are ginsenosides，of which Ginsenoside Rg1 (G-Rg1) is regarded as one of the most important bioactive components responsible for pharmaceutical actions for Ginseng (a traditional Chinese tonic drug) with little toxicity and has been shown to have possibly neuroprotective effects [9]. In recent years, accumulating evidence indicated that G-Rg1 hadpivotal role in protecting the brain from ischemic damage[10]. Zheng et al reported G-Rg1 could improve neurological function outcome and prevent ischemic neuronal death, as well as reduced infarct volume in histology[11]. Moreover, G-Rg1 increased neurogenesis after transientglobal ischemia in the dentate gyrus of adult gerbils and attenuated the blood brain barrier disruption by cerebral ischemic stroke [12]. Meanwhile, numerous studies on specific mechanism of G-Rg1 is now in progress, including potentiatingnervegrowthfactors (NGF),antioxidant,anti-inflammatory, anti-apoptotic, and immune-stimulatoryactivities, inhibiting excitetoxicity and Ca2+ over-influx into neurons, maintainingcellular ATPlevels,and preservingstructural integrity of neurons et al [13].
However, to date, no systematic review hasexplored the adherence of G-Rg1 experimental researchon ischemic stroke models. Meanwhile, the results of the previous preclinical studiesoften root in relatively small sample size and are quitenuances. Systematically reviewing and meta-analysis of all these papers in anobjective and quantitative manner might offer us with credible and solid newevidence on whether or not G-Rg1 treatment exist neuroprotective effect in experimental ischemic stroke, to select the optimal requirementsfor drug administration for clinical trials. Therefore, in this study, we conducted a systematic review and meta-analysis to identify anyevidence of G-Rg1 as a neuroprotectant on ischemic stroke animalmodels.
Methods/design
Inclusion and exclusion criteria
We included all controlled preclinical studies of the effect of G-Rg1 in animal models offocal cerebral ischemia, where the outcome was measured as infarct volume or/and neurological function score (NFS). NFS is mainly used to assess the effect of new therapeutic methods to indicate the muscle force. To prevent bias, inclusion criteria were pre-specified as follows:
(1) The effect of G-Rg1 was tested in animal models of focal cerebral ischemia induced by temporarymiddle cerebral artery occlusion (MCAO) or permanent MCAO.
(2) Infarct volume and/or NFSwere compared with control animals receiving vehicle or no treatment, as well as compared with positive control drug animals.
Pre-specifiedexclusion criteria were:
(1) Case reports, abstracts,comments, reviews, and editorials and clinical trials.
(2) Non-focal cerebral ischemia model (such as global ischemic models, or hypoxic-ischemic models).
(3) Infarct volume and/or NFSwere not the outcome measures.
(4) Nottesting the efficacy of G-Rg1 on AIS.
Types of intervention
The effect of G-Rg1 was tested in animal models of focal cerebral ischemia induced by temporarymiddle cerebral artery occlusion (MCAO) or permanent MCAO.
Types of outcome measure
Our primary outcome measure isinfarct volume and/or NFS.
Search strategy
A computerized literature search and hand searching of abstractsfrom scientific meetingswere performed to find publications studying theeffect ofG-Rg1 treatment on animal models of acute ischemicstroke from PubMed,EMBASE, Chinese National Knowledge Infrastructure (CNKI), VIP informationdatabase, and Wanfang data Information Site. The publication time is from theinception of each database up to March 2014. All searches were limited to studies on animals.Reference lists from the included literature were used to identify further relevantpublications.The following searchstrategy, using the grouped terms, was used for MEDLINE,and was modified to suit other databases.
Medline (Pubmed) search strategy.
1.Ginseng
2. Ginsenoside
3. Ginsenoside-Rg1
4. G-Rg1
5. or/1-4
5. Ischemia
6. Infarction
7. Stroke
8. or/5-7
9. 5 and 8
Data extraction
The following details of the study designwere extracted from each study: (1)first author’s name and the publication year, method of ischemia induction, and ischemia time; (2) individual data were obtained for each study,including animal number, species, sex, and weight; (3)information on treatment was obtained, including timing and dosage for treatment, method of treatment procedure; (4) outcomemeasures and timing for outcomes assessment were also included.If outcomes were presented from the preclinical studies ofanimals at different time points, we extracted data from the last time point to sacrifice. If the datafor meta-analysis were missing or only expressed graphically, we tried to contact theauthors for further information, and where a response was not received, wemeasured data from the graphs using digital ruler software or exclude. Foreach comparison, we extracted data of mean value and standard deviation from every study.Thetime of lesion was set to zero and the time of drugadministration expressed relative to this.All the data wereextracted independently by two participants
Quality Assessment
Two authors independently extracted data andassessed study quality.For each included study, we will provide a description,comment, and judgment of ‘ Yes ’ , ‘ Unclear ’ , or ‘ No ’ foreach of the following items.
Study quality was assessed based on a ten-item modified scale[15]:
(1) Publication in a peer-reviewed journal.
(2)Statements describing control of temperature.
(3) Randomization to treatment group.
(4) Allocation concealment.
(5)Blinded assessment of outcome.
(6) Avoidance ofanesthetics with known marked intrinsic neuroprotective properties.
(7) Use ofanimals with relevant comorbidities.
(8) Sample size calculation.
(9) Compliance withanimal welfare regulations.
(10) Declared any potential conflict of interest. For thecalculation of an aggregate quality score, each item of the ten-item modified scale wasattributed one point.
Pre-stratified subgroupanalysis
To explore the impact of factors modifying on the outcome measures,we performed a pre-stratified subgroupanalysisith experiments grouped according to thefollowing:
(1)Reported quality score.
(2) Type of anaesthetic used.
(3) Time of initial treatment and G-Rg1 dosage.
The significance of differencesbetween groups was assessed by partitioning heterogeneityand using the x2 distribution with n-1 degrees of freedom,where n equals the number of groups. Meanwhile, we anticipate that all subgroup analyses will be challenging, owing to the lack of data and the anticipatedsmall number of included studies.
Dealing with missing data
We will contact the authors of the primary studies foradditional information on missing data. If this approachis not successful, we will analyze only the available dataand discuss the potential impact of missing data on thefindings of the review in the discussion section.
Assessment of reporting biases
We will assess publication bias visually using a funnelplot generated using RevMan 5.1 software, and statistically using the Egger test. If fewer than ten RCTsare included, we cannot reliably assess for publicationbias.
Sensitivity analysis
Sensitivityanalysis was performed according to quality by removing each individual studyin turn from the total and reanalyzing the remainder.
Statistical analysis
NFS and infarct volumewere considered as continuous data.WMD is a standard statistic that measures the absolute difference between the mean values in two groups. It estimates the amount by which the experimental intervention changes the outcome on average compared with the control. It can be used as a summary statistic in meta-analysis when outcome measurements in all studies are made on the same scale. On the contrary, standardised mean difference (SMD) is used as a summary statistic in meta-analysis when the studies all assess the same outcome but measure it in a variety of ways[16]. Meanwhile, normalised mean difference (NMD) is a useful approach because it illustrates the magnitude of the treatment in the treatment group to a normal, healthy animal. The most common method to calculate NMD effect sizes is as a proportion of the mean in the control group. Typically, effect sizes fall between -100% and +100%. The disadvantage of NMD approach is relevant to ratio scales, but sometimes it is not possible to infer what a “normal” animal would score [16].Weused random effects rather than a fixed effects modelbecause of this takes into account the heterogeneitybetween multi-studies. A very common and simple version of the meta-analysis procedure is commonly referred to as the inverse-variance method.The inverse variance method is so named because the weight given to each study is chosen to be the inverse of the variance of the effect estimate. Allanalyses were performed with RevMan version 5.1.Probability value P 0.05 was considered significant.
Discussion
This systematic review will identify and synthesizeevidence examining the potential benefits and harms ofusing G-Rg1 in experimental ischemic stroke.Giventhe emergence of new evidence, and the lack of an up-dated systematic review, this review will help in summarizing the available evidence, both quantitatively andqualitatively.
Competing interests
The authors declare that they have no competing interests.
Acknowledgments
The study was supported by the Projects of National Science Foundation of China (No.81171203, 81171204, 81200871,81400925 and 81471148), and Projects of the Shanghai Committee of Science and Technology, China (12XD1403800).
Reference
1. Kidwell CS, Liebeskind DS, Starkman S, Saver JL (2001) Trends in acute ischemic stroke trials through the 20th century. Stroke32:1349-1359.
2. Simon RP, Greenberg DA, Aminoff MJ (2009) Stroke. Clinical Neurology. 7th Edition. New York: McGraw-Hill:292–327.
3. O'Collins VE, Macleod MR, Donnan GA, Horky LL, van der Worp BH (2006) 1,026 experimental treatments in acute stroke. Ann Neurol 59:467-477.
4. Muir KW, Lees KR (2003) Excitatory amino acid antagonists for acute stroke. CochraneDatabase Syst Rev:CD001244.
5. van der Worp HB, Howells DW, Sena ES, Porritt MJ, Rewell S (2010) Can animal models of disease reliably inform human studies? PLoS Med 7:e1000245.
6. Wright L, Hill KM, Bernhardt J, Lindley R, Ada L (2012) National Stroke Foundation Stroke Guidelines Expert Working Group.Stroke management: updatedrecommendations for treatment along the carecontinuum. Intern Med J42:562-569.
7. Sena E, van der Worp HB, Howells D, Macleod M (2007) How can we improve thepre-clinical development of drugs for stroke? Trends Neurosci30:433-439.
8. Ellis JM, Reddy P (2002) Effects of Panax ginseng on quality of life. AnnPharmacother36:375-379.
9. Jiang B, Xiong Z, Yang J, Wang W, Wang Y (2012) Antidepressant-like effects of ginsenoside Rg1 are due to activation of the BDNF signalling pathway and neurogenesis in the hippocampus. Br J Pharmacol166:1872-1887.
10. Shen L, Zhang J (2007) NMDA receptor and iNOS are involved in the effects ofginsenoside Rg1 on hippocampal neurogenesis in ischemic gerbils. Neurol Res29:270-273.
11. Zhou Y, Li HQ, Lu L, Fu DL, Liu AJ (2014) Ginsenoside Rg1 provides neuroprotection against blood brain barrier disruption and neurological injury ina rat model of cerebral ischemia/reperfusion through downregulation of aquaporin 4 expression. Phytomedicine 21. pii: S0944-7113(13)00480-7.
12. Zheng GQ, Cheng W, Wang Y, Wang XM, Zhao SZ (2011) Ginseng total saponins enhance neurogenesis after focal cerebral ischemia. JEthnopharmacol133:724-728.
13. Rausch WD, Liu S, Gille G, Radad K (2006) Neuroprotective effects of ginsenosides.Acta Neurobiol Exp (Wars) 66:369-375.
14. Panic N, Leoncini E, de Belvis G, Ricciardi W, Boccia S (2013) Evaluation of theendorsement of the preferred reporting items for systematic reviews andmeta-analysis (PRISMA) statement on the quality of published systematic reviewand meta-analyses. PLoS One8:e83138.
15. Macleod MR, O'Collins T, Howells DW, Donnan GA (2004) Pooling of animal experimentaldata reveals influence of study design and publication bias. Stroke35:1203-1208.
16.Vesterinen HM, Sena ES, Egan KJ, Hirst TC, Churolov L (2013) Meta-analysis of data from animal studies: a practicalguide. J Neurosci Methods221:92-102.