Effects of acupuncture on vascular dementia (VD) animal models: a systematic review and meta-analysis

Background Vascular dementia is the second most common type of dementia that causes cognitive dysfunction. Acupuncture, an ancient therapy, has been mentioned for the treatment of vascular dementia in previous studies. This study aimed to evaluate the effects of acupuncture in animal models of vascular dementia. Methods Experimental animal studies of treating vascular dementia with acupuncture were gathered from Embase, PubMed and Ovid Medline (R) from the dates of the databases’ creation to December 2016. We adopted the CAMARADES 10-item checklist to evaluate the quality of the included studies. The Morris water maze test was considered as an outcome measure. The software Stata12.0 was used for the meta-analysis. Heterogeneity was examined using I2 statistics, and we conducted subgroup analyses to determine the causes of heterogeneity for escape latency and duration in original platform. Results Sixteen studies involving 363 animals met the inclusion criteria. The included studies scored between 4 and 8 points, and the mean was 5.44. The results of the meta-analysis indicated remarkable differences with acupuncture on increasing the duration in the former platform quadrant both in EO models (SMD = 1.56, 95% CI: 1.02 ~ 2.11; p < 0.00001) and 2-VO models (SMD 4.29, 95% CI 3.23 ~ 5.35; p < 0.00001) compared with the control groups. Conclusions Acupuncture may be effective in improving cognitive function in vascular dementia animal models. The mechanisms of acupuncture for vascular dementia are multiple such as anti-apoptosis, antioxidative stress reaction, and metabolism enhancing of glucose and oxygen. Electronic supplementary material The online version of this article (10.1186/s12906-018-2345-z) contains supplementary material, which is available to authorized users.


Background
Vascular dementia (VD) is a heterogeneous group of brain disorders of which the cognitive impairment can be ascribed to cerebrovascular pathologies; more than 20% cases of dementia are vascular dementia, making it the second most prevalent form of dementia, second only to Alzheimer's disease (AD) [1]. Advanced age, diabetes, hypertension, smoking and atrial fibrillation are all risk factors for vascular dementia [2]. Level of education, which is considered to be an effective alternative indicator of cognitive reserves, has a significant association with the expression of VD in that higher education appears to be associated with fewer cognitive deficits [3,4]. As the populations of North America and European countries age, the risk of VD in these regions approximately doubles every 5.3 years. However, the current study suggests that effective therapy for vascular dementia has proven to be more difficult than for Alzheimer's disease. While memantine and cholinesterase inhibitor drugs have been proven to be significantly effective in treating Alzheimer's disease and are therefore labeled for this indication, they are not recommended for use in the treatment of vascular dementia by either regulatory bodies or guideline groups due to their overall low effectiveness and possible side effects [5][6][7].
Acupuncture, an economical type of traditional Chinese therapy with minimal side effects, has been used for many diseases in Asian countries for thousands of years and is widely used for rehabilitation after stroke [8]. Usually after a stroke, 15-30% of subjects will develop vascular dementia within three months, and delayed dementia will develop in the long term due to recurrent stroke in 20-25% of subjects [9]. In recent years, more and more studies, and especially animal experiment studies, have been published to illustrate the effectiveness of acupuncture for vascular dementia.
Until now, no systematic meta-analysis has been published to analyze the effects of acupuncture on enhancing cognitive function in vascular dementia animal models. A systematic review of animal experiments can be beneficial for future experimental designs and provide a basis for clinical studies. Additionally, it can provide valuable directions for further research. It is for these reasons that we conducted this systematic review and meta-analysis.

Methods
This systematic review and meta-analysis complied with standard guidelines (See Additional file 1).

Search strategy
The following electronic databases were searched from database inception to December 2016: Embase, PubMed and Ovid Medline (R), and the following keywords were used: acupuncture, electroacupuncture, acupoint, vascular dementia, multi-infarct dementia and multiinfarct dementia. The search language was limited to English, and we also tried to collect records from other sources. The detailed search strategies are shown in Additional file 2.

Study selection and data extraction
One reviewer (ZYZ) generated the search strategy, searched the databases, and made a list of all the records. Two evaluators (HHD, QC) independently evaluated the articles based on the inclusion and exclusion criteria. Disagreements were solved together through discussions (ZYZ, HHD, QC). Two reviewers (HHD, QC) independently extracted the data.
The following data were extracted: publication year, the last name of the first author, model of vascular dementia, weight range of the included animals, number of animals included, method of treatment with timing and duration in the acupuncture and control groups, the assessment of trials, and the results of each article (positive or negative). The final outcomes were extracted if several outcomes were presented. When the outcome data were only shown graphically, we attempted to contact the authors to obtain the detailed data; if we received no response to our request, we used GetData software to measure the data. Differences were solved together through discussions (HHD, QC, ZYZ).
The quality of each study was evaluated by a score from zero to ten. Two evaluators (HHD, QC) independently extracted the data and assessed the quality of each study. Disagreements were resolved through discussion (HHD, QC, ZYZ).

Statistical analysis
For statistical analysis, we used the statistical software package Stata version 12.0. The data of the Morris water maze test, such as escape latency, time in the quadrant in which the former platform was located, and frequency of crossing through the former platform were considered continuous data, and we therefore calculated standard mean differences (SMD) with confidence intervals (CIs) established at 95%. Heterogeneity in the studies was examined using I 2 statistics. If the I 2 statistic was higher than 50%, we considered significant heterogeneity to be present, and we used a random effects model. Otherwise, we used a fixed effect model. When significant heterogeneity existed, the subgroup analysis would be conducted based on animal species, acupuncture methods and modeling methods. We used Egger's test and Begg's test to assess publication bias.

Study characteristics
The 16 studies included involved 363 rats, 174 of which were in an acupuncture group and 189 of which were in a control group. All the studies stated the weight range of the rats, which ranged from 180 to 490 g. A total of 7 of the 16 studies mentioned the age of the animals [11, 14, 16-18, 23, 25], which ranged from 2 months old to 12 months old. Sixteen of the included studies used different subtests of the Morris water maze test; all the studies met the inclusion criteria by using escape latency as outcome data, while two studies used swimming speed [24,26], five studies used duration in the quadrant of the former platform position [12,13,21,24,26], one used swimming distance [19], and three used frequency of crossing former platform [12,15,16]. Two species of rats were used in the 16 studies: seven studies used Sprague-Dawley (SD) rats [11,13,15,[17][18][19]25], and nine studies used Wistar rats [12,14,16,[20][21][22][23][24]26]. The basic characteristics of the studies are listed in Table 1 [28].

Control interventions
Ten studies adopted some interventions in the control groups. The Western medicine nimodipine was used as a control intervention in three studies [11,13,15], and the remaining seven studies adopted placebo-acupuncture as a control intervention [12,14,16,[22][23][24]26].

Morris water maze outcomes analyses Escape latency
Twelve studies adopted escape latency as an outcome index. And all of these studies reported the positive effectiveness of acupuncture on reducing escape latency, except for the one study [20] that was designed with two acupuncture groups, the positive control group and cluster-needling group, which showed negative results and positive results, respectively. Of these 12 studies, six of them provided detailed data regarding the marked effect of acupuncture [11,13,15,[18][19][20], and we extracted the data from the other six studies that demonstrated the data in a graphical representation by using GetData software [16,[21][22][23][24][25][26]. To avoid double-counting [30], the effects of different acupuncture intervention arms included in a single study were averaged and entered once in the analysis [20] [n = 280, SMD = − 3.06, 95%CI (− 4.04~− 2.09), p < 0.00001; heterogeneity I 2 = 87.1%, random effects model, Fig. 2a] [31][32][33]. Three studies compared the effect of acupuncture with nimodipine [11,13,15], and all three of these studies concluded that escape latency was not significantly different between the medication group and the acupuncture group [n = 64, SMD = − 0.07, 95% CI (− 0.56~0.42), p = 0.775 > 0.05; heterogeneity I 2 = 0%, fixed effect model, Fig. 2b].

Duration in original platform
Five studies showed the effect of acupuncture on improving the duration in the quadrant of the former platform compared with the control group (VD group or placebo-acupuncture group) [12,14,21,24,26]. All five of these studies reported positive results, but none of them provided detailed data [13]; we therefore extracted the data from the figures [n = 119, SMD = 2.52, 95%CI (1.34~3.69), p < 0.00001; heterogeneity I 2 = 82.0%, random effects model, Fig. 2c].

Frequency of crossing former platform
Three studies reported the positive results of acupuncture on increasing the frequency of crossing the former platform location [12,15,21] [n = 69, SMD = 1.84, 95% CI (0.95~2.74), p < 0.00001; heterogeneity I 2 = 56.3%, random effects model, Fig. 2d]. There was no significant difference observed in the animals' swimming speed in the water maze in the two studies that measured it, although the detailed data were unavailable [24,26].

Duration in original platform
Modeling methods For all five of the studies that regarded duration in original platform as an outcome measure, adopted electroacupuncture, and used Wister rats, we only conducted a subgroup analysis of the modeling methods (Fig. 2c). Both the EO models (SMD 1.56, 95% CI 1.02~2.11; p < 0.00001) and the 2-VO models (SMD 4.92, 95% CI 3.23~5.35; p < 0.00001) found a remarkable effect with the use of electroacupuncture, and this subgroup analysis significantly reduced the heterogeneity (EO: I 2 = 0.9%, 2-VO: I 2 = 0%).

Publication bias test
We performed a publication bias test for the outcome of escape latency using Egger's test (Pr > |z| = 0.001 < 0.05, continuity corrected) and Begg's test (Pr > |z| = 0.005 < 0.05, continuity corrected, Fig. 3). The results of these two tests indicated the potential existence of publication bias across all of the included studies [34]. We did not conduct a publication bias test for the other outcome measures, because there were fewer than ten included studies for each measure [35].

Signaling pathways
Of the sixteen included studies, fifteen described possible mechanisms of acupuncture in ameliorating cognitive function. The main signaling pathways were summarized in three aspects, oxidative stress damage reduction, nerve apoptosis suppression and neurogenesis. See Table 4 [36,37].

Discussion
To the best of our knowledge, this report describes the first systematic meta-analysis exploring the effect of acupuncture on vascular dementia in animal experiments with the results of the Morris water maze test as the outcome assessment.

Implications
This study indicates that electroacupuncture could increase the duration in the former platform quadrant both in EO models (SMD = 1.56, 95% CI: 1.02~2.11; p < 0.00001; heterogeneity: I-squared = 0.9%) and 2-VO models (SMD = 4.29, 95% CI: 3.23~5.35; p < 0.00001; heterogeneity: I-squared = 0%) compared with the control groups. This finding suggests that acupuncture may play a potential role in ameliorating cognitive dysfunction in animal models. A previous study indicates that the addition of acupuncture therapy to routine care may have beneficial effects on improvements in cognitive status but limited efficacy on health-related quality of life in vascular dementia patients [38]. The high heterogeneity between the studies based on escape latency time cannot be completely explained. We conducted subgroup analyses based on the method of stimulation, the modeling method and the animal species used, but no evident cause was found. Meanwhile, the subgroup analysis of the modeling methods that regarded the duration in former platform as an outcome measure obviously reduced the heterogeneity. We found all five of the studies in this analysis adopted the same method of stimulation, the same animal species, and similar acupoint combinations. Four of the five studies used CV6, CV12, CV17, SP10, and ST36, the other study used ST36. These findings indicate that, in addition to the differences in stimulation method, modeling methods and animal species used, acupoint combinations may be another source of heterogeneity among the studies. Systematic reviews can help promote the methodological quality of preclinical animal studies. Systematic reviews can help promote the methodological quality of preclinical animal studies. Essential methodological details are significant to measure the quality of a body of evidence and to assess the bias risk in animal trials. However, the insufficiency of the methodology is evident in many aspects of the present study. For instance, an adequate target animal model represents an important aspect to improve the quality of the experimental design. All included studies were performed on healthy and young animals. However, vascular dementia [39] generally occurs in aged, diabetic or hypertensive patients; therefore, experiments using young and healthy animals may overestimate the effectiveness of the intervention [10]. Hence, appropriate target animal models (hypertensive, advanced age and diabetic) should be used in future experimental research.
The included studies investigated several signaling pathways to gain a better understanding of the mechanism of improving cognitive function via acupuncture, including modulating the production and degradation of free radicals to reduce brain damage [11], exerting anti-apoptotic effects by up-regulating B-cell lymphoma-2 (Bcl-2) and counter-regulating Bcl-2-associated X protein (Bax) to protect neurons [14], enhancing glucose metabolism in the brain [16], reducing tumor protein P53 and Noxa expression to protect pyramidal cells from apoptosis [17], increasing cerebral blood flow for increased glucose and oxygen supply to neurons [21], exerting neuroprotective effects via an antioxidative pathway mediated by nuclear factor erythroid2-related factor 2 (Nrf2) [24], and increasing complex enzymes to protect neurons from oxidative stress [26]. The present study indicated that acupuncture protects neurons during vascular dementia mainly through enhancing oxygen and glucose metabolism and anti-apoptosis and antioxidant properties. However, the signaling pathways targeted by acupuncture were infrequently and incompletely reported. Therefore, this field should be further explored in future clinical studies.

Limitations
This systematic meta-analysis has several limitations, the first of which is language bias. We limited the language of our searches to English only, which may cause potential publication bias. The results may have differed if we had included studies reported in Chinese, Korean, Japanese or other languages. Second, the total sample size was still not big enough, although we made a concerted effort to search all of the studies that met the inclusion criteria. Third, the quality of the included studies was unsatisfactory, which had an important influence on the results of the systematic meta-analysis. Fourth, evident availability bias was caused by two included studies from which the data cannot be extracted [35]. We tried to contact the authors by e-mail, but we received no reply.  NO nitric oxide, NOS nitric oxide synthase, MDA malondialdehyde, GSH-Px glutathione peroxidase, AVP arginine vasopressin, SS somatostatin, Bcl-2 B-cell lymphoma-2, Bax Bcl-2 associated X protein, P53 Tumor protein P53, P70S6K P70 ribosomal protein S6 kinase, mTOR mammalian target of rapamycin, eIF4E eukaryotic translation initiation factor, Ach acetylcholine, DA dopamine, 5-HT 5hydroxytryptamine, CBF cerebral blood flow, PDE phosphodiesterase, ERK extracellular signal-regulated kinase, cAMP 3′,5′-cyclic AMP/protein kinaseA, PKA protein kinaseA, CREB cAMP/PKA/cAMP response element binding protein, Nrf2 nuclear factor erythroid-related factor 2, TNF tumor necrosis factor, IL interleukin, coxIV cytochrome oxidase IV, ROS reactive oxygen species Fifth, we performed Begg's test and Egger's test for publication bias assessment, and the results indicated potential publication bias. In view of the limitations above, we recommend that non-English language literature should be included in future systematic meta-analyses. Furthermore, blind outcome assessments, use of target animal models (hypertensive, advanced age and diabetic), calculation of sample size, statement of compliance with animal welfare regulations, possible conflicts of interest and detailed data publishing should be considered in future animal model studies.

Conclusions
From a methodological perspective, animal experiments should be standardly and appropriately designed and transparently reported. Acupuncture may play a potential role in ameliorating cognitive dysfunction in animal models. Furthermore, our findings indicates that acupuncture could protect neurons in animal models of vascular dementia through enhanced oxygen and glucose metabolism, as well as antioxidant and anti-apoptosis effects. Thus, this field should be further explored in vascular dementia clinical trials.