Multi-modality FMRI Study On The Correlation Between Negative BOLD Responses And GABA/Glx Elicited By Needling

BackgroundA large number of basic and clinical studies have shown that the key to acupuncture to regulate overall function is the brain center.In the past two decades,functional magnetic resonance imaging(fMRI)has been increasingly used in acupuncture brain effects and mechanism research due to its advantages of non-invasiveness,brain central integrity.Previously,our team found that the common feature of manual needling is to modulating the limbic-paralimbic-neocortical network(LPNN).fMRI studies showed that the deactivated brain regions elicited by acupuncture partially overlapped with the default mode network,but more effects in the limbic system.These limbic brain regions are closely related to activities such as pain,emotion regulation,memory and cognition,and also are the key brain areas for acupuncture analgesia,anti-anxiety,anti-depressive disorder and other effects.Positive BOLD response(activation)reflects neuronal excitation and increased cerebral blood flow,but the underlying mechanism of negative BOLD response(deactivation)is not clear,and further research is still needed.There are currently hypotheses such as "blood stealing" and neuronal inhibition.Whether the deactivation results from down-regulation of excitatory or up-regulation of inhibitory forces remains to be explored.This would require other experimental approaches to probe into the neurochemical processes that underlie the hemodynamic response.Neural activity in general is constituted to a large degree by the excitation-inhibition balance(EIB).Most importantly,the EIB is closely related to the activities of glutamate and GABA(y-aminobutyric acid)as,respectively,the brain’s main excitatory and inhibitory neurotransmitters.GABAergic neurons have an effect on neuronal firing frequency and hemodynamic response and may be related to changes in BOLD signal.The BOLD effect is to a large degree a result of oxygen being used to support energy production and so one would expect that the most energy intensive system-the glutamatergic,would be most related to this measure.Magnetic resonance spectroscopy(MRS)is a noninvasive imaging technique that can measure brain levels of glutamate-glutamine(Glx)and GABA in vivo.Domestic and foreign scholars combined with BOLD and MRS techniques found that there were different correlations between the BOLD signals induced by tasks(emotional perception and judgment,visual stimulation,cognitive tasks,etc.)and GABA/Glx.However,the correlation between the brain effect of needling stimulation and the neurotransmitter GABA/Glx has not been studied.Our research will combine BOLD with MRS techniques to explore the neurochemical(GABA/Glx)basis of acupuncture at right LI 4.Objective1.To establish and optimize BOLD and MRS multimodal fMRI protocol for brain functional effects induced by acupuncture;2.To compare the brain functional response induced by manual acupuncture and Von Frey filament(epidermal stimulation)at the right-side Hegu acupoint;3.To investigate the changes of neurotransmitter GABA/Glx before and after the two stimulation;and analyze the difference in neurotransmitter changes caused by the two stimulation methods;4.To explore the correlation between activation/deactivation and neurotransmitter GABA/Glx induced by acupuncture.MethodsThis study recruited 79 healthy volunteers(24 males,24.42±1.44 yo).Each volunteer received two sessions of fMRI-MRS examination,apart from each other by three or five days.The MRI scan sequence was the same,including high-resolution anatomy,pre-task MRS,resting state BOLD,task BOLD,and task-MRS scans;A volume of interest(VOI)of 35×30×25 mm3 was placed at the bilateral medial prefrontal cortex(including ACC).In the two sessions,Right Hegu point was stimulated,but the stimulation methods were different.Randomized order for manual acupuncture and Von Frey filament(epidermal stimulation).The tasks were designed as the block design.Each block undergoes three intermittent acupoint stimulations,each for 30 seconds stimulation with two minutes pauses.MRS data was processed by LC Model software.fMRI data was preprocessed by SPM 12,REST,Brainnet software,and then analyzed the data within groups,compared the results between groups,and quantificated the BOLD signal in ROI.The processed data was analyzed using SPSS 23.0.Results1.Establishment of the protocolTen healthy volunteers were initially recruited.After repeated experiments,multimodal fMRI research protocol with BOLD and MRS were successfully established and applied to the study of acupuncture brain mechanism.2.Changes in brain functional activity(BOLD responses)induced by two stimulations In the manual acupuncture group,the main activation regions were somatosensory cortex(SI/II),thalamus and insular cortex,and deactivation regions:bilateral frontal cortex,anterior cingulate gyrus,parietal cortex,hypothalamus,putamen,Hippocampus,amygdala,and reticular formation;Von Frey group mainly activated the brain regions of somatosensory cortex(SI/II),insular cortex,negative activation area:bilateral anterior cingulate gyrus,precuneus.The deactivation effect of the manual needling group is more extensive and intensive,mainly in the limbic system.Comparison between the two groups(manual acupuncture group-Von Frey group)showed that in the manual acupuncture group,the deactivation of the medial prefrontal cortex was stronger,and the activation of the occipital cortex was stronger.The deactivation effect of manual acupuncture group was showed the trend of slightly stronger than that of Von Frey group(t=-1.830,p=0.069).The deactivation effect on the subjects with deactivation voxels above 30 induced by needling[BOLD mean(-3.36±0.40)]was stronger than control stimulation[BOLD mean(-1.19±2.58)],,t=-3.59%,p=0.002.3 Changes in neurotransmitters induced by two stimulationsThere was no significant difference between GABA and Glx before and after the two stimulation methods(p>0.05).No significant difference was found between the two stimulation methods in GABA and Glx(p>0.05).4.The gender related differences in GABA/GlxThe results of 47 girls and 19 boys were included in the analysis.The manual acupuncture group:no significant gender differences were detected for baseline and post-task GABA levels(P=0.79,P=0.99),baseline and post-task Glx concentrations were significantly higher in the male participants(P=0.026,P=0.024).Von Frey group:no significant gender differences were detected for baseline and post-task GABA levels,post-task Glx concentrations were significantly higher in the male participants(P=0.04),there is the similar tendency for baseline Glx.5.The correlation between GABA and GlxMRS values shows a positive relationship between GABA and Glx,not only on baseline levels but on stimulated levels in both of two groups.There is no significant correlation between the GABA changes or Glx changes in the manual acupuncture group.In the Von Frey group,the changes of GABA demonstrated a positive correlation with the changes of Glx.6.The relationship between task-induced BOLD signal and GABA/GlxIn the two groups,deactivation or activation BOLD responses were not correlated to the resting GABA or Glx levels.During needling stimulation,the second GABA or Glx levels showed no correlation with positive or negative BOLD response.A trend was found but not significant toward the negative correlation between the second GABA levels and negative BOLD response(r=-0.303,p=0.061).The second GABA levels demonstrated a negative interaction with deactivation when put two groups together.There was a negative correlation between deactivation BOLD signals and ΔGlx in the manual acupuncture group(r=-0.18,p=0.03).ΔGABA levels demonstrated a negative interaction with deactivation BOLD signals in the Von Frey group(r=-0.404,p=0.011).A negative correlation between deactivation when putting two groups together and GABA changes(r=-0.262,p=0.016),and a trend toward negative correlation between deactivation when putting two groups together and Glx changes was also found but not significantly(r=-0.212,p=0.053).ConclusionThe multimodal fMRI can be applied to the study of acupuncture effects in the brain and to improve our understanding of neurochemical base with the brain activity;1)This study further confirmed our hypothesis of needling modulating the limbic-paralimbic-neocortical network.2)Acupuncture produces the relatively specificity in the brain with BOLD effect and GABA,Glx changes comparing with Von Frey touching stimulation.3)There is a negative correlation between ΔGABA/ΔGlx and deactivation during stimulation.These results imply that the negative BOLD response seems a result of inhibitory activity related to GABA/Glx.Acupuncture may affect the function of anterior cingulated cortex by regulating GABA and Glx levels,so as to achieve brain regulation effects on pain,emotions,and the other effects.