| IntroductionMemory is an area that everyone would like to improve. From supplements to exercises, products and services have been marketed to people with claims of helping boost mental capacity and memory; but few have been shown to be effective.Working Memory (WM) was originally described by Baddeley and Hitch. WM is understood as short-term memory (STM) plus attentional control. It consists of three constituent systems:a Central Executive (CE), which is in charge of allocating mental resources and attention; a Phonologic Loop; and a Visual Sketchpad where audio or visual data are kept in STM respectively.WM has been associated with reading comprehension and arithmetic calculation. The Span Tasks that have been developed to measure WM have been associated with predicting such diverse capabilities as:Note taking, language comprehension, playing bridge, learning a computer language, learning to spell, following directions, building vocabulary, writing, complex learning, and reasoning ability. Working memory capacity (WMC) can be a major factor in determining one's success in many areas. So WM is an area that most, if not all, people would want to improve.Anxiety has been shown to disrupt WM. Studies show it disrupts the ability of the CE to focus attention; and that it limits the ability of the CE to ignore extraneous stimuli; and that anxiety may impair all facets of WM and not just the CE.Anxiety has been shown to impair performance in math, reading and OSPAN task measures of working memory. Anxiety can also impair achievement in other areas as well, such as athletic performance. Acupuncture has been shown to reduce anxiety. Studies have shown that acupuncture can reduce generalized anxiety, depressive anxiety, and pre-operative anxiety. See Pilkington, Kirkwood, Rampes, Cummings, and Richardson for a review of the literature regarding acupuncture and anxiety.If anxiety impairs memory and acupuncture can reduce anxiety, can acupuncture improve memory? Some research has been done with mice, gerbils, monkeys, and rats who suffered cognitive impairment for a variety of reasons. Some research has also been directed at examining acupuncture's ability to help cognitively-impaired humans as well. But to date, no study has examined whether acupuncture can improve memory in healthy human subjects.This study examines the effect of one acupuncture treatment on State-Anxiety (SA) and performance on the Automated Operation Span Task (AOSPAN).MethodParticipants90Subjects were recruited from local universities. The Institutional Review Board of the National University of Health Sciences granted full approval for the study.Inclusion criteria was that all subjects must:Be undergraduate university students aged18-30; be wiling to receive acupuncture; have not received acupuncture in the past three months; be free of any serious medical problems; not be taking any psychoactive medication; not be pregnant or breastfeeding; and be fluent in English. All subjects who did not meet the inclusion criteria were excluded from the study. Subjects were compensated between$10-$20for their participation.SettingThe study was conducted at two private acupuncture clinics around Chicago, Illinois, USA. Consent and demographic questionnaires were filled out in the waiting room. The remainder of the procedures was performed in an8x10foot treatment room with a massage table, a laptop computer on a desk, and one chair.Instruments and Measures The State-Trait Anxiety Inventory (STAI) by Spielberger is a widely-used self-report anxiety instrument. It contains two separate20-item subscales that measure trait (baseline) and state (situational) anxiety. Form Y-1measures State Anxiety (SA) and form Y-2measures Trait Anxiety (TA). Each form contains20statements and subjects rate how much each statement reflects their true feelings. Twenty is the minimum score and80is the maximum. The STAI has shown test-retest reliability and external validity. It is one of the most widely-used anxiety measurement instruments in the world. Some authors even refer to the STAI as being the "gold standard" of anxiety measurement instruments.The Automated Operation Span Task (AOSPAN) by Unsworth et al is a computerized test of working memory that has shown good internal reliability and external validity. It provides several pieces of data to analyze. Subjects are presented with a math problem to perform in their head and then are shown a letter to remember. Then there is another math problem and another letter. After a series of between three and seven of these math-letter pairs, subjects must recall the letters they were shown in the correct order. There are a total number of75letters and math problems each. The AOSPAN Absolute Score and the Total Correct Score both reflect the recall of the letters. The Total Correct score counts all correct responses. Absolute Score only gives credit for letters recalled correctly when the entire set is recalled correctly. For example, in a set of seven math problems and letters, if a subject correctly recalls six of the letters, the Total Correct score would be six and the Absolute score would be zero. Studies that use the AOSPAN to measure memory most often compare the Total Correct Score. The AOSPAN also tracks performance on the math questions broken down into accuracy and speed errors.Stainless steel acupuncture needles from the DBC company were used for the Acupuncture group. The type of needle were Spring Singles, size0.20mm width by30mm length for body points; and0.20mm diameter by15mm length for points on the head.Independent VariableThe independent variable was whether or not the subject received acupuncture for20minutes at Sishencong (EX-HN1), Yintang (EX-HN3), Shenting (Du24), Shenmen (Ht7), Neiguan (PC6), and Taixi (Kd3) while they lay on a massage table for20minutes. All needles were inserted with even method. Sishencong (EX-HN1), was needled transversely with all needles pointing towards Baihui (Du20) at a depth of0.5cun. Shenting (Du24) was needled in an inferior direction to a depth of0.5cun. Yintang (EX-HN3) was needled transversely with an inferior direction to a depth of0.3cun. Shenmen (Ht7) was needled perpendicularly to a depth of0.5cun. Neiguan (PC6) was needled perpendicularly to a depth of1cun. Taixi was needled perpendicularly to join with Kunlun (UB60). Acupuncture was administered by an experienced, professional acupuncturist. Subjects in the control group had the same points touched and swabbed with alcohol but were not needled. Dependent VariablesThe dependent variables that were measured were the scores on the STAI and the AOSPAN.ProcedureSubjects were randomized by computer ahead of time into either Acupuncture or Control groups. All subjects were tested one at a time and had only one appointment to keep. At the start of the appointment, subjects had the study design partially explained to them and then they completed the Demographic Questionnaire and Informed Consent Forms.Next, all subjects were administered the STAI forms Y-1and Y-2. After the initial administration of the STAI, all subjects were instructed to remove their shoes and socks and lay on a treatment table. The treatment room employed soft lighting and relaxing music.Subjects in the Acupuncture Group then received acupuncture, at:Sishencong (EX-HN1), Shenting (Du24), Yintang (EX-HN3), Shenmen (Ht7), Neiguan (PC6), and Taixi (Kd3). All points were be needled bilaterally with even method and needles were retained for20minutes.Acupoints were chosen in an effort to calm the spirit and improve mental function. According to the Manual of Acupuncture by Peter Deadman et al.: Sishencong (EX-HN1), benefits the eyes and ears; calms the spirit; and is indicated for poor memory. Shenting (Du24) benefits the brain and calms the spirit. Additionally, the Du channel goes to the brain. Yintang (EX-HN3) calms the Shen and is indicated to calm anxiety and agitation. Shenmen (Ht7) calms the spirit, regulates and tonifies the Heart; and is indicated for poor memory, fear and fright. Additionally, the Heart organ houses the mind. Neiguan (PC6) is indicated for poor memory, apprehension, fear and fright. The Pericardium is the Heart Master and also benefits the Heart. Taixi (Kd3) was chosen because the kidney is associated with the brain and because its low position on the body balances the effect of all the points on the upper body. In this treatment, Taixi (Kd3) helps to ground the treatment and the subjects'consciousness.Subjects in the Control Group were laid on the same table for20minutes and had the acupoints touched and swabbed with alcohol, but were not needled. After this variable period, all subjects followed the same protocol.Subjects completed the Y-1form again. Then they were directed to a laptop computer where they received automated instructions in performing the AOSPAN. Subjects were informed that a strong performance on the test would enter them into a drawing for a cash prize and were encouraged to do their best.Care was taken to reduce the likelihood that subjects in the control group would realize that they were in the control group. Subjects were not told the order of events of the study. They were told,"You will fill out some self-evaluation questionnaires, take some computerized tests of memory, and may receive acupuncture at some point." When they were administered the AOSPAN, the researcher told them,"Now we will have you take the first memory test." This was intended to raise the possibility in the subjects'mind that they could still receive acupuncture before taking another memory test.The length of time that the researcher spent with each group and the amount of conversing and the amount of physical contact were kept uniform in order to minimize the placebo effect as described by Finniss et al, who showed that these differences can affect outcomes.Statistical analysisThe unpaired T-test was utilized for statistical comparison of mean values between Acupuncture and Control Groups, and between subgroups for STAI and AOSPAN data using www.graphpad.com online software. Regression analysis was performed to examine interactions between all measured parameters of STAI and all measured parameters of the AOSPAN using online software from www.easycalculation.com.ResultsControl group had46subjects:22males,24females, and a mean age of21.28years. Acupuncture group had44subjects:16males,28females, and a mean age of20.53years. There were no significant differences in gender makeup or age between the two groups. There were no adverse reactions reported from any subjects in the acupuncture group. Results are reported as mean value±standard deviation.STAIThe STAI yielded numeric values for:Initial State-level anxiety (SA1), Trait level anxiety (TA), and State-level anxiety after the variable period (acupuncture or rest, SA2). The difference between SA1and SA2was calculated and termed ASA. Unpaired t-tests were performed to compare Acupuncture and Control groups and subpopulations within groups.There were no significant differences in SA1or TA between the two groups. The mean ΔSA was-6.35±7.49in the Control group and-7.61±5.65in the Acupuncture group (p=-0.33, not significant). The mean SA2was29.63±8.2in the Control group and26.14±4.5in the Acupuncture group (p=0.0146, significant).The values obtained for TA ranged from23to63. Using a median split, subjects with TA below43were classified as Low-Anxious (LA, n=62) and those with TA at or above43were considered as High-Anxious (HA, n=28). Within the Acupuncture group, the reduction in SA was greater for those considered to be HA (9.93,±6.28, n=14) compared to those considered LA (6.64±5.11, n=30), but this was not quite statistically significant (p=0.0625).AOSPANThe AOSPAN provided numeric values for:Absolute Score, Total Correct Score, Total Math Errors, Math Accuracy Errors, and Math Speed Errors.Subjects who received acupuncture performed better than control. For the Total Correct Score, subjects in the Acupuncture group scored9.5%higher than those in the Control group (65.39±7.38compared to59.70±13.1, p=0.0134, significant), raising the percentage correct to87.2%for the Acupuncture group from79.6%for the Control group. The mean AOSPAN Absolute score was45.87±18.36(61.2%correct) in the Control group and52.20±14.28(69.6%correct) in the Acupuncture group (p=0.072, approaching significance). For the subgroup of males, the AOSPAN Absolute score was44.14±16.73in the Control group (n=22) and55.13±15.01in the Acupuncture group (n=16, p=.044, significant). The mean total number of math errors was36%less in the Acupuncture group (2.68±2.31) than the Control group (4.22±3.44, p=0.0153, significant). Mean number of math speed errors was1.24±1.59in the Control group and was0.80±1.3in the Acupuncture group (p=0.153, not significant). The mean number of math accuracy errors was2.98±2.52in the Control group and1.89±1.71in the Acupuncture group (p=0.0188, significant).Regression analysis was performed and no significant correlations were found between: STAI and AOSPAN scores.Overall, there was a trend that subjects with HA performed worse on the AOSPAN than LA subjects. When broken down into Control and Acupuncture groups, HA subjects performed below the LA subjects in the Control group; but this detriment was reduced or eliminated in the acupuncture group.There was also a trend noticed that the improvement in scores on the AOSPAN that was found in the acupuncture group was more pronounced for males than for females. Discussion20minute needle retention at points:Sishencong (EX-HN1), Shenting (Du24), Yintang (EX-HN3), Shenmen (Ht7), Neiguan (PC6) and Taixi (Kd3) improves memory and decreases anxiety immediately after administration. However, the improvement in memory was unrelated to both SA and ΔSA. This does not prove that acupuncture cannot improve memory through the reduction of anxiety. It is possible that another protocol could support such a hypothesis. This study shows that acupuncture may improve memory via a different mechanism than specifically through reduction of anxiety.This particular acupuncture treatment also resulted in a reduction of anxiety that was greater than resulted from rest alone, but that difference was not quite statistically significant (7.61points for Acupuncture group compared to the Control group's6.35). Due to the large standard deviation, this trend was not statistically significant. If only the final state-level anxiety is compared, the subjects in the acupuncture group had lower anxiety (p<0.05).This research also supports the existing evidence that HA individuals do not perform as well as LA on tests of working memory. In all categories that AOSPAN measures, HA individuals scored lower than their LA counterparts, although not significantly. When examined as a whole, HA subjects' Absolute score was11%less than HA's, the Total Correct Score was5.6%less, and they made33%more math errors compared to their LA counterparts. The use of the median split to define HA and LA groups has been questioned by Conway et al and it is true that there are some practical limitations to this procedure. The median split is still widely used though and in this case yields some interesting trends.The administration of acupuncture negated some of the deleterious effects of HA. When broken down between Control group and Acupuncture group, the effect of HA was much less for those who received acupuncture.Without acupuncture, HA subjects performed12.5%worse than LA on the Absolute Score, whereas with acupuncture the HA subjects performance was impaired by only7.6%. Total Correct Score for HA subjects was8.6%worse for Control subjects compared to LA Control, but for HA Acupuncture group the score was reduced by only1.5%compared to LA Acupuncture subjects. HA subjects without acupuncture made52.2%more math errors than LA without acupuncture, but HA subjects with acupuncture made13.2%fewer math errors than LA acupuncture subjects. In fact, HA subjects who received acupuncture scored higher on all measures of the AOSPAN than the LA subjects in the control group. While these findings are not statistically significant, this is likely due to the small sub-population size and may become significant in a larger study. The trend seems clear though. Untreated, HA impairs performance of WM. Acupuncture reduces or eliminates that impairment.It is not just the HA subjects that benefit though. Comparing LA Control to LA Acupuncture groups, the treatment group outperformed the control group in all measures with the exception of Math Speed Errors.Alternate research designsAnother way to design this study would be to have subjects take the AOSPAN before and after the variable period. For this study, this design was rejected because it was felt that taking the test twice resulted in measuring subjects' ability to learn a task rather than testing memory. Additionally, the AOSPAN requires subjects to go through a lengthy practice period where their response time is measured (unbeknownst to the subjects at the time) and then used as the basis for timing the actual measures of the test. In a second administration, subjects would know their response times were being measured and could manipulate them in order to give them more time during the actual test. If it were possible to alter the AOSPAN so that the first round practice time measurements could be used for the second round of testing; and that the second round could begin without the practice sessions, then it may be possible to run the study this way. However, no data exists testing the reliability nor validity of the AOSPAN's measurement when administered a twice in such a short time span.Another way to design this study would be to have subjects take the AOSPAN, then receive six to twelve weeks of treatment, and then retake the AOSPAN. This may give subjects sufficient time to forget the procedure and yield valid measures on the second administration of the AOSPAN.Both the one-time protocol and the6-or12-week protocols could be performed using different points. This study focused on the Heart, Pericardium, Kidney, Du channels and extra points on the scalp. The Spleen is also involved in cognition, particularly in its association with the Yi, which is roughly translated as the Will. The addition of Sanyinjiao (Sp6) or other points on the Spleen channel are worthy of investigation. There is no reason to believe that another protocol could not have a greater effect. Auricular points with acupuncture or acupressure could also be investigated. Acupressure could be compared with acupuncture.Baihui (Du20) has similar functions and indications as Sishencong (EX-HN1). Replications could substitute Baihui for Sishencong (EX-HN1), and investigate whether one scalp needle would be as effective as four. In fact, this study could be replicated with any or several points eliminated to investigate how minimal an intervention could be used to gain the same result. Perhaps only a few needles are necessary.This experiment could also be run using different populations. There is no reason to assume that the beneficial effects shown in this study are limited to subjects under the age of30. Future research could examine a broader age range such as18-65, or just look at seniors (65or older), or any subgroup.Why no sham group? Sham acupuncture does not exist. It is not an inert intervention. The two most common methods for administering sham acupuncture are either cutaneous stimulation/superficial needling or needling points away from major (or indicated) acupuncture points.Superficial needling still affects the flow of energy in the channels. Superficial needling has been shown to induce physiologic changes in the limbic system and those changes are different between different subject types. For example, superficial needling will increase activity within the limbic system in subjects who have no pain, but it will reduce it in those with pain. Some studies have shown that sham acupuncture is as effective as true acupuncture; and that both are more effective than placebo medication. Other studies have shown that the addition of both verum and sham acupuncture to standard medication provide superior benefit than standard medication alone, but that the addition of verum acupuncture is more beneficial than sham. Others have shown that, while both sham and verum may be beneficial, that they may work through different mechanisms. At least one study even concluded that sham was more effective than verum.Secondly, there is no place on the body into which you can insert a needle that is energetically inert. There are the defined acupoints, but every spot on the body lies within an acupuncture meridian and will therefore have some effect on the energy flow of the subject. Studies have shown an effect where placebo (or no treatment) has some degree of effect, sham acupuncture has more effect, and true acupuncture has even more effect.Lundeberg et al present an excellent review of the literature regarding so-called "placebo" acupuncture. They explained that minimal acupuncture does cause physiologic changes as measured by fMRI. They conclude that placebo acupuncture does not serve to elucidate the effects of acupuncture but rather introduce a potential bias, which interferes with understanding acupuncture's true effects. Did the subjects in the Acupuncture group know that they were in the treatment group? Yes. Did those in the Control group know that they were in the Control group? No. Is it possible that those in the treatment group knew that they were supposed to do better and therefore did perform better on the AOSPAN? Perhaps. All subjects were told that a strong performance on the AOSPAN would enter them into a drawing for a cash prize, so it is assumed that all subjects gave their best effort. It is possible that knowing they had received a treatment gave Acupuncture subjects greater confidence and focus. For this reason, future studies may incorporate a sugar pill for the control group. With this addition, everyone may think that they had received some sort of treatment prior to taking the AOSPAN.Acupuncture has been shown to be an effective treatment for anxiety. This study showed its effect on state level anxiety, but does not address the trait anxiety. The common interventions for addressing anxiety in the long term are medications and psychotherapy. Acupuncture is clearly a better choice than anti-anxiety medications because it carries much less risk of side effects and has been studied long enough to ascertain its safety as a long-term therapy. Acupuncture may be used in conjunction with psychotherapy.Lastly, the aging population of the world is experiencing profound problems with memory loss. Future study should focus on using this treatment and other point combinations to affect and improve memory and cognitive function in the elderly.Acknowledgements:The author would like to thanks to my support team in China:Dr. Xu Nenggui for taking me on as a student and advising me with great patience and wisdom. Thanks to Dr. Li Min, our liason, friend and great helper. And thanks to the Guangzhou University of Chinese Medicine. Thanks also goes to the support team in the USA:Dr Hui Yan Cai, Dr. Patricia Rush, Jeanie Bussell, Christopher Martiniano, Judith Schlaeger, Tricia Miller, Melissa Lee Grein, Jia Xu, Long Huynh, and Julia Kravitz, in addition to my family. Thanks to National University of Health Science and Judy Pocious for their assistance with Institutional Board Review and approval.
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