Electroacupunture contributes to recovery of neurological deficits in experimental stroke by activating astrocytes

Abstract

Background:

Neurological deficits is one of the most prevalent clinical manifestation after stroke. The effects of astrocytes activated by electroacupunture (EA) after stroke on the neurological recovery in middle cerebral artery occlusion (MCAO) rats was not clear and definite.

Objective:

Our previous study showed that treatment with EA for 7 days contributed to the activation of astrocytes in MCAO rats. The purposes of this study were to 1) confirm the effects of EA for 14 days on activation of astrocytes in MCAO rats, and 2) test the relationships between activation of astrocytes and neurological functional recovery induced by EA in MCAO rats.

Methods:

All rats were randomly divided into five groups: naïve control group, sham control control group, MCAO, MCAO/EAn, MCAO/EAd (n = 8, for each group). Rats in MCAO/EAn group received EA treatment at acupoints of Neiguan (PC06). MCAO/EAd group received EA stimulus at acupoints of Diji (SP08). The primary indicators were locomotor recovery, histopathology, immunohistochemistry, RT-PCR and Western blot.

Results:

The neurological deficit and histopathological improvements and activation of astrocytes were observed after EA treatment at acupoints PC06. Parametric correlation analyses revealed a cubic correlation relationship between activation of astrocytes and neurological recovery of MCAO rats treated with EA.

Conclusion:

EA treatment at the acupoints of Neiguan involved in the regulation of activation of astrocytes, which our data suggested has a cubic correlation relationship with the neurological recovery of MCAO rats.

Keywords

EA treatment astrocytes functional recovery MCAO rats

Abbreviations

BWT

Beam-walking test

CNS

Central nervous system

Electroacupunture

GFAP

Glial fibrillary acidic protein

MCAO

Middle cerebral artery occlusion

tPA

Tissue plasminogen activator

1 Introduction

Currently, acute cerebrovascular disease is one of the major threats to human health. The incidence of ischemic stroke in China is about 2, 000, 000. Stroke remains a significant concern in human health. Ischemic stroke has been becoming one of the most common causes of death and disability in China. Although tissue plasminogen activator (tPA), the only FDA approved treatment for stroke, is an effective therapeutic option in stroke, only 3–5 % of patients are able to receive and potentially benefit from this treatment, due in part to the narrow time window and neurovascular toxicity (Lansberg, Albers, & Wijman, 2007; Marder, Jahan, Gruber, Goyal, & Arora, 2010; Mozaffarian et al., 2016). Thus, finding an effective therapeutic strategy for stroke remains a high priority.

In the CNS, the most abundant subtype of glial cells is astrocytes, which by several fold outnumber neurons. Nevertheless, astrocytes have not been a major therapeutic target for the treatment of stroke, most of which research emphasis was on neurons. In mammalian brain, spinal cord, and retina, astrocytes have multitude of functions. Astrocytes situated between ischemic blood vessels and neurons could induce the formation of neuronal synapses and are involved in the control of the energy supply to neurons and the turnover of neurotransmitters (Belanger, Allaman, & Magistretti, 2011; Benarroch, 2005; Schousboe, Bak, & Waagepetersen, 2013). Therefore, the idea that astrocytes play a central and fundamental role in the pathogenesis of ischemic neuronal death has been developed in recent years (Burda, Bernstein, & Sofroniew, 2016; Eroglu & Barres, 2010; Pellerin et al., 2007; Suzuki et al., 2011). An abundance of data suggests that, under pathophysiological conditions, highly dynamic and complex astroglial-neuronal interactions affect brain function and survival. Neuroprotective efforts targeting the functional activation of astrocytes may constitute a superior strategy for neuroprotection.

Acupuncture is a Chinese traditional medicine therapy method which has been used for more than 3000 years. EA delivering electrical stimulation to acupuncture points through acupuncture needles, has been recommended as a complementary therapy in stroke treatment and rehabilitation in both Asian and Western countries. Several studies investigating the effectiveness of EA showed beneficial effects on protection against cerebral ischemia damage (Chen & Fang, 1990; Lan et al., 2013; Lo, Cui, & Fook-Chong, 2005; Lu et al., 2016; Wang, Liu, Yu, Jiang, & Han, 2009).

Our previous study has demonstrated that treatment with EA could prevent the ischemia-induced impairment of experimental stroke (Han et al., 2015; Lu et al., 2014, 2015; Zhao et al., 2013). The data revealed that EA for 7 days contribute to the activation of astrocytes. However, the effects of EA for 14 days has not been examined. In addition, the role of activation of astrocytes after stroke is controversial. Just like a double-edged sword, reactive astrocytes exert not only beneficial but also detrimental effects on neuroprotection and neurorestoration. On one hand, reactive astrocytes were confirmed to protect central nervous system (CNS) cells and tissue by performing functions such as uptake potentially excitotoxic neurotransmitters, reducing vasogenic edema, and limiting the spread of inflammatory cells or infectious agents from areas of damage or disease into healthy CNS parenchyma, and so on. On the other hand, mice deficient in glial fibrillary acidic protein (GFAP) and Vimentin also exhibited positive outcomes such as better post-traumatic synaptic regeneration (Wilhelmsson et al., 2004), improved axonal regeneration (Cho et al., 2005), better regeneration and better functional recovery after spinal cord trauma (Desclaux et al., 2015; Menet et al., 2003). Other evidences suggested that activated astrocytes could form a dense glial scar eventually. This means that the extension of axonal regeneration was impeded. Activated astrocytes secrete a variety of nerve regeneration inhibitors, such as chondroitin sulfate proteoglycans, oxygen free radicals, which result in not only a negative impact on the recovery of nerve structure and function, but also secondary injury of CNS by releasing such pro-inflammatory factors as TNF-α, IL-6 (Fitch& Silver, 2008).

To our knowledge, no study has investigated how activated astrocytes affected the restoration of neurological deficits induced by EA in MCAO rats. In this study, we examined the effects of EA treatment at the Neiguan acupoint on activation of astrocytes and functional outcome in a rat model of MCAO. As a result, we found that the repeated needling could modulate the activation of astrocytes which has a cubic correlation relationship with the neurological recovery of MCAO rats treatedwith EA.

2 Materials and methods

2.1 Animals

Adult male Wistar rats weighing 220 to 250 g were obtained from the Beijing Vital River Experimental Animals Laboratory (Beijing, China) (license No. SCXK (Jing) 2012-0001). The experimental protocol was approved by the Ethics Committee for Animal Experimentation of the Shandong University of Traditional Chinese Medicine (Jinan, China) in accordance with the guidelines of Institutional Animal Care and Use Committee and the regulations. All animals received humane care in compliance with the guidelines. The rats were housed under the same conditions: 12 h/12 h-light/dark circle, 22 °C temperature, and 45–50 % humidity with free access to pellet chow and water.

2.2 MCAO model and experimental groups

The Longa’s method was used to copy the model of MCAO of rat (Han et al., 2015; Longa, Weinstein, Carlson, & Cummins, 1989; Lu et al., 2014). Prior to MCAO surgery, rats were anesthetized with 10 % chloral hydrate administered intraperitoneally (at a dose of 350 mg/kg). A midline incision was made on the ventral surface of the neck. Following the subcutaneous tissue blunt dissection, the right common carotid artery, the external carotid artery and the internal carotid artery were isolated and exposed. The right common carotid artery and the external carotid artery were ligated with 6.0 silk suture. The right external carotid artery and the common carotid artery were both simultaneously ligated. The internal carotid artery was temporarily occluded with a microvascular clip. Arteriotomy was performed in the common carotid artery approximately 5 mm proximal to the bifurcation. A silicone-coated 4-0 nylon suture (Xinong Corporation, Beijing, China) was inserted into the internal carotid artery through the incision of the common carotid artery. The filament was advancedapproximately 20 mm distal to the carotid bifurcation. The core body temperature of animals was maintained at 37 °C all the time by means of a feedback regulated heating blanket. The incision was sutured layer by layer.

After rats recovering consciousness from MCAO surgery, their neurological function deficits would be graded on a scale of 0–4 (Table 1). The rats with the scores of 2-3 were divided randomly into three groups: MCAO, MCAO/EAn, MCAO/EAd (n = 8, for each group). Subsequently, the rats returned to their respective home cages to eat and drink freely. Sham control rats received the same procedure except that the filament was advanced approximately 5 mm distal to the carotid bifurcation.

Table 1
Standards of Neurological Function Scores

Neurobehavioral performance right after awaking from anesthesia Scores

Normal walk 0

Failure to extend left forepaw fully 1

Circling toward the paretic side 2

Falling down to the paretic side 3

No spontaneous walking and loss of consciousness 4

Neurobehavioral performance right after awaking from anesthesia	Scores
Normal walk	0
Failure to extend left forepaw fully	1
Circling toward the paretic side	2
Falling down to the paretic side	3
No spontaneous walking and loss of consciousness	4

2.3 EA stimulation

Acupuncture was performed once daily for 14 days at the corresponding acupoints bilaterally with two stainless-steel 0.20 mm-diameter needles which were connected to a HANS 100A electro-stimulator (Jisheng Medical Instrument Co., Nanjing, Jiangsu, China) at a frequency of 2/15 Hz and an intensity level of 1 mA for 30 min. In the MCAO/EAn group, acupuncture stimulated bilaterally the point of Neiguan which is located in the interosseal muscles between the radius and ulna of the forelimb 3 mm proximal to the wrist crease. The needles were inserted vertically into Neiguan in depth of 2 mm. In the MCAO/EAd group, an off-meridian point Diji was chosen to be inserted in view of no evidence of therapeutic effects on cerebral ischemia. Diji was located on the medial aspect of the lower leg, 3 cun (Cun used in traditional Chinese medicine is a proportional unit corresponding to the middle segment of one’s middle finger.) below SP 9, on the line connecting the tip of the medial malleolus and SP 9 (Fig. 1). In the both sham and model group, rats were restrained as those in both MCAO/EAn and MCAO/EAd with no acupoint inserted.

Fig.1

Rat schematic showing the location of the acupuncture points used in the study. PC06 stands for “Neiguan”, which is located in the interosseal muscles between the radius and ulna of the forelimb 3 mm proximal to the wrist crease; SP8 stands for “Diji”, which was located on the medial aspect of the lower leg, 3 cun below SP9, on the line connecting the tip of the medial malleolus and SP9.

2.4 Assessment of functional outcome

The experiment was performed between 10:00 a.m. and 4:00 p.m. All animals underwent neurobehavioral testing before MCAO and days 1 and 14 after MCAO. Tests including neurological deficit score, beam-walking test (BWT) were conducted by a blinded examiner.

Neurological deficit scores Neurological deficit evaluation was performed in 30 minutes after EA treatment of 14th day in accordance with the standard of Table 1.

BWT Prior to MCAO operation, all of rats were trained to walk through a beam (60 cm long, 4.5 cm wide and 40 cm above a foam pad horizontally) for a week. At both end of the beam there was a box, respectively. Animals were placed on the beam and given voice and optical stimulus. As a result, all of rats could walk through it successfully within 1 minute after the training. BWT was conducted when the rats recovered fully from anesthesia and on the day sacrificed. They were given a score from 1 to 7 according to the standard of Table 2.

Table 2
Standard of BWT Scores

Neurobehavioral performance right after awaking from anesthesia Scores

Falls off, no attempt to balance or hang onto the beam <20 seconds 1

Attempts to balance on beam but falls off >20 seconds 2

Attempts to balance on beam but falls off >40 seconds 3

Hugs the beam and two limbs fall down from the beam, or spins on beam >60 4

Hugs the beam and one limb falls down from beam 5

Grasps side of beam 6

Walks through and balances with steady posture 7

Neurobehavioral performance right after awaking from anesthesia	Scores
Falls off, no attempt to balance or hang onto the beam <20 seconds	1
Attempts to balance on beam but falls off >20 seconds	2
Attempts to balance on beam but falls off >40 seconds	3
Hugs the beam and two limbs fall down from the beam, or spins on beam >60	4
Hugs the beam and one limb falls down from beam	5
Grasps side of beam	6
Walks through and balances with steady posture	7

2.5 Tissue collection, sample preparation, histologic and immunohistochemical assessment

2.5.1 Histologic and immunohistochemical assessment

After completing the behavior tests, rats were euthanized by being given pentobarbital (50 mg/kg) administered intraperitoneally. Five of each group were transcardially perfused with cold 0.9% saline followed by 4 % paraformaldehyde in 0.1 M phosphate buffer. The brains were removed and fixed in 4 % (w/v) phosphate buffer-paraformaldehyde. A coronal brain slice about 1 cm-thickness from the starting point of middle cerebral artery to rostral 0.5 cm and caudal 0.5 cm of the brain. Afterwards, the slices were embedded in paraffin and sectioned coronally in 5-μm-thick slices, which was followed by the steps of deparaffinization in xylene. After several rehydratations the slices were stained by hematoxylin and eosin, successively. Photographs of each whole slide were taken with a digital camera (ISH300; Tucsen, Fuzhou, China) mounted on the stereomicroscope (SZX7, Olympus). The images were captured on a computer with the Viewfinder software (Tucsen, Fuzhou, China). The infarct areas showing reduced staining under light stereoscopic microscopy, were traced and quantified with the Image-Pro Plus 4.0 (IPP 4.0). Infarct volumes were expressed as a percentage of the contralateral side±SEM. The rest photographs were taken with a digital camera (DMD500, Danjier, Ji’nan, China) mounted on the microscope (Eclipse CI-L, Nikon, Japan). The images were captured on a computer with the Viewfinder software (Danjier, Ji’nan,China).

The remaining 3 rats in each group were deeply anesthetized to harvest the brains, each of which was sliced into 3 parts as described above. The central slices about 1 cm-thickness were grinded in liquid nitrogen and divided into 3 aliquots for qRT-PCR test and Western blot.

2.5.2 Immunohistochemistry were performed for the staining of GFAP

The sections were dewaxed and dehydrated. Then the sections were treated with 0.3% H2O2-methanol for 10 min at room temperature to suppress the endogenous peroxidase activity. After pretreatment with 3% normal bovine serum albumin for 30 min at room temperature in PBS to block nonspecific binding the sections were incubated with cocktail containing mouse anti-GFAP monoclonal antibody (1:1500, cat# sc-58766, from Santa Cruz Biotechnology, Inc. Dallas, TX, USA) in 0.1 M PBS at 4 °C for 24 h. The sections were treated with a cocktail containing biotin-conjugated goat anti-mouse secondary antibody (1:200; Zhongshan Gold Bridge Biotechnology, Beijing) in 0.1 M PBS for 20 min at room temperature. After several buffer rinses the sections were treated with SABC (Zhongshan Gold Bridge Biotechnology, Beijing) and DAB. Controls included omitting incubation with primary or secondary antibodies. For the quantitative analysis of the intensity of GFAP, four fields of each section were chosen. The intensity was calculated with IPP 4.0 and expressed as mean optical density (IOD).

2.6 Quantitative real-time reverse transcription-PCR of mRNA of GFAP (qRT-PCR)

To detect the mRNA levels of GFAP in brain tissue RT-PCR was performed. Total RNA was extracted from one aliquot brain tissue using Trizol reagent (Invitrogen) in line with the manufacturer’s protocol. The target gene sequence was obtained from NCBI gene library, and the primers were designed by Beijing Xinlilai Technology Co. Ltd. (Beijing, China) with Primer Premier 5.0 (Canadian Premier Life Insurance Company) software. Glyceraldehyde-3-phosphate dehydrogenase (GADPH) was set as an internal reference. Total RNA (1μg) was taken to reverse transcribe using the SuperScript III Reverse Transcriptase (Invitrogen, CA) with primers. Primers of GFAP in PCR reaction are synthesized as follows: 5’-AGAAAACCGCATCACCATTC-3’ (forward), and 5’-GCACACCTCACATCACATCC-3’ (reverse). Quantitative real-time polymerase chain reaction was performed with a SYBR green core reagent kit (Applied Biosystems) in a 7500 fast real-time polymerase chain reaction system (Applied Biosystems). The expression of GFAP mRNA was determined by Step-One Plus Real Time PCR System (Applied Biosystems). PCR reaction was performed under the following conditions: 95 °C for 2 min, 94 °C for 20 s, 60 °C for 20 s, and 72 °C for 30 s. The run ended after 40 cycles.

2.7 Western blot analysis of GFAP protein

The aliquots for Western blot were homogenized in RIPA lysis buffer (Beyotime, Nantong, China) mixed with 1×complete protease inhibitor cocktail (Beyotime, Nantong, China) and centrifuged. The supernatants were harvested and used for sodium dodecylsulfate-polyacrylamide gel electrophoresis. Equal amounts of protein were diluted in 5×loading buffer. Such steps concerned with proteins as boiling, loading onto 10% polyacrylamide gels and transfering to nitrocellulose membranes were committed in sequence. The membranes were incubated in Tris-buffered saline containing 0.05% Tween-20 and 5% nonfat milk powder for 1 h at room temperature to block the non-specific sites. Subsequently, the membranes were subjected to be immersed fully in the solutions containing monoclonal mouse antibody against GFAP (1:80,000) overnight at 4 °C with rinse, incubation in horseradish peroxidase-labeled goat anti-mouse IgG (1:3,000) for 1 h at room temperature following. After washed with Tris-buffered saline, the membranes were immersed in the electrochemiluminescent reagent, and exposed to Electrochemiluminescence film (Eastman Kodak Company, USA). The strips of target proteins were analyzed by QuantityOne software with β-actin as the internal reference.

2.8 Statistical analysis

The software SPSS 19.0 for Windows (SPSS, Inc., Chicago, IL, USA) was used to analyze all of the data. All results were presented as Mean±SD and were analyzed by one-way analysis of variance, and between group differences were detected with LSD method for homogeneity of variance, and Tamhan’s T2 method for heterogeneity of variance. Values of P < 0.05 were considered statistically significant. And all the figures were performed with GraphPad Prism6.0 Software (GraphPad Software Inc., San Diego, CA). The regression model tested an association between BWT scores (dependent variable) and GFAP levels obtained from immunohistochemistry (independent variable) by curve estimation ofSPSS 19.0.

3 Results

3.1 EA treatment reduces infarct size after MCAO

The infarct size of EA-treated animals and the others are presented as a percent of the contralateral hemisphere (Fig. 2A). EA at Neiguan significantly (P < 0.05 vs both MCAO group and EAd group) reduced infarct size by 37 % compared to the animals of MCAO group (Fig. 2).

Fig.2

EA at Neiguan reduced the cerebral infarct size of rats. (A). Representative HE-stained sections were harvested from the same coronal plane. (B). Graphic representation shows significant reduction in size of infarct following EA treatment at Neiguan. ^* P < 0.05: Group EAn versus Groups naïve control and sham control; ^# P < 0.05: Group MCAO /EAn versus Group MCAO; ^* P < 0.05: Group MCAO /EAn versus Group MCAO /EAd.

3.2 EA treatment induced improvement of neurological deficit in the MCAO rats

3.2.1 EA treatment induced improvement of neurological function scores in the MCAO rats

The surgery of MCAO led to increasing of neurological deficit scores of the rats to 3.06±0.32. For the sham-operated rats there was no difference between before and after surgery. Treatment with EA at Neiguan for 14 days improved significantly the neurological scores compared with the MCAO group (P < 0.05). The score of MCAO group at the end of the experiment was similar to that of MCAO/EAd, and there was statistical no difference between the MCAO group and MCAO/EAd despite slight recovery from cerebral ischemia for the rats of these two groups (Fig. 3).

Fig.3

EA treatment induced improvement of neurological deficit in the MCAO rats. ^* P < 0.05: Group EAn versus Groups naïve control and sham control. ^# P < 0.05: Group EAn versus Group MCAO. ^* P < 0.05 Group MCAO /EAn versus Group MCAO /EAd.

3.2.2 EA treatment induced improvement of the BWT outcome

The rats of both the naïve control and sham control group could walk across the beam swimmingly. The EAn group showed a better BWT performance than that of both MCAO and EAd group (P < 0.05). The results indicated that EA treatment accelerate functional recovery compared to natural recovery. There was no significant difference between the Diji group and the MCAO group (P > 0.05) (Fig. 4).

Fig.4

EAn group showed a better BWT performance than that of both MCAO and EAd group. ^* P < 0.05 Group MCAO /EAn versus Groups naïve control and sham control. ^# P < 0.05 Group EAn versus Group MCAO. ^* P < 0.05 Group EAn versus Group MCAO /EAd.

3.3 EA treatment alleviate the histopathologic changes

In the brains of sham-operated animals, the astrocytes of the cerebral cortex, hippocampus and other parts of the grey matter showed a normal distribution with no morphological changes. In the groups of both model and Diji, the cerebral cortex showed a large necrotic area with a decrease of staining intensity in the H-E sections. The ischemic penumbra showed remarkable intra- and intercellular edema. A large number of inflammatory cells, mainly foam cells derived from macrophages infiltration with neovascularization were seen in the ischemic penumbra. In the Neiguan group, the degree of edema was significantly smaller than that in the both model and Diji groups. The cortical necrosis of Neiguan were decreased obviously with larger scope of repair (Fig. 5A).

Fig.5

EA treatment alleviate the histopathologic injury. The cortical necrosis of group of EAn were decreased obviously with larger scope of repair. (A) 4×. (B) 40×. Treatment with EA at Neiguan resulted in a preventive effect on the pathologic changes of pyramidal neurons in the CA1 area of hippocampus. (C) 4×. (D) 40×.

In addition, neurons in the hippocampus CA1 are selectively vulnerable to ischemia and hypoxia (Nikonenko, Radenovic, Andjus, & Skibo, 2009). In both naïve and sham control group, the hippocampal CA1 pyramidal neurons were tightly arranged, orderly and intact, nuclei were round or oval, stained light blue, with clear nucleolus, uniform chromatin. However, histopathologic analysis of the sections of model rats showed that hippocampal neurons in the CA1 area appeared such histopathologic damages as loss, atrophy, and dark staining compared with those of the naïve and sham control rats. Treatment with EA at Neiguan resulted in a preventive effect on the pathologic neuronal changes. In contrast, treatment with EA at Diji did not show any morphological difference from the animals in MCAO group (Fig. 5).

3.4 EA treatment contributes to modulation of astrocytes activation

The expression and localization of GFAP was examined by immunohistochemical staining. The expression of GFAP was markedly increased in more astrocytes with hypertrophied somata, coarse processes in the region of the ischemic hippocampus at 14 days in the rats treated with EA at Neiguan compared with the other groups (Fig. 6). The relative level of GFAP was obtained by using IPP 4.0 (Fig. 6). To confirm the results of immunohistochemical staining, Western blot methods were used. The semiquantitative analysis of Western blot indicated that EA treatment at Neiguan significant increased the expression of GFAP protein compared with that in both sham and MCAO groups (Fig. 7). We further explored the mRNA expression of GFAP. The results showed a concordance of the increased mRNA expression with protein expression of both genes in the MCAO/EAn comparing with those in model group (P < 0.05) (Fig. 8).

Fig.6

EA treatment contributes to modulation of astrocytes activation. (A). The expression and localization of GFAP of hippocampus was examined by immunohistochemical staining. (B). The relative level of GFAP was obtained by using IPP 4.0. ^* P < 0.05 Group EAn versus Groups naïve control and sham control. ^# P < 0.05 Group MCAO/EAn versus Group MCAO. ^* P < 0.05 Group MCAO/EAn versus Group MCAO/EAd.

Fig.7

(A). The expression of GFAP was examined with Western Blot. (B). The semiquantitative analysis of Western blot indicated that EA treatment at Neiguan significant increased the expression of GFAP protein compared with both sham control and MCAO groups. ^* P < 0.05 Group EAn versus Groups naïve control and sham control. ^# P < 0.05 Group EAn versus Group MCAO. ^* P < 0.05 Group MCAO/EAn versus Group MCAO/EAd.

3.5 The neurorestorative effects of EA is correlated with the level of GFAP

The expression levels of GFAP obtained from immunohistochemical staining and the BWT outcome are shown in Fig. 8. BWT outcome was dependent variables, and the expression levels of GFAP were independent variables. The regression models of association between the neurorestorative effects of EA and the level of GFAP was shown in Table 1 and Fig. 9. These results elucidated that the expression of GFAP have an impact on the outcome of BWT. In the term of pattern of influence, it was likely to be curvilinear relationship between the expression of GFAP and the outcome of BWT. The results of curve estimation showed that the cubic regression model was better fitted with the data of GFAP (R² = 0.776, p = 0.000) compared with the linear regression model and the quadratic model which coefficients of determination (R²) were 0.068 (P = 0.221) and 0.682 (P = 0.000), respectively. The regression model demonstrated that the level of GFAP was involved in the motor neural function restoration with consecutive EA treatments.In case of the level of GFAP was more than 100, the BWT score would increase on the same trend to GFAP. However, when this tendency would continue until the level of GFAP was more than 350, the BWT score would decline with the increase of GFAP(Fig. 9).

Fig.8

mRNA expressions of GFAP and MCT1 in the ischemic brain tissue of each group. ^* P < 0.05 Group EAn versus Groups naïve control and sham control. ^# P < 0.05 Group MCAO/EAn versus Group MCAO. ^* P < 0.05 Group MCAO/EAn versus Group MCAO/EAd.

Fig.9

The neurorestorative effects of EA is correlated with the level of GFAP with a quadratic model which coefficients of determination (R²) were 0.068 (P = 0.221) and 0.682 (P = 0.000), respectively.

4 Discussion

To the best of our knowledge, this study is the first to provide correlation analysis between the tissue levels of the brain specific astroglial protein GFAP in rats of MCAO with the motor neural function restoration with consecutive EA treatments.

The Neiguan point - the Luo point of the pericardium meridian of hand jueyin, which exists in a close physiological and pathological relationship with the brain-is one of the eight confluences that regulate the Qi. Therefore, acupuncture at the Neiguan point is commonly chose for treating cerebral ischemic diseases (Chang et al., 2012; Ren, Wang, Fang, Zhang, & Li, 2010). EA, as one form of acupuncture, delivering electrical stimulation to the acupoints through acupuncture needles, has been recommended for the cerebral ischemia rehabilitation in both Asian and Western countries. A considerable number of studies have been conducted to investigate the effectiveness of EA on stroke. Positive outcomes of acupuncture have been well known as a treatment for achievement of functional recovery after stroke (Wu, Mills, Moher, & Seely, 2010; Yu, Liu, Zhang, & Han, 2005).

Most studies report EA to be effective on acute reduction in lesion size and behavioral impairments, but less is known about its long-term neurorestorative effects following stroke. To address this, we examined EA’s effects on a panel of behavioral tests (Bederson score and beam walking test) sensitive to unilateral ischemic insult.

In the present study, the acupoint PC06 was selected based on previous findings showing the beneficial effects of EA on cerebral ischemia (Lu, et al., 2014, 2015). It was found that functional improvement during treatment with EA consecutive for 7 days after MCAO by up-regulating astrocytic monocarboxylate transporter 1 (MCT1) expression which benefits the energy metabolisms of neurons in the ischemic region. We now document a sustained motor recovery for 14 days, and we further elucidate the possible mechanisms involved. Consistent with the earlier observations, we found that behavioral deficits caused by MCAO were significantly improved by EA treatment for 14 days consecutively.

We reported that EA has the effects on both the activation and proliferation of astrocytes, which are the most abundant cell type in the central nervous system. For long, astrocytes have been considered as a constituent of the brain glue tiling the entire CNS and have remained out of the spotlight well into the 1980 s (Han et al., 2010). Traditionally, they have been viewed as cells that serve supportive roles. Increasing evidence up to now suggests that astrocytes play a critical role in various brain functions. In addition to being involved in the induction and maintenance of the blood brain barrier and neuronal synapses, astrocytes modulate the extracellular ionic osmolarity (Honsa et al., 2014), buffer neurotransmitters (Burda et al., 2016), and maintain water and blood flow (Pekny & Pekna, 2014).

In response to any kind of CNS injury, astrocytes exhibit structural, molecular and functional changes. Astrocytes undergo a characteristic hypertrophy of cell bodies and their processes. Experimental evidence indicates that these astrocytes exert such functions as neuroprotective barriers to inflammatory cells and infectious agents, and formation into scar in particular along borders to severe tissue damage, necrosis, infection or autoimmune-triggered inflammatory infiltration (Bush et al., 1999; Drogemuller et al., 2008; Faulkner et al., 2004; Herrmann et al., 2008; Sofroniew & Vinters, 2010; Voskuhl et al., 2009). The data from many studies using transgenic or another kind of experimental animals confirm that reactive astrocytes protect CNS cells and tissue by such functions as uptake of potentially excitotoxic glutamate, protection from oxidative stress via glutathione production, neuroprotection via adenosine release, protection from NH4⁺ toxicity, neuroprotection by degradation of amyloid-beta peptides, facilitating blood brain barrier repair, reducing vasogenic edema after trauma, stroke or obstructive hydrocephalus, stabilizing extracellular fluid and ion balance and reducing seizure threshold, and limiting the spread of inflammatory cells or infectious agents from areas of damage or disease into healthy CNS parenchyma (Sofroniew, 2014). In addition, other evidences have shown that activated astrocytes promoted by EA are involved in such functions as energy homeostasis through enhancing the astrocyte–neuron lactate shuttle implemented by MCT1 expressed in astrocytes. (Lu et al., 2015), regulation of blood flow in the brain (Allan, 2006), and adjustment of synaptic activity and function (Sofroniew, 2014). In addition, reactive astrocytes induce and release various neurotrophic factors that affect neuronal survival in a paracrine fashion (Han et al., 2010). Our results concerned with astrocytes showed that behavior assessment and molecular data are consistent with the neurorestorative response. After 14 day treatments, the motor functions of the limbs were better in the MCAO/EAn than in control group, suggesting that the EA treatment is quite effective for hemiplegia and disequilibrium caused by acute middle cerebral artery infarction.

GFAP is a hallmark intermediate filament protein uniquely found in astrocytes in CNS. Physically, it plays an essential role in maintaining shape and motility of astrocytic processes and contributes to white matter architecture, myelination and blood-brain barrier integrity. This signature astrocyte cytoskeletal protein is highly regulated in disease. Experimental evidence showed that mouse model eliminating GFAP might decrease the resistance of the brain tissue to severe mechanical stress (Pekny & Pekna, 2014). In mice, lacking GFAP and another intermediate filament proteins Vimentin would impact functional recovery, axonal remodeling after stroke, and increasing the infarct volume by 2.1–3.5 fold larger than in wild-type (WT) mice (Li et al., 2008; Liu et al., 2014). In addition, evidence from an in vitro study demonstrated that GFAP and Vimentin deficiency resulted in increased cell death and confer lower degree of protection to co-cultured neurons than WT astrocytes. However, the functional role of activation of astrocytes after stroke is controversial. Mice deficient in GFAP and Vimentin also exhibited positive outcomes such as better post-traumatic synaptic regeneration (Wilhelmsson et al., 2004), improved axonal regeneration (Cho et al., 2005), and better regeneration and functional recovery after spinal cord trauma (Desclaux et al., 2015; Menet, Prieto, Privat, & Gimenez y Ribotta, 2003).

Therefore, reactive astrocytes exert not only beneficial but also detrimental effects on neuroprotection and neurorestoration. This means that there should be some balances between these two absolutely opposite effects of astrocytes activated after cerebral ischemia. An important question that have to be addressed is to what extent of astrocytes activation is beneficial or detrimental.

The present study employed two tests, Bederson score and BWT, to estimate the neurological outcome. Our observations of behavioral outcomes showed that EA substantially contribute to neurological recovery after cerebral ischemia. Further study results indicated the activation of astrocytes demonstrated by upregulated expression of GFAP is involved in the neurological recovery. These EA beneficial effects of activation of astrocytes in our study seem to be inconsistent with some mentioned above. This difference may suggest that the diversified roles of astrocytes reaction between cerebral ischemia and spinal cord injury. After spinal cord injury, extension of axons owing to reduced scar formation to bridge damage may enhance functional recovery, while it is not necessary to promote neurite extension since there is no neural cell survival in the ischemic infarct core area (Liu et al., 2014). Our data suggest that the restorative effects of EA may be relevant to the activation of astrocytes exhibiting up-expression of GFAP. On the other hand, the relationship between positive effects of EA and the expression level of GFAP is not linear. With the aggravation of GFAP accumulation, its contribution to the improvement of neural function is getting smaller and smaller. This facet of GFAP seems to be involved in the negative effects of astrocyte activation and reactive astrogliosis. Based on the above results, it is clear that EA could provide a preferable modulation of reactive astrocytes to maintain moderate responds in a cerebral ischemia context.

Although reactive astrocytes could form an inhibitory glial scar following stroke, they also perform functions important in neural repair. Our findings suggest the involvement of astrocytes in functional recovery of EA after stroke. The activation of astrocytes represents a therapeutic mechanism for neurorestorative effects of EA. The EA therapy is an approach to modulate astrocytes reactivity and augment their protective functions involved in improvement of neurological recovery after stroke.

5 Conclusions

These findings suggest that EA treatment at the acupoints of Neiguan involved in the regulation of activation of astrocytes, which our data suggested a cubic correlation relationship with the neurological recovery of MCAO rats.

Footnotes

Acknowledgments

This work was supported by the National Natural Scientific Foundation (No. 81202765, No. 81303053, and No. 81373723) and by the China Postdoctoral Science Foundation (No. 2014M561959).

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