Objective Electroacupuncture (EA) has been widely used for treatment of stroke, but there is little information on the effect of EA on the neuroprotective function in traumatic brain injury (TBI). The aim of the present study was to investigate the protective effects and mechanisms of EA treatment in a TBI rat model.
Methods Male Sprague–Dawley rats were randomly divided into four groups: sham operation, TBI control, TBI+EA treated for 30 min or TBI+EA treated for 60 min. The animals were treated with EA immediately after TBI. The EA was applied at acupuncture points GV20, GV26, LI4 and KI1 with a dense-dispersed wave, frequencies of 0.2 and 1 Hz, and amplitude of 1 mA for 30 or 60 min. Regional blood flow, cell infarction volume, extent of neuronal apoptosis, expression of cell apoptosis-associated factor transforming growth-interacting factor (TGIF) were studied, and functional outcome was assessed by running speed test. All tests except regional blood flow were performed 72 h after TBI onset.
Results Immediately after TBI, compared with the TBI control groups, the regional blood flow was significantly increased by EA treatment for 60 min. Compared with the TBI controls 72 h after TBI, the TBI-induced run speed impairment, infarction volume, neuronal apoptosis and apoptosis-associated TGIF expression were significantly improved by EA treatment.
Conclusions The treatment of TBI in the acute stage with EA for 60 min could increase the regional blood flow and attenuate the levels of TGIF in the injured cortex, might lead to a decrease in neuronal apoptosis and cell infarction volume, and might represent one mechanism by which functional recovery may occur.
- TRAUMA MANAGEMENT
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Traumatic brain injury (TBI) is a significant public health problem globally. The annual TBI-related death rate is 19.4 per 100 000 persons,1 and TBI affects up to 2% of the population per year.2 However, patients who experience TBI are still inadequately treated because of the lack of effective treatments.3 ,4 TBI survivors often experience severe neurological deficits.5 TBI has been suggested to trigger a series of secondary brain injuries, such as regional ischaemia, blood flow decrease, DNA changes and eventually apoptotic cell death.6 Recently, transforming growth-interacting factor (TGIF) was reported to be involved in the signalling pathway of cell apoptosis.7 Therefore, the prevention and treatment of secondary brain injuries, such as regional ischaemia and cell apoptosis, represent major aspects of the therapeutic management of TBI.3 ,8
Electroacupuncture (EA) stimulation, an application of electrical current via acupuncture needles, is one of the most popular forms of this traditional therapy. According to the 2002 National Health Interview Survey, acupuncture is performed on 2.1 million adults per year in the USA.9 At present, EA is widely used experimentally and clinically to treat disorders of the nervous system, such as stroke.10–,15 However, there are few studies and reports on the inﬂuence of EA after TBI, especially regarding its effect on the regional blood flow and neuronal TGIF expression involved in cellular apoptosis during the acute stage.
In this study, we test the hypothesis that EA treatment may attenuate TBI-induced cerebral injury and improve neurological outcomes by increasing the regional blood flow and reducing cell apoptosis factor TGIF expression after TBI. To examine this hypothesis, experiments were conducted to assess the therapeutic effects of EA on regional blood flow immediately after TBI in a rat model. In addition, neuronal apoptosis and TGIF expression in the neuronal cells and astrocytes in the area of ischaemia cortex were measured 72 h after TBI. We also compared the motor deficits and cerebral infarction volume during TBI in rats with or without EA treatment.
Adult male Sprague–Dawley (SD) rats (a total of 132 males) weighing 280±20 g were used in these experiments. The animals were kept under a 12/12-h light/dark cycle and allowed free access to food and water. All experimental procedures conformed to the National Institute of Health (NIH) guidelines and were approved by the institutional Animal Care and Use Committee (IACUC) of Chi Mei Medical Center (IACUC approval no. 10012722) to minimise discomfort in the animals during surgery and the recovery period. For each group of measurement parameters we used six SD rats. At the end of the experiments, the control rats and any rats that survived TBI were killed with an overdose of urethane. An endpoint 3 days after TBI was selected because lateral ﬂuid percussion causes motor and cognitive dysfunction from 3 days to 1 year after TBI.16
Traumatic brain injury
During surgery, EA and blood flow testing, the rats were in an anaesthetised state without body and limb movement. The animals received generalised anaesthesia with sodium pentobarbital (25 mg/kg, intraperitoneally; Sigma Chemical Co.) and a mixture containing ketamine (44 mg/kg, intramuscularly; Nankuang Pharmaceutical, Taiwan), atropine (0.02633 mg/kg, intramuscularly; Sintong Chemical Ind. Co., Taiwan) and xylazine (6.77 mg/kg, intramuscularly; Bayer, Germany). They were placed in a stereotaxic frame, and their scalp was incised sagittally. The animals were subjected to a lateral fluid percussion injury (FPI) to induce TBI.17 After an incision was made in the scalp, a 4.8 mm circular craniotomy was performed midway between the lambda and bregma and 3.0 mm to the right of the central suture. A modified leurlock connector (trauma cannula) with a 2.6 mm inner diameter was secured into the craniotomy with cyanoacrylic adhesive and dental acrylic. A moderate FPI (2.0–2.2 atm) was produced by rapidly injecting a small volume of saline into the closed cranial cavity with a fluid percussion device (VCU Biomedical Engineering, Richmond, Virginia, USA). The animal was removed from the device, the dental acrylic was removed and the incision was sutured.
The right femoral artery of the rats was cannulated with polyethylene tubing (PE50) under sodium pentobarbital anaesthesia for blood pressure monitoring. All recordings were made with a four-channel Gould polygraph. The core temperature (Tco) was monitored continuously by a thermocouple, and the mean arterial pressure (MAP) and heart rate were monitored continuously with a pressure transducer.
The rats were randomly divided into four groups: sham operation, TBI control, TBI+EA for 30 min and TBI+EA for 60 min, immediately after TBI. Electroacupuncture was applied at acupuncture points Baihui (GV20), Shuigou (GV26), Hegu (LI4) and Yongquan (KI1) using WHO standard locations, with a dense-dispersed wave, frequencies of 0.2 and 1 Hz, and amplitude of 1 mA (low frequency five-channel transcutaneous electrical nerve stimulation (TENS) unit and electrical needle stimulator, model 05B, Ching Ming Medical Device Co, Ltd, Taiwan) for 30 or 60 min.
Regional cerebral blood flow monitor
Laser Doppler flow (LDF) was used to monitor the cerebral blood flow (CBF) in the injured region of the subjects. The LDF probe was inserted into the location at anterior–posterior −0.8 mm and lateral+4 mm from the bregma and was installed on the stereotaxic frame after surgery. The animal was retained in the stereotaxic frame used for the operation. Changes in CBF were continuously monitored in all animals at the same timepoint from 30 min before TBI to 120 min after TBI. Blood perfusion as measured by the monitor is automatically expressed as a value of perfusion units (PU), which represent the product of the relative number of moving blood cells and their relative velocities and is a standard index of CBF. The CBF was recorded by a laser Doppler CBF monitoring apparatus (OxyLab P02/OxyLab LDF, Oxford Optronix Ltd, UK) connected to a personal computer, which was used to collect and analyse the data.
Cerebral infarction assay
The triphenyltetrazolium chloride (TTC) staining procedures followed those described previously.18 Six animals in each group were killed at 72 h after TBI. The volume of infarction, as revealed by negative TTC stains indicating dehydrogenase-deficient tissue, was measured in each slice and summed using computerised planimetry (PC-based Image Tools software). The volume of infarction was calculated as 2 mm (thickness of the slice)×(sum of the infarction area in all brain slices (mm2)).
Motor function evaluation: run speed
All the test and sham groups animal were run on a motor-driven treadmill at constant speeds 72 h post TBI to evaluate run speed by a TreadScan gait analysis system. In the TreadScan, each paw was automatically colour coded and measured over 20 s of locomotion, and the gait parameters were automatically calculated.19
Neuronal apoptotic assay
The cellular identification of apoptotic cells was performed by double staining with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling (TUNEL) and the neuronal nuclear marker Neu-N at 72 h after TBI.20 The procedures followed those described previously.18 The number of TUNEL/Neu-N-positive cells in the samples was measured in each slice and summed using computerised planimetry (PC-based Image Tools software). The monoclonal mouse anti-Neu-N antibody (MAB377, Chemicon Millipore Corporation, Billerica, Massachusetts, USA) was used in this study, which was then detected with the Alexa-Fluor 568 anti-mouse (IgG) antibody (A11031, Life Technologies Co. Grand Island, New York, USA).
Apoptosis-associated factor TGIF expression in neuronal cells and astrocyte
Adjacent 50-µm sections corresponding to coronal coordinates 2.0–7.0 mm posterior to the bregma were sliced consecutively. The procedures followed those described previously.18 The following antibodies were used in this study: monoclonal mouse anti-Neu-N (MAB377, Chemicon Millipore Corporation, Billerica, Massachusetts, USA) and Alexa-Fluor 568 anti-mouse (IgG) (A11031, Life Technologies Co.), monoclonal anti-glial fibrillary acidic protein (GFAP) antibody (Abcam, 1 : 200). The polyclonal rabbit anti-TGIF antibody (H-172, Santa Cruz, Inc., California, USA) was used, followed by detection with the Alexa-Fluor 488 anti-rabbit (IgG) antibody (A11034, Life Technologies Co., Grand Island, New York, USA). The number of labelled cells was calculated from five coronal sections/rat and expressed as the mean number of cells per section. For the negative control sections, all of the procedures were performed in the same manner in the absence of the primary antibody.
Data for immunoreactive cell counting and lesion volumes were evaluated for Gaussian (normal) distribution and are expressed as the means±SE. These data were analysed with one-way analysis of variance (ANOVA) and, if p<0.05, then analysed using a Newman–Keuls post-hoc test. The difference was considered significant when p<0.05. All data were analysed with Sigma Plot V.11.0 for Windows (Systat Software, San Jose, California, USA).
Treatment with EA for 60 min, but not for 30 min, significantly reduced the FPI-induced cerebral infarction volume. As shown in figure 1, the TTC-stained sections at 72 h after TBI showed a significant increase in the infarcted area of the TBI compared with those of the sham rats (+p<0.05; n=6). The TBI-induced infarction volume was significantly decreased by the EA treatment for 60 min (136±6 vs 91±7) (*p<0.05; n=6) but not by the EA treatment for 30 min (136±6 vs 121±7) (p=0.25; n=6).
Compared with the TBI group, the treatment with electroacupuncture for 60 min after TBI, during and after treatment, significantly increased the regional blood flow but not the MAP compared with the TBI group (#p<0.05; n=6) (figures 2 and 3).
The motor function tests evaluated by run speed showed that the TBI rats after TBI performed significantly less well than did the sham controls (141±19 vs 217±11) (*p<0.05; n=6). The TBI-induced run speed deficits were significantly ameliorated in the 60 min EA rats compared with the TBI rats (141±19 vs 214±16) (#p<0.05; n=6) (data not shown).
In the TUNEL assay, the number of positive apoptotic cells in the ischaemic cortex was significantly increased (*p<0.05; n=6) compared with the sham controls (61±3 vs 2±1). The increased number of positive apoptotic cells in the ischaemic cortex induced by FPI was significantly reduced (#p<0.05; n=6) by 60 min EA treatment (27±2 vs 61±3) (figure 4).
The positive TGIF expression in neuronal cells in the ischaemic cortex was significantly increased (*p<0.05; n=6) at 72 h after TBI compared with the sham controls (64±7 vs 48±2). However, this increased number of TGIF-positive neuronal cells in the cortex induced by FPI was significantly decreased (#p<0.05; n=6) after 60 min EA treatment (64±7 vs 45±1) (figure 5).
The positive TGIF expression in astrocytes in the ischaemic cortex was significantly increased (*p<0.05; n=6) compared with the sham controls (43±2 vs 29±1) at 72 h after TBI. However, this increased number of TGIF-positive astrocytes in the cortex induced by FPI was significantly decreased (#p<0.05; n=6) after EA treatment (43±2 vs 33±2) (see online supplementary figure).
In the present study, we found treatment of TBI with EA for 60 min at the acute stage was associated with increased regional blood flow and attenuated levels of TGIF in the neurons and astrocytes in injured cortex, a decrease in neuronal apoptosis and cell infarction volume, and improved functional recovery.
To the best of our knowledge, this study is the first to present neuroprotective effects of EA on regional blood flow and neuronal apoptosis-associated factor TGIF expression in traumatic central nervous system (CNS) injury. These findings will hopefully serve as a foundation for future studies on EA treatment in the acute stage of TBI.
Traumatic head injury may cause direct mechanical damage, such as vessel distortion and cell damage initially, followed by changes in cerebral blood ﬂow and finally apoptotic cell death.21
In the present study, EA treatment with 1 and 0.2 Hz, 1 mA EA for 60 min after TBI increased the regional blood flow. These results were consistent with those in a previous report on acupuncture points GV20 and/or GV26 in an ischaemia-reperfusion rat model.13 ,22 ,23
In our study, the EA-induced increase in regional CBF was not related to systemic blood pressure changes or movement of the rat's body and limbs. As figure 3 shows, a uniform injury was created among the animals due to the use of a standard and reproducible FPI device. Additionally, the frequencies of 1 and 0.2 Hz and intensity of 1 mA of the stimulus were standardised, and there was no difference in the systolic blood pressure among the groups throughout the course of the study as shown in figure 2. Finally, the rats were under a general anaesthetic state without body or limb movement during the experimental course. These findings are similar to those of Hsieh et al24 and Zhou et al,22 which were reported in an ischaemia-reperfusion rat model.
The main limitation of this study is the absence of a sham operation and EA group to demonstrate the specific effect of EA to protect brain function in TBI.
In the current study, the increase in regional CBF occurred during EA treatment and persisted for 60 min afterwards. As with the previous study, the mechanism of the EA-induced increase in the blood flow must take account of the rapid response.22 ,25 We speculate that the current through the GV20 and GV26 acupuncture points directly stimulated the cerebral vessels and that the central integration of afferent EA signals transmitted from the limbs, such as the LI4 and KI1 acupuncture points, contributed to the increase in the regional blood flow. Additionally, because the increase in the regional CBF persisted after EA ended in our study, we consider that it is also likely mediated via humoral regulation, which is a relatively slow and prolonged process. However, the exact underlying mechanism for this remains unclear.
Apoptosis is one of the secondary injury mechanisms after TBI. It is known to contribute to cell death in ischaemic brain tissue.5 ,26 In the current study, we found numerous apoptotic neurons in the ischaemic cortex of TBI animals; this was significantly reduced in the EA treatment group, suggesting that EA treatment alleviates neuronal apoptosis. Moreover, the TGIF levels in the ischaemic cortex correlated well with the number of apoptotic neuronal cells in the ischaemic cortex, suggesting a possible role of TGIF inhibition in the alleviation of neuronal apoptosis by EA.
Several studies have demonstrated that EA affects neuronal apoptosis by activating the phosphoinositide-3-kinase (PI3K)/Akt pathway,27 downregulating caspase 3 expression in the brain tissue surrounding the haematoma,28 upregulating the mitochondrial membrane potential in focal cerebral ischaemia/reperfusion injury,29 promoting the inducible expression of heat shock protein 70 (Hsp70) and the improvement of the inhibition of Hsp90 expression in ischaemia/reperfusion injury,30 inhibiting nuclear factor (NF)κB expression in cerebral ischaemia-reperfusion injured rats,14 downregulating hippocampal phospho-c-Jun N-terminal kinase (p-JNK) levels in a depression rat model,31 and decreasing the amounts of cyclooxygenase 2 (COX-2) and NFκB and enhancing transforming growth factor β1 (TGFβ1) expression in the brain tissues in ischaemia-reperfusion injury.32 We further demonstrated the effects of EA on the expression of the apoptosis-associated factor TGIF in neurons and astrocytes.
As a pro-apoptotic factor, TGIF has been demonstrated to be an essential component of the tumour necrosis factor α (TNFα) cytotoxic programme and to contribute to the execution of the TNFα apoptotic programme.7 TNFα is a proinflammatory cytokine that can be produced by activated microglia and astrocytes to induce neuronal insults.33 ,34 Consistent with our recent study on hyperbaric oxygen effects (Wang CC, Lin KC, Chio CC, et al, unpublished results), the present study showed that EA could attenuate TGIF expression in neurons and was associated with neuronal apoptosis after TBI. These results might support the role of TGIF in apoptosis in secondary brain injury after TBI. However, the relationship between TGIF in astrocytes and neuronal apoptosis needs to be clarified. Taken together, we propose that reduced TGIF expression in neurons and astrocytes might be a promising strategy to rescue neurons following TBI.
In the present study, motor function was examined by gait analysis using the TreadScan device to evaluate the spatial and temporal aspects of limb function changes during overground locomotion after TBI. Consistent with a previous study,35 the velocity of the run speed was significantly reduced after TBI but significantly increased after EA treatment.
In addition to the antiapoptotic effects, EA treatment reduced secondary brain damage by activating α7 nicotinic acetylcholine receptor (α7nAChR), with its anti-inflammatory effects, in cerebral ischaemic injury.36 This reverses the increases in the neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) levels in the hippocampus of rats with penicillin-induced epileptic seizures,37 stimulates angiogenesis in ischaemic stroke,13 ,38 upregulates the endogenous insulin-like growth factor 1 expression following middle cerebral artery (MCA) occlusion,39 and regulates the neurotransmitters and modulating brain glutamate release in the ischaemic condition.12 ,15 The potential effect on heat shock protein40 enhances brain-derived neurotrophic factor expression.41 Therefore, EA may be a useful treatment option for patients who experience TBI because of its multiple effects, and its use in the acute stage might have clinical benefits worth studying.
Treatment of TBI with EA for 60 min using low frequencies of 0.2 and 1 Hz and an intensity of 1 mA during the acute injury phase can increase the CBF and decrease cell ischaemia and damage. We also suggest that the decreased levels of TGIF in the injured cortex might lead to a decrease in neuronal apoptosis, and might represent one mechanism by which functional recovery may occur.
Acupuncture, used in stroke, may influence traumatic brain injury.
In an animal model, 60 min of electroacupuncture given immediately after injury significantly reduced the consequences of brain trauma.
The authors thank Chi-Mei Medical Center, Tainan, Taiwan, for instrument support.
This web only file has been produced by the BMJ Publishing Group from an electronic file supplied by the author(s) and has not been edited for content.
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Contributors CHC and YCH both contributed to the writing of this manuscript; CHC was the main researcher who provided the innovative idea that was the focus of this study; CYH and CCW revised the manuscript and coordinated the study of related research; JRK was responsible for the final submission after full revision, acceptance of the manuscript and for organising the research group.
Funding This work was supported by Chi-Mei Medical Center, grant number CMFHR10108.
Competing interests None.
Provenance and peer review Not commissioned; externally peer reviewed.