Background
Neuroinflammation is the major cause of disability and death after traumatic brain injury (TBI). Activated astrocytes and microglia are markers of neuroinflammation after TBI [
1,
2]. These activated cells can release tumor necrosis factor-alpha (TNF-α) and can signal neuronal apoptosis and impair brain function [
3,
4]. Therefore, attenuating reactive microgliosis and astrogliosis may be a promising strategy for the treatment of the neurological sequelae of TBI.
Electroacupuncture (EA), a highly popular traditional Chinese therapy, is also widely used in the USA, with 2.1 million adults undergoing EA per year [
5]. Previous studies have demonstrated the beneficial effects of EA on stroke [
6‐
8], spinal cord injury [
9], arthritis [
10], and sciatica [
11]. Recently, we have demonstrated that application of EA 60 min post-TBI has neuroprotective effects on neuronal cells. These effects might be attributable to the anti-apoptotic effects of EA, as demonstrated in the injured cortex in a fluid-percussion model of TBI [
12]. However, the effects of EA on neuroinflammation after TBI still require clarification.
In this study, we tested the hypothesis that EA therapy attenuates TBI-induced cerebral injury and improves neurological outcomes by inhibiting activation of microglia and astrocytes, as well as TNF-α expression in activated microglia and astrocytes, after TBI. To this end, we assessed neuronal apoptosis and TNF-α expression in activated microglia and astrocytes in the ischemic cortex at 72 h after TBI. We also compared motor deficits and cerebral infarction volume after TBI in rats that did or did not receive EA therapy for 60 min per day for 3 days.
Discussion
In the current study, treatment of TBI with EA for 60 min per day for 3 days, using low frequencies of 0.2 and 1 Hz and an intensity of 1 mA, during the acute injury phase, was shown to decrease neuroinflammation and the expression of factors associated with neuronal apoptosis. This may represent a mechanism by which functional recovery may occur after TBI.
Most acupuncture-related research in Chinese medicine employs “acupoint groups”, which comprise two or more acupoints. Therefore, the therapeutic roles and mechanisms of single, specific acupoints are difficult to discern in these studies. Rather, the majority of acupuncture experimental research describes the synergistic effects of “acupoints groups”. Xu et al. investigated the effects of acupuncture at the acupoints Baihui (GV 20) and Zusanli (ST36) in an ischemia − reperfusion injury model after middle cerebral artery occlusion. They found that TNF-α expression was lower in the EA group than in the model and sham-operated groups [
7]. In a study by Cheng et al., acupuncture at the Baihui (GV 20) and Dazhui (GV14) acupoints significantly downregulated the expression of TNF-α, GFAP, S100B, and nuclear factor-kB in the ischemic cortical penumbra [
8]. Jiang et al. selected Shuigou (GV 26) and Fengfu (DU16) for acupuncture treatment of traumatic spinal cord injury and found that EA had anti-oxidative, anti-inflammatory, and anti-apoptotic effects as indicated by reduced expression of inflammatory cytokines, including TNF-
α [
9]. Gu et al. treated patients that had undergone laparoscopic cholecystectomy (LC) at Hegu (LI 4), Neiguan (PC6), Zusanli (ST 36), and Yanglingquan (GB 34), and found that the TNF-α levels decreased significantly at 3 days after LC [
20]. In the current study, EA was applied at the acupuncture points GV20, GV26, LI4, and KI1; we found that this significantly attenuated neuroinflammation in a TBI model.
Related studies on EA therapy have employed different EA parameters, including EA frequency, waveform, and intensity. Liu et al. have reported that EA at a frequency of 2 and 5 Hz, 0.4–10 mA, with an intermittent waveform, was more effective for treatment of sciatica [
11]. Chan et al. previously reported that EA at 2 Hz (low frequency) can provide neuroprotection by preserving retinal function in glaucomatous rats [
21]. Kuai et al. compared the effects of EA between different waveforms (continuous, intermittent, and sound-electric waves); EA treatment of arthritis with intermittent waves increased the β-endorphin content in tissues with local inflammation [
10]. Chuang et al. demonstrated that 60 min of EA treatment in the acute stage of TBI could show a better outcome than a 30-min treatment, as determine from an increase in the regional blood flow and attenuation of neuroinflammation-associated parameters [
12].
In the current study, EA with sparse-dense wave of low frequency (0.2 Hz/1 Hz) and intensity of 1 mA was applied for 60 min daily for 3 days. Therefore, the therapeutic time used was 2–3 times that used in previous studies. This design was consistent with that used by Gu et al. [
20], who used the same sparse-dispersed wave. Results of both studies showed that TNF-α levels were decreased in the injured tissues after EA treatment. In future, the efficacy of intermittent and sparse-dense waveforms should be compared, and the correlation between TNF-α levels and different EA waveforms should be investigated.
The timeline of TNF-α release varies, ranging from 1 h to months after TBI [
22,
23]. Our findings on TNF-α expression and neuroinflammation at 72 h after TBI are in line with many previous results. TNF-α expression was significantly higher in the lesion boundary zone in TBI-control rats at 72 h post-TBI than in rats with TBI who were treated with simvastatin [
24], etanercept [
2,
25], hyperbaric oxygen therapy [
26], or EA [
12]. Similarly, in the current study, we found numerous Caspase-3- and TUNEL-positive neurons in the ischemic cortex of TBI animals; these were significantly reduced in the EA treatment group, suggesting that EA treatment alleviates neuronal apoptosis. Based on these results, we propose that TNF-α is produced by activated microglia and astrocytes after TBI, thus activating the neuronal apoptosis pathway, and that these adverse effects could be attenuated by EA treatment [
27].
Besides affecting glial TNF-α expression, as shown in this study, EA has multiple other effects in several animal models. For example, EA activates the α7 nicotinic acetylcholine receptors to attenuate inflammatory processes, thereby providing protection against cerebral ischemic injury [
6]. EA also increases brain-derived neurotrophic factor expression in heat stroke [
28], modulates the NF-E2 related factor 2/antioxidant response element pathway to provide protection against endotoxic shock-induced acute lung injury [
29], and inhibits the ERK1/2-Egr-1 signaling pathway, thereby protecting cardiomyocytes in a mouse model of myocardial ischemia − reperfusion [
30]. Thus, we believe that EA therapy may be useful for patients with TBI because of these effects. We suggest that application of EA in the acute stage of TBI may have clinical benefits.
Silver [
31] demonstrated that, after TBI, glial scar formation, particularly those involving astrocytes, interfered with functional neuronal regeneration. In the present study, the TBI-induced astrogliosis was significantly attenuated by EA therapy at 72 h after TBI. Therefore, we propose that EA may have beneficial effects on neuronal regeneration.
In order to avoid interference of the effects of different treatments, recent acupuncture studies have used a sham acupuncture group as the control against which to compare the results of the experimental group. The non-acupoints are usually situated adjacent to actual acupoints, and in several experimental animal models, they have been separated by a distance of <5 mm [
29,
32,
33] or have been far away from the actual acupoints [
34]. For example, Zhang et al. showed that the therapeutic effects of EA applied to actual acupoints on TNF-α expression were better than the effects of EA administered at non-acupoints in a Wistar rat abdominal adhesion model [
32], and Yu et al. reported the same effects in an endotoxic shock-related lung injury model in rabbits [
29]. Furthermore, in SD rat models equivalent to those used in our study, Du et al. demonstrated the same results in an abdominal adhesion model even though the rats’ weights were less than those in our study [
33]. Finally, Eshkevari et al. described the same results in a cold stress model in rats with weights similar to those in our study [
34]. Therefore, our study did not include a non-acupoint group, but focused on comparing whether EA delivered at acupoints could notably improve the injured cortex after TBI.
Some limitations of the current study should be considered. First, only male rats were investigated. Future studies should evaluate whether EA protects female rats from TBI-induced neurobehavioral and pathological changes. Second, only one method (the inclined plane test) was used to evaluate functional outcomes, due to limited equipment availability. Third, we were unable to characterize changes in the injured brain that occurred on each day within the 3-day EA treatment window after TBI. Therefore, a time-series imaging study using this experimental TBI model/EA treatment paradigm should be conducted in future. Fourth, we did not perform EA at non-acupoints. Results from an appropriate control groups are required to clarify the specific effects of EA stimulation of acupoints and other influences.
Acknowledgements
The authors thank Chi-Mei Medical Center, Tainan, Taiwan, for instrument support.