Laser Acupuncture at HT7 Improves the Cerebellar Disorders...

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Available online at www.sciencedirect.com

Journal of Acupuncture and Meridian Studies

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J Acupunct Meridian Stud 2017;--(-):--e--

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RESEARCH ART ICLE
72 737475767778798081828384
Laser Acupuncture at HT7 Improves theCerebellar Disorders in Valproic Acid-RatModel of Autism

Jurairat Khongrum 1,2, Jintanaporn Wattanathorn 2,3,*
85868788899091
1 Department of Physiology (Neuroscience Program), Faculty of Medicine, Khon KaenUniversity, Khon Kaen, Thailand2 Integrative Complementary Alternative Medicine Research and Development Center, KhonKaen University, Khon Kaen, Thailand3 Department of Physiology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand

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Received: Feb 6, 2017Revised: Jun 5, 2017Accepted: Jun 7, 2017

KEYWORDS

autism;GABAergic function;IL-6;laser acupuncture at

*

104105106107

PA

It

Y

HT7 acupoint;oxidative stress;Purkinje cell

Corresponding author. DepartmentDevelopment Center, Khon Kaen UE-mail: [email protected], jint

108109110111112

lease cite this article in press as: Khcid-Rat Model of Autism, Journal of

SSN 2005-2901 eISSN 2093-8152tp://dx.doi.org/10.1016/j.jams.2012017 Medical Association of Pharma-NC-ND license (http://creativecom

AbstractThe novel therapeutic strategy against autism is essential due to the limited therapeuticefficacy. Based on the benefit of laser acupuncture at HT7 acupoint on the neurologicaldisorders related with oxidative stress and inflammation, its benefit on oxidative stress,neuroinflammation, and GABAergic/glutamatergic imbalance in cerebellum of autismhave been considered. To elucidate this issue, male rat pups were induced autistic-likeconditions by valproic acid (VPA) and treated with laser acupuncture at HT7 acupointonce daily between postnatal Day 14 and Day 40. At the end of study, the changes ofoxidative stress markers, the expressions of cytokines interleukin 6 (IL-6) and glutamicacid decarboxylase (GAD) proteins (65 kDa and 67 kDa) together with gamma-aminobutyric acid transaminase (GABA-T) activity and density of Purkinje cell in the cer-ebellum were assessed. The results showed that laser acupuncture HT7 decreased oxida-tive stress, IL-6 expression, and GABA-T activity but increased the expressions of GAD 65kDa together with the density of Purkinje cells in the cerebellum. Therefore, laseracupuncture at HT7 is the potential strategy to improve the cerebellar disorders inVPA-rat model of autism. The mechanism may occur partly via the decrease of oxidativestress status, inflammation, and the improved GABAergic function.

of Physiology, Faculty of Medicine and Integrative Complementary Alternative Medicine Research andniversity, Khon Kaen 40002, Thailand. Q2 Q3

[email protected] (J. Wattanathorn).

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ongrum J, Wattanathorn J, Laser Acupuncture at HT7 Improves the Cerebellar Disorders in ValproicAcupuncture and Meridian Studies (2017), http://dx.doi.org/10.1016/j.jams.2017.06.006

7.06.006copuncture Institute, Publishing services by Elsevier B.V. This is an open access article under the CCmons.org/licenses/by-nc-nd/4.0/).

mailto:[email protected]

mailto:[email protected]

http://dx.doi.org/10.1016/j.jams.2017.06.006

http://creativecommons.org/licenses/by-nc-nd/4.0/

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1. Introduction

In recent decades, numerous lines of evidence point outthat the cerebellum is one of the key brain regions affectedin autism [1] and may serve as an important etiology ofautism [2e5]. Data obtained from postmortem studies haverevealed that most autistic patients show cerebellar hy-poplasia and the decreased number of Purkinje cells [6e8].In addition to the structural changes of the cerebellum, thereduction of the GABAergic function [9,10] and theincreased oxidative stress status [11,12] in the cerebellumare also reported. It has been suggested that the cerebellardisorders are linked to sensorimotor, language, and socialinteraction deficits in autism [13].

Despite the advancement of technology, no therapeuticstrategy can cure autism. Most current therapeutic strate-gies focus on the symptomatic treatment. Therefore, thenovel effective therapeutic strategy is still required.Accumulative lines of evidence have demonstrated thatlaser acupuncture, an alternative stimulation acupoint withlaser light, can improve many neurological disorders such asthe early phase of Alzheimer’s disease [14] and Parkinson’sdisease [15]. In addition, our previous work has demon-strated that laser acupuncture at HT7 acupoint can miti-gate autistic-like symptoms partly via improved oxidativestatus [16]. As the cerebellum has been proposed to play animportant role on the pathophysiology of autism, it isinteresting to understand the effect of laser acupuncture atthe HT7 acupoint on both structural and functional changesof the cerebellum. Therefore, this study aimed to deter-mine the effect of laser acupuncture at the HT7 acupointon the structural and biochemical changes of the cere-bellum in valproic acid-rat model of autism. The possibleunderlying mechanism was also investigated.

2. Materials and methods

2.1. Animals and experimental protocol

All experimental protocols used in this study had beenapproved by the Institutional Animal Care and Use Com-mittee Khon Kaen University, Khon Kaen, Thailand (AEKKU56/2556). On postnatal Day 14, the experimental animalswere housed together in a cage, maintained in a 12:12 hourlightedark cycle, and given ad libitum access to food andwater.

2.2. Experimental protocol

Male rat pups (18e30 g) were induced with autistic-likesymptoms by valproic acid or VPA (Sigma-Aldrich, USA) at adose of 400 mg/kg BW via intraperitoneal injection on postnatal Day (PND) 14 [16,17]. Then, the experimental animalswere divided into four groups (n Z 6) as following: (1)Group I, control group: rat pups received no treatment; (2)Group II, VPA group: rat pups were injected VPA alone; (3)Group III, VPA þ sham laser acupuncture group: rat pupswere injected VPA and received laser acupuncture treat-ments at a point 2e4 mm lateral to HT7; and (4) Group IV,VPA þ laser acupuncture HT7 group: rat pups were injected

Please cite this article in press as: Khongrum J, Wattanathorn J, LaserAcid-Rat Model of Autism, Journal of Acupuncture and Meridian Studi

VPA and received acupuncture treatment at the HT7 acu-point bilaterally.

Between PND14 and PND40, rats in Groups IIeIV weresubjected to the assigned treatments once daily. On PND41, they were sacrificed and the cerebellum hemisphereswere isolated for determining oxidative stress markers,including malondialdehyde (MDA) level and the activities ofsuperoxide dismutase (SOD), catalase (CAT), and gluta-thione peroxidase (GSH-Px) enzymes. Moreover, Purkinjecell’s density, gamma aminobutyric acid transminase(GABA-T) activity and the expressions of glutamate decar-boxylase and interleukin-6 (IL-6) were also investigated.The schematic diagram showing the experimental protocolsis presented in Fig. 1.

2.3. Laser acupuncture treatment

Five minutes prior to treatment, the rat pups were sepa-rated and housed individually. A continuous blue laser beam(Xinland International Limited, Hongguang Rd, Lianhu Dis-trict, Xi’an, Shaanxi, China) with a wavelength of 405 nm,an output power of 100 mW (0.100 J/s), and a spot diam-eter of 500 mm was applied to each animal for 10 minutesonce daily between PND14 and PND40. The animals inGroup IV received laser application at HT7 acupoint (thetransverse crease of the wrist of the forepaw, radial to thetendon of the muscle flexor carpi ulnaris) whereas those inGroup III were exposed to a laser at a point 2e4 mm lateralto the HT7 acupoint [18].

2.4. Preparation of tissue homogenate and proteindetermination

After the last laser administration, the rat pup was anes-thetized with intraperitoneal injection of sodium pento-barbital. Cerebellum hemisphere was isolated and keptcool and prepared as cerebellar homogenate. In brief,cerebellar hemisphere was homogenized in 4% of 0.2M PBSbuffer pH 7.4 and subjected to a 12,000 rpm centrifugationQ

at 4�C for 15 minutes. The supernatant was harvested andproteins determined by the method of Lowry [19] usingbovine serum albumin as standard solution.

2.5. Determination of oxidative stress status

To determine the oxidative stress status, the de-terminations of malondialdehyde (MDA) level and the ac-tivities of main scavenger enzymes such as superoxidedismutase (SOD), catalase (CAT), and glutathione peroxi-dase (GSH-Px) were carried out.

The measurement of malondialdehyde (MDA), a lipidperoxidation process marker, was performed using the thio-barbituric acid reactionmethod. In brief, 750mL of 20%aceticacid, 100 mL of 8.1% sodium dodecyl sulfate, and 750 mL 0.8%of thiobarbituric acidweremixedwith 100mL of supernatant.Then, the mixture was boiled in a water bath at 95

�C for 1

hour. After cooling to the room temperature, 500 mL ofdistilled water (DW) and 2500 mL of n-butanol and pyridinewere added and centrifuged at 4000 rpm for 10 minutes.Absorbance was determined at wavelength 532 nm viaspectrophotometer. MDA level was calculated using a series

Acupuncture at HT7 Improves the Cerebellar Disorders in Valproices (2017), http://dx.doi.org/10.1016/j.jams.2017.06.006

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Figure 1 Schematic diagram showing all experimental procedures. (A)VPA injection schedule for autistic-like condition induc-tion. (B) Experimental protocol of laser acupuncture and the determination of various parameters. IL-6 Z interleukin 6; PND Zpost natal day; VPA Z valproic acid.

Laser Acupuncture at HT7 Improves the Cerebellar 3

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of standard solution of 1,1,3,3-tetramethoxypropane andexpressed as nmol/mg protein [20].

SOD activity was assessed based on the reaction in axanthine/xanthine oxidase system. In brief, 20 mL of su-pernatant and 20 mL of 5 units XOD were mixed with 200 mLof the solution containing 0.1M PBS pH 7.8, 10.7mM EDTA,1.1mM cytochrom C and 0.108mM xanthine. The absorbanceat 550 nm was measured via spectrophotometer and theactivity of SOD was presented as units per milligram ofprotein [21].

Catalase activity was measured based on the rate ofH2O2 decomposition. In brief, 10 mL of supernatant wasmixed with 150 mL of 50mM potassium phosphate, 25 mL of5N H2SO4, and 50 mL of 0.1M H2O2 at room temperatureunder dark conditions. Absorbance at 490 nm was readusing a spectrophotometer. Activity was presented as unitsper milligram of protein [22].

GSH-Px activity was determined as previous described[16] based on the original method [23] with a slight modi-fication. In brief, 10 mL of sample supernatant was mixedwith the solution containing 10 mL of 48mM sodium phos-phate, 0.02mM DL-dithiothreitol, 100 mL of 0.95mM sodiumazide, 10 mL of 1mM glutathione, and 100 mL of 30% H2O2.Then, the mixed solution was shaken for 10 minutes beforeadding 10 mL of DTNB. The absorbance at 412 nm wasrecorded using a spectrophotometer and GSH-Px activitywas presented as units per milligram of protein.

2.6. Determination of GABA transaminase activity

The cerebellum hemisphere was homogenized with coolbuffer consisting of 0.5% Triton X-100, 5mM dithiothreitol,1mM pyridoxal phosphate, and 10mM sodium phosphate


buffer pH 7. The homogenate was centrifuged at 2000 rpmfor 20 minutes at 0�C. The supernatant was harvested andused for the determination of GABA transaminase (GABA-T). In brief, 10 mL of supernatant was mixed with 40 mL ofsolution containing 20mM GABA, 10mM a-ketoglutarate,and 0.5mM NAD in 0.05M sodium phosphate buffer pH 8.0.After the 30-minute incubation at 30�C, the absorbance at340 nm was recorded using a spectrophotometer. Theenzyme activity was presented as mMol/milligram ofprotein [24].

2.7. Western blot analysis

The cerebellar hemisphere was homogenized in M-per re-agent containing protease inhibitors (Thermo Fisher Scien-tific, Pierce Biotechnology, Inc., USA Q). The homogenate wassubjected to a 20-minute centrifugation (10,000 rpm) at4�C. Supernatant was collected for determining total pro-tein using NANO drop spectrophotometer (NanoDrop ND-1000 Spectrophotometer V3.5 User’s Manual, NanoDropTechnologies Inc., USA Q). The supernatant protein wasseparated via 10% sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDSePAGE) and transferred onto apolyvinylidene difluoride (PVDF) membrane (Bio-Rad Labo-ratories, USA Q). The membrane was incubated in a blockingbuffer (5% skim milk in Tris-buffer saline with 0.05% Tween-20) for 1 hour at room temperature. After the blocking, themembrane was incubated with primary antibody for 2 hoursat room temperature (1:5000 anti GAD65/67 or 1:2000b-actin, Sigma-Aldrich) followed by a 1-hour incubation withsecondary antibody (HRP conjugated antirabbit IgG 1:2000,Abcam, USA Q). The expression of GAD 65/67 was visualizedby using the ECL plus substrate (Thermo Fisher Scientific,


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Pierce Biotechnology, Inc.) and ImageQuant LAS 4000 (GEHealthcare, UK). Band density was analyzed using Image-Quant TL software (GE Healthcare) [25].

2.8. Immunohistochemical evaluation

Frozen sections of cerebellum hemisphere were cut 10 mmthick. The slides were washed with 0.1M phosphate buff-ered saline (PBS) pH 7.4 for 5 minutes and exposed to highpower (800 watts) microwave for 5 minutes. The blocking ofendogenous peroxidase activity was performed by incu-bating sections with 0.5% H2O2 in methanol for 10 minutes.Then, the sections were incubated with mouse monoclonalIL-6 antibody (1:100, Abcam) at 4�C overnight followed by a1-hour incubation with secondary biotinylated goat anti-mouse IgG antibody (1:2000, Abcam) at room temperature.The immunoperoxidase reaction product was visualized byusing a 30-minute incubation at room temperature withDAKO kit (The DAKO EnVisionþ System, Biocompare Inc.,USA). The color was developed by incubating with 0.05%diaminobenzidine tetrahydrochloride (DAB) (Sigma-Aldrich)for 5 minutes. Finally, all slides were dehydrated withethanol and xylene before mounting. Density of IL-6 posi-tive neuron was determined under light microscope(Olympus BH-2 BHS/BHT System Microscopes, 100� magni-fication) [26] by ImageJ program (Java Software, USA) [27].

2.9. Histological study

Cerebellar hemisphere was embedded in 4% para-formaldehyde and postfixed in 30% sucrose in phosphatebuffer overnight. Serial of brain frozen sections at 10 mmthick were prepared via cryostat and stained with hema-toxylin and eosin [28]. The evaluation of Purkinje density incerebellum was performed under light microscope at 40�magnification. Counts were made in three adjacent fieldsand the mean number was calculated and expressed asdensity of neurons per 255 mm2.

Table1 Effect of laser acupuncture on oxidative stress statustivities of superoxide dismutase (SOD ), catalase (CAT), and gluta

Cerebel

Group MDA level(nmol/mg.protein)

SOD act(unit/mg.p

Control 0.143 � 0.01 6.179 �VPA 0.297 � 0.05z 1.509 �VPAþsham laseracupuncture

0.373 � 0.04z 1.320 �

VPAþlaseracupuncture HT7

0.172 þ 0.05y,k 2.409 �

The results are expressed as mean� SEM of oxidative stress status marbetween group.* p < 0.05, compared with the VPA group.y p < 0.001; compared with the VPA group.z p < 0.001; compared with the control group.x p < 0.01; compared with the control group.k p < 0.05; compared with the control group.ANOVA Z analysis of variance; CAT Z catalase; GSH-Px Z glutathionthe mean; SOD Z superoxide dismutase; VPA Z valproic acid.


2.10. Statistical analysis

Data were presented as mean � standard error of the mean(SEM). All statistical analyses were carried out using SPSSversion 15.0 (SPSS Inc., Chicago, IL, USA Q). The distributionsof the variables were assessed using the Kolmogor-oveSmirnov test. The one-way analysis of variance (ANOVA)with post hoc LSD test was performed to compare contin-uous variables. The statistical difference was regarded at p< 0.05.

3. Results

3.1. Oxidative stress status

Table 1 showed that VPA-rat pups increased MDA levels butdecreased SOD, CAT, and GSH-PX activities in cerebellarhemisphere (p <0.001, p < 0.001, p < 0.001, and p < 0.05,respectively Q, compared with the control group). Sham laseracupuncture failed to modify all parameters mentionedearlier. However, laser acupuncture at HT7 acupointsignificantly attenuated the changes induced by VPA. It wasfound that VPA-rats which received laser acupuncture atHT7 acupoint decreased MDA levels but increased CAT ac-tivity in cerebellar hemisphere (p <0.001 and p < 0.05,respectively, compared with the VPA group).

3.2. GABAergic system

The alteration of GABAergic system was determined bymeasuring the activity of GABA-T, a key enzyme on GABAdegradation, and the expression of two isoforms of gluta-mic acid decarboxylase, a crucial enzyme for GABA syn-thesis. In Fig. 2, it was found that VPA-rat pups increasedGABA-T activity (p < 0.01, compared with the controlgroup). Sham laser acupuncture produced failed to producea significant change of this parameter in VPA-rats.

markers including malondialdehyde (MDA) level and the ac-thione peroxidase (GSH-Px) in the cerebellar hemisphere.

lum

ivityrotein)

CAT activity(unit/mg.protein)

GSH-Px activity(unit/mg.protein)

0.88 24.95 � 2.25 3.87 � 0.250.30z 12.02 � 4.90z 2.87 � 0.36k

0.81z 14.88 � 5.90z 2.21 � 0.12k

0.31x 20.62 � 4.09*,k 2.85 � 0.52k

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Figure 2 Effect of laser acupuncture on the GABA transaminase activity in the cerebellum. The results are expressed as mean �SEM of GABA transaminase activity, n Z 6/group, one-way ANOVA and post hoc LSD test in comparing between group. *p < 0.05;compared with the VPA group. y p < 0.01; compared with the control group Q26. ANOVA Z analysis of variance; GABA Z gamma-aminobutyric acid; SEM Z standard error of the mean. Q27


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However, this elevation was attenuated by laseracupuncture at the HT7 acupoint (p < 0.05, compared withVPA group).

Our results also showed that VPA-rat pups significantlydecreased the expression of GAD 65 and GAD 67 kDa (p <0.01 and p< 0.001, compared with the control group). Shamacupuncture failed to modify GAD 65 and GAD 67 expres-sions in cerebellar hemisphere of VPA rats. Interestingly,laser acupuncture at HT7 acupoint significantly enhancedthe expression of GAD 65 kDa in VPA-rats (p < 0.05

Figure 3 Effect of laser acupuncture on the expression of GAD 65expressed as mean � SEM of relative density of protein GAD 65/67 ein comparing between group.*p < 0.05; compared with the VPA ganalysis of variance; GAD Z glutamic acid decarboxylase; SEM Z


compared with the VPA group). Unfortunately, no change inGAD 67 kDa expression was observed in VPA-rats as shown inFig. 3.

3.3. Histological and immunohistological changes

The interleukin-6 (IL-6) expression in cerebellar hemi-sphere was also determined based on its role on immuneand inflammatory responses [29] and its modulation effecton autism-like behaviors through the impairments of

kDa and GAD 67 kDa in cerebellar hemisphere. The results arexpression, n Z 6/group, one-way ANOVA and post hoc LSD test

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Figure 4 The effect of laser acupuncture on the expression of IL-6 positive neuron in cerebellar hemisphere of VPA- rats. Theresults are expressed as mean � SEM of IL-6 positive neuron expression, n Z 6/group, one-way ANOVA and post hoc LSD test incomparing between groups.* p < 0.05; compared with the VPA group.y p < 0.001; compared with the control group Q31. ANOVA Zanalysis of variance; IL-6 Z interleukin 6; SEM Z standard error of Q32the mean; VPA Z valproic acid.


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synaptic formation, dendritic spine development, andneuronal circuit balance in autism [30]. In Fig. 4, it wasfound that VPA rats enhanced IL-6 positive neuron densityin cerebellar hemisphere (p < 0.001, compared with thecontrol group). Interestingly, laser acupuncture at HT7acupoint attenuated the elevation of IL-6 positive neurondensity induced by VPA in this area (p < 0.05 comparedwith VPA group). No significant change of the mentionedparameter was observed in VPA rats which received shamlaser acupuncture.

The effect of laser acupuncture at HT7 acupoint onPurkinje cell density in the cerebellum was also performedand results were shown in Fig. 5. The results showed thatVPA decreased the density of Purkinje neuron (p <0.001,compared with the control group). This change was coun-teracted only by laser acupuncture at HT7 acupoint (p<0.01, compared with VPA group). No change was observedin VPA-rats with sham laser acupuncture.


4. Discussion

As Purkinje cell loss in cerebellum has been reported to belinked to autism-like behavior [31,32], cerebellar changes intheautismmodelhas gainedmuchattention.Ourdata showedthat VPA-rat pups decreased antioxidant activities butincreased MDA level in the cerebellum. These data are inagreement with a previous study [33] which shows that Pur-kinje cell is vulnerable to oxidative stress resulting in degen-eration [34,35].Therefore, the decreased Purkinje celldensity in VPA-rat pups might be due to the decreased SOD,CAT, and GSH-Px activities leading to an excess oxidativestress which in turn enhanced the attack of oxidative stress atlipid component of membrane resulting in the elevated MDAlevel and Purkinje cell degeneration. Besides oxidative stress,inflammation especially IL-6 also partially plays a role on thepathogenesis of autism [36,37]. The current data also showthat IL-6 expression increases in cerebellum of VPA-rat pups


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Figure 5 The effect of laser acupuncture on Purkinje cell in rats subjected to VPA treatment. The results are expressed as mean� S.E.M. of density of Purkinje cell, n Z 6/group, one-way ANOVA and post hoc LSD test in comparing between groups.*p < 0.01;compared with the VPA group.y p < 0.001; compared with the control group. ANOVA Z analysis of variance; SEM Z standard errorof the mean; VPA Z valproic acid. Q33


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which is in agreement with the change that is observed in anautistic brain. Our study has clearly demonstrated that laseracupuncture at HT7 acupoint decreased IL-6 in cerebellum ofVPA-rats. Although low level laser therapyhas beenpreviouslyshown to improve brain oxidative stress status [38] andinflammation [39,40], we found that the laser application atnonacupoint failed to show the improved oxidative stressstatus and inflammation. Therefore, the positive modulationeffect of laser acupuncture at HT7 acupoint observed in thisstudymightnotdue to thebenefitof laser alone.However, thepossible explanation of how laser acupuncture at HT7 acu-point can improve oxidative stress status and inflammation incerebellum still requires further exploration. The improvedoxidative stress status and inflammation induced by laseracupuncture at HT7 might be responsible partly for theenhanced Purkinje cells leading to the increased GABAergicfunction.


Purkinje cell axons are regarded as the sole output of thecerebellar cortex and all synapses on cerebellar or vestibularnuclei. Cerebellar nuclei in turn connect broadly to variousregions of the brain including motor cortex, association, andparalimbic cortices which contribute the crucial roles onmotor coordination and balance, cognitive, and emotionalfunctions [41]. Therefore, the increasedPurkinje cell survivalin cerebellum induced by laser acupuncture at HT7 acupointmight contribute to the role on the improved autistic-likebehavior [15].

5. Conclusion Q

The present study is the first study to demonstrate thatlaser acupuncture at HT7 acupoint improves oxidativestress status and inflammation which in turn improves


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Purkinje cell loss and the reduction of GABAergic functionin cerebellum. Therefore, laser acupuncture at HT7 acu-point is the potential strategy to improve cerebellar dam-age in VPA-rats model of autism.

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Disclosure statement

The authors declare that they have no conflicts of interestand no financial interests related to the material of thismanuscript.

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Acknowledgments

This study was supported by the National Research Council,Thailand, Integrated Complementary Alternative MedicineResearch and Development Center, Khon Kaen University,Invitation Research of Faculty of Medicine, and GraduateSchool, Khon Kaen University, 40002.

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Laser Acupuncture at HT7 Improves the Cerebellar Disorders...

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