Acupuncture for behavioral changes of experimental depressive disorder: a systematic review and meta-analysis

Acupuncture is considered to be a promising alternative therapy for depression. Nevertheless, up to now, it remains controversial on the effects exerted by acupuncture on behavioral changes in depression models. Consequently, it’s necessary to develop a systematic review and meta-analysis to assess the effect of acupuncture for model rats of depression. Ultimately, 90 studies involving 1861 models were identified. Behavioral indicators including the number of crossings (NC) and the number of rearings (NR) in open field test (OFT), the capacity of sucrose intake (CSI) and the rate of sucrose intake (RSI) in sucrose intake test (SIT), final weight (FW) and gain weight (GW) were employed as main outcomes in depression model rats. The pooled results showed that acupuncture had not less effect than western medicine on improving NC, NR, FW, GW, RSI (P > 0.05). However, the CSI improvement was poorer compared with west medicine (P < 0.05). In conclusion, acupuncture has not less effect on behavior changes than western medicine, including improving NC, NR, RSI, FW and GW in depression models.

Study quality and publication bias. The score of each study ranged from 2 to 7 out of a total 10 points quality checklist. Eleven studies got 2 points, forty-one studies got 3 points, twenty-nine studies got 4 points, eight studies got 5 points, one study got 6 points, two studies got 7 points (Table 2). Twenty-four studies utilized anesthetics which have no effect on depressive symptoms. Forty-four studies described the control of temperature, including control of the room and rats' anal temperature. Fifty-six studies were published in peer-reviewed journals. None of the studies described the sample size calculation, allocation concealment, blinded assessment of outcome. Twelve studies described compliance with animal welfare regulations and three studies declared potential conflicts of interest. Only two studies did not describe randomization.   Liu 2012 46 CUMS  SD rats  Male  200-250  8/8  mention  not  mention   NOT  SAME  EA  LI4,LR3  mention  30 min  10  21d  Fluoxetine  OFT:NC,NR;  x W:FW; SI:CSI.   Lu 2008 47  CUMS &  Solitary  SD rats  Male  200 ± 20  8/8  mention  not  mention  SAME EA  GV20,GV29,  ST36,ST40  mention  15 min  11  22d  Maprotiline  OFT:NC,NR;  W:FW; SI:CSI.   Lu 2013 48  CUMS  SD rats  Male  180-200  10/10 mention  not  mention  SAME MA  GV20,PC6  mention  10 min  14  28d  Paroxetine  OFT:NC,NR;  W:FW; SI:RSI.   Lu 2016 49  CUMS  SD rats  Male  180-200  8/8  mention  not  mention  SAME MA  GV20,PC6  mention  10 min  14  28d  Fluoxetine  OFT:NC, Outcome and effect estimates. Based on various behavior indicators of the included studies, different pooled data of 69 studies in 67 articles were applied in meta-analysis and 21 studies in 20 articles were not applied in meta-analysis because they did not provide raw data. The data lacked details like observing time or specifications were also not pooled in the analysis because it indicated that the data is not precise enough. All continuous data were presented as mean ± SD.
Investigation of heterogeneity. Sensitivity analysis: sensitivity analysis showed that heterogeneity reduced to be acceptable after removing two studies, three studies and one study of which coincidence degree was poor among other studies pooled in the meta-analysis of NR, CSI and RSI, respectively. So, to a large extent, these studies may be the origination of heterogeneity in NR, CSI and RSI. Tests for overall effects in these cases showed that acupuncture had not less effect than western medicine on improving NR (Z = 1.14 (P = 0.25)), CSI (Z = 1.00
Scientific RepoRTS | 7: 9669 | DOI:10.1038/s41598-017-09712-1 (P = 0.32)), RSI (Z = 0.68 (P = 0.50)) in depression model rats. The heterogeneity was not substantially altered after dismissing any study pooled in the meta-analysis of NC and GW. Subgroup analysis: to further investigate the source of heterogeneity among the studies pooled in the meta-analysis of NC and GW, a subgroup analysis was conducted. Study characteristics, including different types of acupuncture (acupuncture or electroacupuncture), different stimulation acupoints (scalp acupoints, body acupoints, or scalp acupoints and body acupoints), different intervention time (modeling and intervention at the same time or intervention after or in the process of modeling) and different duration of treatment (more than 21d or less than 14d) are thought to be potential factors affecting the effect of acupuncture. The subgroup analysis was done on the basis of these characteristics. It's failed to subgroup the results by acupuncture method and duration on the basis of GW, since only one study selected manual acupuncture and all the studies' duration were more than 21d. Unfortunately, we did not find the exact source of heterogeneity among studies by subgroup analysis.
Sensitivity analysis and subgroup analysis were not developed because heterogeneity among studies that were pooled in the meta-analysis of FW (I 2 = 35% (P = 0.01)) is acceptable. Details of sensitivity analysis and subgroup analysis were shown in Table 4 and the raw data were shown in Figs S1-S9. Assessment of publication bias. Funnel plot showed asymmetry and it indicated a potential publication bias (Fig. 8). Begg's test showed there was no significant publication bias (p = 0.548), but the Egger's test (p < 0.001) indicated publication bias possibly existed (Fig. S10). In addition, all of the studies published by Chinese authors and 47 studies published in Chinese journals [11][12][13]15

Discussion
Efficacy of acupuncture. To our knowledge, this is the first systematic review and meta-analysis of English and Chinese literatures to investigate the efficacy of acupuncture for animal model on depressive disorder with behavioral changes as the outcome. The present study indicate that compared with western medicine, acupuncture has not less effect on behavior indicators including NC, NR, RSI, FW and GW for depression model rats, but the evidence on whether acupuncture is as effective as western medicine for depression model rats on CSI   effective in treating functional dyspepsia 98 , in which poor appetite is one of the core symptoms. In our analysis, Baihui (GV20) and Yintang (GV29) were most frequently used, while acupoints of stomach are scarcely used. Therefore, we consider that acupoints may be the main influencing factor in effecs of acupuncture on symptoms of depression.

Explanations for indicators, models and interventions.
In depression research, the OFT is a commonly indicator to assess the general locomotor activity and willingness to explore 9 . The SIT is a behavioral indicator that can assess the degree of anhedonia in models 10 . In addition, obvious change in weight is one of the core depressive-like symptoms. Moreover, some studies had doubt about the effects of acupuncture on these indicators in depression models 3,7,8 , which suggesting whether acupuncture has effects on these indicators needs to be resolved. Therefore, we selected NC, NR, CSI, RSI, FW and GW as behavioral indicators to evaluate the effects of acupuncture on depressive-like symptoms.   95 . Four studies made models on the basis of underlying pathogenesis of depression, included IA models 22 (IA can simulate effects of the endogenous excitatory amino acids on the onset of depressive symptoms), L-AAA models (L-AAA can destroy astrocytes) 64 , EAAT1 antagonist models and EAAT2 antagonist models 77 (EAAT1 and EAAT2 antagonist can inhibit functions of glutamate transporter). Results of these studies showed that the models were all successful. Nevertheless, it needs further researches to demonstrate which models have the highest validity, reliability and utility.
A Cochrane review has suggested that there is not enough evidence to demonstrate acupuncture is effective for depression and it has recommended future research should employ comparative designs and compare acupuncture with structured psychotherapies such as standard care 101 . Therefore, we compared acupuncture with western medicine, which can be considered as standard treatment in animal models at present. Current available anti-depressants are limited by lagged therapeutic time and undesirable side effects including acute nausea and headaches as well as chronic sexual dysfunction, weight gain, and diminished REM (rapid eyes movement) sleep 2 . We expected that acupuncture may have not less effect than anti-depressants on depression and have an advantage on less side effects 5, 102 , which is helpful for patients to adhere to the treatment.
Mechanisms of acupuncture. The mechanisms of acupuncture on depression remain unclear. Main possible mechanisms of acupuncture include: (1) regulating the level of neurotransmitter such as monoamine neurotransmitter (5-HT, DA, NE 11,71,84,93 ), Glu and GABA; (2) regulating neuroendocrine system (HPA axise 88,93 , HPT axise 52 , MT 59 ); (3) regulating inflammatory cytokines such as IL-1β, IL-2, IL-6, TNF-α, NO, PGE2, iNOS, COX-2, NF-kB 21,27,49 ; (4) promoting neuronal regeneration and neurotrophy through several signaling pathways (including BDNF, CREB, PKA, JNK, ERK, etc) 15,19,25,32,37,46,89 . In addition, acupuncture can modulate brain-gut peptide hormone (including GAS, NPY, CGRP, NT, etc) 55, 69 , inhibit over-activation of RAS and suppress oxidative stress 59,62 . We consider that acupuncture treat depression by multi-targets through neuroendocrine-immune system and the future researches should enlarge the sample size and further clarify the pathophysiological effects of different acuopints or acupoint combinations. Table S1, acupoints that were most frequently used (over 10 studies) include scalp acupoints (GV20, GV29) and body acupoints (LR3, PC6, LI4, SP6). We expected that acupoints may be a potential source of heterogeneity among these studies. Hence, subgroup analysis according to different stimulation acupoints (scalp acupoints, body acupoints, or scalp acupoints and body acupoints) based on NC and GW was conducted (Table 4). Nevertheless, results showed that different stimulation acupoints may not be the source of heterogeneity. Due to some unexpected influences of acupuncture parameters (such as the stimulation dose of acupuncture, different meridians, and so on) and small sample size of included studies, it is difficult to deny acupoints' contribution to the heterogeneity among the included studies.

Inspirations for further studies. As shown in
It is also interesting to find that methods for selection of acupoints included on the basis of traditional Chinese medicine theory (49 studies), clinical experience (6 studies), preliminary clinical and/or experimental researches (19studies), summary of previous research reports (15 studies). This means that it doesn't have a standard theoretical guidance for selection of acupoints. We suggest that future studies should clarify the physiological effects of different acupoints or acupoint combinations and the differences in their effects on depression. Decreased IL-1β, IL-10 in hippocampus.

Ji 2013 30
Increased the GFAP and astrocyte in hippocampus.
Decreased CRF in hypophysis.
Down-regulated expressions of CRF 1 mRNA, POMC mRNA in hypophysis.
Up-regulated expressions of CREB,BDNF in hippocampus.
Jing 2016  Increased the mRNA and protein expression of IL-1β, IL-6, and TNF-α in the hippocampus and prefrontal cortex and cytokine concentrations in serum.
Luo 2016 51 Regulated GS,EAAT1,EAAT2 of astrocyte cell in prefrontal cortex. Table 3. Proposed mechanisms of acupuncture in behavior changes in depression models of studies included. Notes: β-EP, β-endorphin; p-CREB, phosp-horylated cAMP response element binding protein; 5-HT, 5-hydroxytryptamine; 5-HT1A: 5-hydroxyindoleacetic acid; 5-HTT, 5-hydroxytryptamine transporter; ACEI, Angiotensin converting enzyme I; acH3K9, acetylation of histone 3 lysine 9; ACTH, adrenocorticotropic hormone; AIF, apotposis inducing factor; AngII, angiotensin II; AT1R, angiotensin II Type 1 Receptor; Bax, Bcl-2 Assaciated X protein; Bcl-2, B-cell lymphoma 2; BDNF, brain derived neurotrophic factor; bFGF, basic fibroblast growth factor; CaMKII, calcium-calmodulin-dependent protein kinase II; cAMP, adenosine 3' , 5'-cyclic monophos-phate; CGRP, calcitonin generelated peptide; CNTF Rα, ciliary neurotrophic factor Methodological quality of the included studies was generally low and only one of them got 6 points and two got 7 in our assessment. Randomization, blinding and sample-size estimation serve as the core standards of rigorous study design. In our analysis, only two studies did not describe randomization, but none of the studies described the sample size calculation, allocation concealment, blinded assessment of outcome. Blinding should also be utilized in intervention process. Research shows that the visual impact of needling is a potential factor that leads to the placebo effect of acupuncture 103 . Therefore, we suggest that animals should be prevented from seeing themselves or their companions being needled in intervention process. And more attention should be paid to the methodological quality in future studies.
In the sensitivity analysis, we considered that Jing2016 36 , Fan2013 21 were the major sources of heterogeneity in NR, Zhang2008 88 , Fan2013 21 ,Yang2013 81 were the major sources of heterogeneity in CSI and Sun2016 66 were the major sources of heterogeneity in RSI. We found that quality of Zhang2008 88 , Fan2013 21 and Yang2013 81 are considerable poor (one got 2 points and the other two got 3 points). We believe that avoiding publication bias and improving methodological quality of studies play an important role in reducing the heterogeneity in studies.
In the subgroup analysis, we did not find the exact factor that accounts for the heterogeneity among studies pooled in the meta-analysis of NC or GW. We also developed the meta regression and the result showed that factors of acupuncture treatment did not account for the heterogeneity in studies (Figs S11 and S12). More possible factors that can influence outcomes, such as experimental models, should be considered in later researches to interpret the heterogeneity among studies.

Sensitivity analysis
Removed studies Changes of I 2 (P)

Conclusions
Acupuncture has not less effect than western medicine on behavior indicators including NC, NR, FW, GW, and RSI. These indicators can be utilized to evaluate acupuncture effectiveness in experimental depressive disorder. Nevertheless, the results of this meta-analysis need to be interpreted with caution and high-quality researches are urgently needed. Future studies should pay more attention to the methodological quality, especially in sample size calculation, allocation concealment, blinding in intervention and assessment. Additionly, we suggest that future studies should further clarify the physiological effects of different acupoints or acupoint combinations and the differences in their effects on depression. Data extraction. We extracted the key contents of the studies in reference to the ARRIVE guideline 104 , and the following details were extracted: (1) publication year and the first author's name; (2) animal species, sex, weight; (3) the randomization and blinding; (4) interventions and modeling; (5) outcome indicators. When studies set up two or more acupuncture groups, the group which had greater effects on the outcome indicators was extracted. All continuous data of outcome indicators which was presented as mean ± standard deviation (SD) were extracted if reported. Missing data or further information was sought from the primary authors via e-mail if necessary.

Methods
Quality assessment. We evaluated the methodological quality of the included studies by applying the list of Collaborative Approach to Meta-Analysis and Review of Animal Data from Experimental Stroke (CAMARADES) 105 which is modified on the basis of the characteristics of acupuncture treatment in depression researches when assessing the quality of studies, the items included: (1) sample size calculation; (2) randomization to treatment group; (3) allocation concealment; (4) blinded assessment of outcome; (5) correctness of methods of modeling; (6) avoidance of anesthetics with resistance to depressive; (7) describing control of temperature; (8) compliance with animal welfare regulations; (9) publication in a peer-reviewed journal; (10) declared any potential conflict of interest. For the calculation of an aggregate quality score, each item was attributed one point. Two authors independently extracted data and assessed study quality. Disagreements were solved after discussion over the details of the studies. Statistical Analysis. Meta-analysis and subgroup-analysis were performed by RevMan V.5.3, and analysis of publication bias and Meta-regression were conducted with STATA/SE 12.0. We considered all behavioral indicators as continuous data, and then an estimate of the combined effect sizes utilizing standard mean difference (SMD) with the given random effects model. We used the random model rather than fixed since heterogeneity between multi-studies has to be taken into account. Publication bias was assessed with a funnel plot and Egger's test. To assess heterogeneity, the I 2 statistic was used. To clarify the impact of factors potentially modifying the outcome measures, we also conducted sensitivity analysis and subgroup analysis according to the following variables: different types of acupuncture, different stimulation acupoints, different intervention time, different duration of treatment. The difference between groups was assessed by partitioning heterogeneity and using the χ 2 distribution with n-1 degrees of freedom (df), where n indicate the number of groups. Probability value p < 0.05 was considered significant. Data Availability. All data generated or analysed during this study are included in this published article (and its Supplementary Information files).