Neuroprotection of Catalpol for Experimental Acute Focal Ischemic Stroke: Preclinical Evidence and Possible Mechanisms of Antioxidation, Anti-Inflammation, and Antiapoptosis

Neuroprotection is defined as using a therapy that affects the brain tissue in the still-viable ischemic penumbra to salvage or delay the infarction. Catalpol, the main active principle of the root of Radix Rehmanniae, was reported to have pleiotropic neuroprotective effects in neurodegenerative diseases including ischemic stroke. Here, we evaluated the neuroprotective effects of catalpol in experimental acute ischemic stroke. Studies on catalpol in animal models of acute ischemic stroke were identified from 6 databases. Twenty-five studies involving 805 animals were included. Twelve comparisons showed significant effects of catalpol on decreasing infarct size according to 2,3,5-triphenyltetrazolium chloride staining compared with the control (P < 0.05). One study reported significant effect of catalpol on reducing infarct size according to magnetic resonance imaging scan compared with the control (P < 0.05). Meta-analysis of these studies indicated that catalpol significantly improved the neurological function score according to Zea Longa score, Bederson score, balance beam-walking test, adhesive removal test, bar-grasping score, and corner test compared with the control (P < 0.05). In conclusion, catalpol exerted neuroprotective effects for experimental acute focal ischemic stroke, largely through reducing oxidative reactions, inhibiting apoptosis, and repressing inflammatory reactions and autophagy. However, these apparently positive findings should be interpreted with caution because of the methodological flaws.


Introduction
Neuroprotection refers to the concept of using a therapy that affects the brain tissue in the still-viable ischemic penumbra to salvage or delay the infarction [1,2]. Possible mechanisms of neuroprotective treatments are to prevent local inflammation, excitotoxicity, free radical damage, neuronal apoptosis, and calcium influx into cells, resulting in both improvement of functional outcomes and reduction of infarct size [3]. In the past decades, a wealth of research has been conducted into the development of numerous neuroprotective treatments capable of reducing brain damage following ischemic stroke of animal models [4]. However, up to now, clinical trials have not identified efficacious neuroprotective therapies for stroke patients [5]. Thus, given the huge translational gap between these animal studies and clinical trials, seeking or developing innovative neuroprotectants is urgently needed. Radix Rehmanniae (Latin name), rehmannia root (English name), Dihuang (Chinese name), the roots of Radix Rehmanniae Recens, was first recorded in the book of Shennongbencaojing (Shennong's Classic of Materia Medica)-the earliest complete pharmacopoeia of China. In modern times, Radix Rehmanniae and Radix Rehmanniae-based prescriptions are still widely used for treatment of various diseases in China and elsewhere worldwide [6,7]. Radix Rehmanniae exerts its pharmacological actions on the endocrine system, blood system, immune system, nervous system, cardiovascular system, and so forth [8]. Catalpol (Figure 1), an iridoid glucoside, is the main active principle of the root of Radix Rehmanniae. Recent studies reported that catalpol had pleiotropic neuroprotective effects against hypoxic/ischemic injury, Alzheimer's disease, and Parkinson's disease in both in vivo and in vitro models [9]. Catalpol had been found to have antioxidation, anti-inflammation, antiapoptosis, and other neuroprotective properties [9], suggesting the potential neuroprotective effect of catalpol on stroke [10].
Systematic reviews are considered as the highest level of medical evidence; only data from systematic reviews will be proposed as 1a-evidence according to the levels of evidence from the Centre of Evidence-Based Medicine in Oxford [11]. Preclinical systematic reviews are a novel approach to appraise and synthesize results from animal research into a single and useful document that can indicate the direction for further basic research, reduce and refine the experimental studies, and enhance the rate of success in future clinical trials [12]. However, no systematic analysis has yet been conducted to assess the efficacy of catalpol for experimental ischemic stroke. Therefore, we aimed to identify the current evidence of catalpol as neuroprotective agent in animal models of acute focal ischemic stroke.

Search Strategy.
Experimental studies of catalpol for acute focal ischemic stroke were identified from PubMed, Web of Science, Excerpta Medica Database (EMBASE), Wanfang Data information site, Chinese National Knowledge Infrastructure (CNKI), and VIP information database. All searches were performed from inception to April 2017. Chinese databases were searched by using the following search terms: "Catalpol" AND ["ischemic stroke" OR "cerebral infarct" OR "middle carotid artery occlusion (MCAO)" OR "cerebral ischemia/reperfusion"]. The term used in English databases was merely "Catalpol." We manually searched dissertations, conference proceedings, and reference lists of identified publications relevant to this topic.

Eligibility.
Experimental studies on catalpol for acute permanent MCAO or temporary MCAO models and compared with vehicle or no treatment were included. Meanwhile, the primary outcome measurements should be neurological function score (NFS) and/or infarct volume (IV). Exclusion criteria were prespecified as follows: (1) the article was a review, case report, comment, only an abstract, or editorial; (2) the article was not an animal study; (3) the article was not a research about acute focal cerebral ischemia model, such as traumatic, global, chronic cerebral ischemic models or not cerebral ischemic models; (4) catalpol was not used as a monothrapy; (5) neither NFS nor IV was used as one of the outcome measurements; (6) there was not a control group in the study; (7) the article was a duplicate publication.
2.3. Quality Assessment. The methodological quality of each included study was evaluated by using Collaborative Approach to Meta-Analysis and Review of Animal Data from Experimental Studies (CAMARADES) 10-item checklist [13]: (1) peer-reviewed publication; (2) statements describing control of temperature; (3) randomization to treatment group; (4) allocation concealment; (5) blinded assessment of outcome; (6) avoidance of anesthetics with known notable intrinsic neuroprotective properties; (7) use of animals with relevant comorbidities; (8) sample size calculation; (9) compliance with animal welfare regulations; (10) declared any potential conflict of interest. For calculating an aggregate quality score, each item of this scale was attributed one point. Two authors (ZXW and YWT) independently extracted information and evaluated quality study. Disagreements were solved after discussing the details of the studies.

Data Extraction.
The following information of each included study was extracted: (1) the first author's name and publication year, permanent or temporary MCAO, ischemic time, the anesthetic used, and random method; (2) characteristics of animals, including sex, species, weight, and animal number; (3) treatment information, including the drug used, method of treatment, timing for initial treatment, and duration of treatment; (4) outcome measurements, timing for outcome assessments, and corresponding data of mean value, standard deviation, and between-group differences. NFS and/or IV was extracted separately. If outcomes were presented at different time points, we extracted data from the last time point. If studies utilized dose gradient of the drug, we extracted data from the highest dose of catalpol because of the prespecific criteria and the dose-response relationship. If the data were incomplete or presented in graphs, we tried to contact the authors for data needed or calculated using relevant software.
Information of the mechanism studies of catalpol for experimental ischemic stroke among the included articles and other compounds from Rehmanniae Radix was extracted as the following: the first author name, publication year, models used in experiment, interventions in experimental group and control group, observation, and possible mechanisms.
2.5. Statistical Analysis. All data of NFS and IV were considered as continuous variables. Meta-analysis was performed with RevMan version 5.0. The random effect model and standard mean difference (SMD) were utilized herein. The I 2 statistics were chosen for the assessment of heterogeneity. Furthermore, to explore potential sources of high heterogeneity, subgroup analyses were performed according to timing for outcome assessments and sex of animals. Difference between groups was determined by partitioning heterogeneity and utilizing the χ 2 distribution with degrees of freedom (df). When probability value was less than 0.05, the difference was considered significant.

Study Inclusion.
We identified 2322 papers after systematical searches of six databases. After removing duplicates, 1749 records remained. By reading the titles and abstracts, 1591 articles were excluded for at least one of the following reasons: (1) the article was a review, case report, comment, only an abstract, or editorial; (2) the article was not an animal study; (3) the article was not a research about cerebral ischemia or stroke. After reviewing the full text of the remaining 158 papers, 80 studies were excluded because catalpol was not used as a monothrapy; 36 studies were excluded because the animal model was not acute focal cerebral ischemia; 11 studies were removed because the outcome measurement was neither NFS nor IV; 6 studies were excluded because they are duplicate publications. Ultimately, 25 eligible articles were identified   (Figure 2).

Effectiveness.
Eleven studies (15 comparisons) used IV based on TTC staining as outcome measurement. Fourteen comparisons of these studies [14, 16, 18-22, 25, 29, 31, 38] reported that catalpol could significantly reduce IV compared with the control (P < 0 05); one comparison [18] showed no significant effect of catalpol for decreasing IV compared with the control, according to TTC staining. Two studies adopted IV based on MRI scan as outcome measurement. One study [33] reported significant effects of catalpol for reducing IV according to MRI scan compared with the control (P < 0 05), whereas the other one [24] reported no significance.

Discussion
4.1. Efficacy of Catalpol. To our knowledge, it is the first systematic review that investigated the efficacy of catalpol for experimental acute focal ischemic stroke. Our analysis of 25 studies with 805 animals showed that catalpol significantly reduced IV and improved NFS, suggesting the potential neuroprotective functions of catalpol in experimental acute focal ischemic stroke. However, given the methodological flaws, the overall available evidence from the present study should be interpreted cautiously.

4.2.
Limitations. Some limitations should be considered while interpreting our study. First, we only included studies from Chinese and English databases. The absence of studies written in other languages may, to a certain degree, generate selective bias [48]. Second, only 5 out of 25 studies were English papers and the remaining ones were all Chinese papers, thereby limiting generalization of the findings. Third, the quality scores ranging from 2 to 7 points revealed low methodological quality of included studies. Most of the research had flaws in aspects of randomization, allocation concealment, and blinding and sample size calculation, which are the core standards of study design [49]. In addition, none of the included studies used animals with relevant comorbidities, which would have created more relevant models for human pathology [49]. Thus, the present study should be interpreted cautiously.

Implications.
There is a wealth of evidence showing the poor design of animal research [50], which is considered as a roadblock to translate animal research into promising preclinical drug treatments for human disease [51]. In the present study, the low quality of included studies rests with inherent limitations in the primary studies. Thus, some measurements have been developed to directly or indirectly overcome methodology quality issues for animal researches. The animal research: reporting in vivo experiments (ARRIVE) [52] is a reporting guideline consisting of a 20-item checklist for the Introduction, Methods, Results, and Discussion. We recommend to use the ARRIVE guidelines when designing animal research on catalpol, in order to improve the methodological quality. The Stroke Therapy Academic Industry Roundtable (STAIR) meetings [53] provide recommendations on dose, time window, design, outcome assessment, animal species, and model of preclinical studies of acute stroke. We also suggest utilizing the STAIR recommendations specifically for the study of catalpol treatment for experimental stroke. It is disappointing that many drugs that showed significant effects and looked promising in animal researches failed to translate into clinical drug treatments [54]. The application of excessive drug doses and the timing of drug administration in animal models, which are inapplicable for human disease, are considered to be two of the main reasons for the failure to translate from animal models to human [54]. In the present systematic review, doses of catalpol and timing for initial administration in animal models were inconsistent among the 25 included studies. Thus, we suggest further studies to determinate the optimal gradient doses and timing of administration in animal models of acute ischemic stroke.
The molecular and biological mechanisms of the neuroprotective effects of catalpol have not been fully elucidated. The present study showed that catalpol had neuroprotective effects for ischemic stroke through different mechanisms as follows: (1) reduction of oxidative reactions by increasing the activity of SOD, GSH-PX, and catalase, increasing the expression of NOX2 and decreasing the concentration of MDA and NO; (2) inhibition of apoptosis by increasing bcl-2 expression and decreasing the expression of cleaved caspase-3, caspase-9, and Bax; (3) repression of inflammatory reactions by decreasing the expression of IL10; (4) repression of autophagy by increasing LC3 expression; (5) relief of energy exhaustion by decreasing lactic acid content, increasing pyruvic acid content, and improving the activity of Na + , K + -ATPase and Ca 2+ , Mg 2+ -ATPase; (6) promotion of survival, reparation, and regeneration of neural cells through upregulating the expression of VEGF, bFGF, TrKA, TrkB, AKt, and PI3K; (7) enhancement of angiogenesis by upregulating the expression of EPO, EPOR, VEGF, JAK2, pJAK2, STAT3, and Ang-1; (8) neuroprotection through GLP-1R/β-endorphin pathway. Besides, other compounds from Radix Rehmanniae were reported to have antioxidation, anti-inflammation, and antiapoptosis activities. However, the efficacy of catalpol in terms of cellular and molecular alteration mechanisms along with functional improvement is worthy of further studies.
A total of 18 measuring methods for NFS were used in the 25 included studies, which indicated that the measuring methods for NFS were diverse and inconsistent. Whether and how the different measuring methods for NFS would affect the result of animal studies of acute ischemic stroke is expected to be further studied. Moreover, it is necessary to explore the accuracy of different measuring methods for NFS to filtrate optimum standards for NFS.

Conclusion
The present study demonstrated that catalpol could improve NFS and reduce IV, exerting potential neuroprotective effects on experimental acute focal ischemic stroke, mainly through reducing oxidative reaction, inhibiting apoptosis, and repressing inflammatory reactions and autophagy. In addition, catalpol may be a promising candidate for clinical trials. Future rigor-randomized controlled trials are needed.