Heat-sensitization responses occurred in certain patients while exposed to suspended moxibustion. The response often indicated that the efficacy of moxibustion to those with it tended to triumph over those without. However, its mechanism remains to be explained. Our previous fMRI and EEG studies confirmed the changes of activities in cerebral certain regions accompanied with heat-sensitization responses, especially in prefrontal cortex. Therefore, we hypothesize that neurological system is involved in moxibustion-induced heat-sensitization responses. In the present study, phosphorylation of Cofilin representing long-term potentiation in synapse of prelimbic cortex of medial prefrontal cortex in stroke rats over suspended moxibustion was assessed, and the size of phosphorylated Cofilin positive spine in synapse was also measured. The result showed that heat-sensitization responses were observed to augment cerebral ischemic stroke-induced phosphorylation of Cofilin in prelimbic cortex of rats and increase the numbers of large synapses. This indicated that long-term potentiation of prelimbic cortex was attributed to heat-sensitization responses that were certain neurological responses of medial prefrontal cortex to suspended moxibustion.
Moxibustion is a traditional Chinese medicine approach to healing, with burning dried plant materials (
Through our clinical observations, moxibustion-induced heat-sensitization responses vary with the state of a disease [
50 mature male Sprague-Dawley rats (220 to 250 g) were purchased from Shanghai Slaccas Laboratory Animals Co., Ltd., in Shanghai, China. They were housed in the cage at temperature 20~25°C and humidity 40~70%. The rats were habituated to the cage for 6 days prior to test. Then they were randomly stratified into 4 groups. Normal Group (N, n=10) Sham-Operated Group (S, n=10) Ischemic Control Group (C, n=10) Ischemia over moxibustion exercise Group (M, n=20)
The usages of animal in the experiments were conducted in accordance with NIH Guidelines and approved by the Animal Use and Care Committee for Jiangxi University of TCM for Scientific Purposes.
The rats were injected intraperitoneally with Sodium Pentobarbital (3%) at a dose of 30 mg/kg to be anesthetized. Core body temperature was monitored using a rectal probe and maintained at 37±0.5°C by heating lamp and a heating pad. The values of pH, PaO2, PaCO2 of arterial blood gas, and blood pressure were closely monitored via catheterizing the right femoral artery. The Middle Cerebral Artery Occlusion (MCAO) was obtained by the Intraluminal Filament method as previously described [
Rats were treated with suspended moxibustion exercise in this study, precisely, hanging moxa strip at 3 cm higher than the target area, untouched upon the recipient. Moxa strip made of moxa powder was 12 cm in length, 0.6 cm in diameter, provided by the Affiliated Hospital of Jiangxi University of TCM, China. The fur on the rats neck was shaved off to uncover the acupoint dà zhuī (DU14) that is the location of the seventh cervical vertebrae; DU14 is considered very important for brain functions in acupuncture and moxibustion philosophy [
Rats were given halothane to be anesthetized and then perfused with 4% paraformaldehyde in 0.1 M sodium phosphate buffer (PB), pH 7.2. Brains were postfixed for 2 h, cryoprotected in 20% sucrose/PB, and then sectioned (25
Laser scanning confocal microscopy was applied to take an image of PrL of mPFC in unaffected hemisphere. LSM 880 Axio Observer (Carl Zeiss AG, Jena, Germany) and a Plan Apochromat 63x/1.40 Oil DIC M27 objective were employed. Optical section is 0.6
Data was analyzed with a
Change in tail temperature induced by suspended moxibustion exercise on the acupoint DU14 in MCAO rat model. Because the change of tail temperature was similar among the three consecutive testing days, data of the first day were presented as a representative. Data were expressed as mean ± SD.
All the rats receiving moxibustion exercise showed very quiet instead of irritated during the moxibustion treatment. Among the 18 rats of group M, 11 rats showed the tail temperature increased more than 1°C (3°C on average), and 7 rats were seen at the tail temperature increase ≤1°C on average. Therefore, there were 11 rats in TTI subgroup and 7 rats in non-TTI subgroups. The incidence of tail temperature increase was similar to that of the previous experimental results [
The change of synaptic structure requires the participation of structural protein actin fiber, and the change of action fiber is regulated by Cofilin. The Cofilin protein that is mostly restricted to dendritic spines removes actin monomers from the sharp end of growing actin filaments. The phosphorylation of Cofilin suppresses this activity [
Cofilin, pCofilin, and PSD-95 immunostaining in PrL of mPFC. (a) Cofilin immunoreactivity was abundant in discrete puncta but was absent from cell bodies (data not shown) and dendrites. (b) PCofilin immunoreactivity puncta were present in smaller numbers compared to those labeled for total cofilin (a). (c) PSD-95 immunoreactivity profiles were numerous. (d) Merged images show that pCofilin immunoreactivity profiles were associated with PSD-95 immunoreactivity puncta. The arrows point to the same sites in (b)–(d) to show spatial relationship and overlap. Scale bar: 5
In the contralateral prelimbic cortex, the number of synapses positively expressed by pCofilin (pCof+) was significantly higher in group C compared to groups N and S (
MCAO model increased the number of pCofilin positive (pCof+) spines, and moxibustion-induced heat-sensitization responses in rat’s model characterized by tail temperature increase had enhanced this effect. PCof+ spines were counted and values for a rat in a given cohort were normalized to the mean score for ischemic control rats in that cohort. (a) MCAO rats either or not treated with suspended moxibustion exercise (TTI, non-TTI, and C groups) had more pCof+ spines than did normal rats (N group); this effect was absent in sham rats (S group); differences in pCof+ puncta between TTI versus non-TTI or C groups were significant. (b) The increase in numbers of pCof+ puncta in MCAO rats either or not treated with suspended moxibustion exercise was not accompanied by an increase in total number of PSD-95 puncta. (c) The number of PSD-95 puncta that were colocalized with pCofilin immunoreactivity was expressed as a percentage of the total PSD-95 puncta for each rat in the study (values were then normalized to the within-cohort ischemic control rat mean). The C group had a higher percentage of PSD-95 puncta colocalized with pCofilin than did either N or S groups; differences in pCof+ puncta colocalized with pCofilin between TTI versus non-TTI or C groups were also significant. Data were expressed as mean ± SEM. #
pCof + spines
PSD95
pCof + PSD95
PSD-95 is a protein that is specific to and uniformly distributed within PSDs of excitatory synapses. This experiment showed that the total number of PSDs was not affected by moxibustion intervention and MCAO model. However, 3-D measurement of PSD-95 showed that PSD-95 positive profiles were larger on pCofilin positive (pCof+) spines than on pCofilin negative (pCof-) spines (
PSDs on pCof+ spines were larger than those on pCof- neighbors. Data were expressed as mean ± SEM.
Through our clinic practice, we found that certain subhealthy individuals or patients were able to sense a certain special heat in the body while exposed to suspended moxibustion exercise, which was not part of moxibustion heat radiation. The sense of the special heat was described as heat expansion, heat penetration, and heat transmission, and those were not clarified by neuroanatomy or physiology. In clinic practice, suspended moxibustion exercise with special heat awareness has demonstrated good healing over time, and the sense of moxibustion-induced special heat has drawn more attention in clinic practice. Accordingly, we named the sense as heat-sensitization responses [
The current theory is not sufficient to explain the mechanism of heat-sensitization responses. For example, a patient with fibromyalgia in the back of the head and neck was exposed to moxibustion exercise on the acupoint Zhì yáng (GV9) (Supplementary Figure
Synaptic plasticity refers to changes in synaptic transmission efficiency and synaptic structure under certain conditions. Long-term potentiation (LTP) is a key manifestation of synaptic plasticity [
PrL of mPFC was selected as the observation region of brain tissue by reasons such as the fact that PrL is the key region of mPFC, a widely studied brain region that has extensive and complex connections with other brain regions, and plays an important role in cognitive processes, decision-making tasks, emotional regulation, pain regulation, and many other undefined functions [
In our previous experiments, we observed that the temperature in certain rats’ tail midpoints rose more than 2°C when moxibustion exercise was exerted upon the acupoint DU14 of cerebral ischemia-reperfusion injury rats [
The results of this study show that the MCAO model itself (representing the disease state) significantly increased phosphorylation of Cofilin protein in the PrL of rats. It indicates that the disease state could induce LTP-like plasticity changes in the PrL of rats. Many studies have confirmed that disease states promote changes in certain cortical functions. For example, Rainville et al. found that visceral pain changed activities of the anterior cingulate cortex [
To sum, rats’ MCAO model induced phosphorylation of Cofilin in PrL of mPFC and enlarged synapses. Moxibustion-induced heat-sensitization responses complemented this aggregation. The increase of pCofilin positive spines in PrL represents LTP of PrL. Therefore, we point out that LTP of PrL was attributed to heat-sensitization responses. It is worthy to run a further study on exploring a way simulating the heat sensitive moxibustion to enhance LTP of PrL for cerebral ischemic stroke.
Medial prefrontal cortex
Prelimbic cortex
Tail temperature increase
Long-term potentiation
Phosphorylated Cofilin
Electroencephalogram
Functional magnetic resonance imaging
Middle Cerebral Artery Occlusion.
The data used to support the findings of this study are available from the corresponding author upon request.
The authors declare that they have no conflicts of interest.
Rixin Chen designed and carried out the study and supervised the project. Zhimai Lyu established the models. Dingyi Xie and Yanjun Chen performed the moxibustion, temperature measurement. Dandan Huang performed the immunocytochemistry and laser scanning confocal microscopy operation. Rixin Chen, Zhimai Lyu, and Chunmei Wu collected and analyzed the data, discussed the interpretation of the results, and wrote the paper. All authors read and approved the final paper. Rixin Chen and Zhimai Lyu contributed equally to this work.
This study was supported by the Major State Basic Research Development Program of People’s Republic of China
Supplemental Figure S1: clinical operation of suspended moxibustion. Supplemental Figure S2: the rat’s tail temperature increase when acupoint DU14 was exposed to suspended moxibustion. Supplemental Figure S3: the sense of heat in a patient with fibromyalgia was transmitted along the spine or paraspinal muscles to the muscles of posterior part of the head and neck when acupoint GV9 was exposed to suspended moxibustion.