Xiao-Ying Liu , Xiao-Hong Dai , Wei Zou , Xue-Ping Yu , Wei Teng Ying Wang, Wei-Wei Yu Hui-Hui Ma Qiu-Xin ChenPeng Liu Rui-Qiao Guan Shan-Shan Dong

1 Heilongjiang University of Chinese Medicine, Harbin, Heilongjiang Province, China

2 The First Affiliated Hospital of Heilongjiang University of Chinese Medicine, Harbin, Heilongjiang Province, China

3 Clinical Key Laboratory of Integrated Traditional Chinese and Western Medicine of Heilongjiang University of Chinese Medicine, Harbin,Heilongjiang Province, China

4 Department of Pharmacology of Dali University, Dali, Yunnan Province, China

Abstract Inflammation plays an important role in nerve defects caused by intracerebral hemorrhage. Repairing brain damage by inhibiting the macrophage‐inducible C‐type lectin/spleen tyrosine kinase (Mincle/Syk) signaling pathway is a potential new target for treating cerebral hemorrhage. In this study, we aimed to determine whether acupuncture through Baihui (DU20) to Qubin (GB7) is an effective treatment for intracerebral hemorrhage through the Mincle/Syk signaling pathway. An intracerebral hemorrhage rat model was established by autol‐ogous blood infusion into the caudate nucleus. Acupuncture through Baihui to Qubin was performed for 30 minutes, once every 12 hours,for a total of three times. Piceatannol (34.62 mg/kg), a Syk inhibitor, was intraperitoneally injected as a control. Modi fied neurological severity score was used to assess neurological function. Brain water content was measured. Immunohistochemistry and western blot assay were used to detect immunoreactivity and protein expression levels of Mincle, Syk, and CARD9. Real‐time polymerase chain reaction was used to determine interleukin‐1β mRNA levels. Hematoxylin-eosin staining was performed to observe histopathological changes. Our re‐sults showed that acupuncture through Baihui to Qubin remarkably improved neurological function and brain water content, and inhibited immunoreactivity and expression of Mincle, Syk, CARD9, and interkeukin‐1β. Moreover, this effect was similar to piceatannol. These find‐ings suggest that acupuncture through Baihui to Qubin can improve neurological impairment after cerebral hemorrhage by inhibiting the Mincle/Syk signaling pathway.

Key Words: nerve regeneration; intracerebral hemorrhage; acupuncture; Baihui (DU20); Qubin (GB7); in flammation; Mincle/Syk signaling pathway; nerve protection; neural regeneration

Introduction

Approximately 2 million individuals worldwide are diagnosed with intracerebral hemorrhage (ICH) each year (Sudlow and Warlow, 1997; Broderick et al., 2007; Lloyd‐Jones et al., 2009;Qureshi et al., 2009). Furthermore, ICH is one of the most fatal types of stroke, and results in severe mental dysfunction generated by secondary nerve damage in patients who survive the primary stroke (Aronowski and Zhao, 2011). Current therapies for ICH have a poor impact on prognosis (Mayer and Rincon, 2005; Keep et al., 2012). Cytotoxicity, oxidative stress, and especially, in flammation play key roles in second‐ary injury after ICH (Keep et al., 2012; Zhou et al., 2014).Consequently, regulation of inflammation after ICH may provide insight for future ICH therapies (Zhao et al., 2007).

Acupuncture is one of the most important components of traditional Chinese medicine and widely used in the treatment of various diseases (Lou et al., 2016). Moreover,its curative effect has been widely recognized and accepted worldwide (Meng et al., 2011; Liu et al., 2016, 2017) by the in‐ternational medical community. Our previous study revealed that acupuncture throughBaihui(DU20) toQubin(GB7)has a “reparative” function by inducing expression of endog‐enous glial cell line‐derived neurotrophic factor during acute cerebral hemorrhage (Zhang et al., 2012). More speci fically,acupuncture can improve recovery of neural stem cells by suppressing expression of both Notch1 and Hes1 (Zou et al.,2015). Acupuncture can also antagonize in flammatory brain injury generated by cerebral hemorrhage by suppressing the classical nuclear factor‐κB pathway (Liu et al., 2017). Thus,acupuncture is an effective means of lowering expression of in flammatory mediators in the nervous system. Furthermore,according to current reports, acupuncture has greatly con‐tributed to reducing the rate of stroke‐induced disability and improving recovery of neural function. Studies investigating acupuncture have found that it is a highly potent therapy for reducing neural in flammation (Liu et al., 2016), suppressing cell apoptosis, and alleviating nerve dysfunction (Ma et al.,2016) after stroke. Altogether, these studies show that acu‐puncture has great potential for treating cerebral hemorrhage by inhibiting in flammation.

The immune system plays an important role in the in‐flammatory response. Compared with the adaptive immune system (which is highly pathogen specific), innate immune receptors recognize a variety of pathogens with similar struc‐tures (Park et al., 2006; Tang et al., 2007; Shichita et al., 2012).Toll‐like receptors are a classical example of innate immune receptors, and are a “hot topic” of research because of their participation in inflammation induced by neural system disease, including ischemic stroke and cerebral hemorrhage(Fadakar et al., 2014; Lan et al., 2017). Macrophage‐inducible C‐type lectin (Mincle) is a recently discovered innate immune receptor that was originally recognized as a macrophage tar‐get in the peritoneum (Matsumoto et al., 1999). Toll‐like re‐ceptors recognize ligands expressed on necrotic cells, and in‐teract with downstream spleen tyrosine kinase (Syk) to active a pathway that can induce generation of several in flammatory cytokines, including interleukin (IL)‐1β (Brown, 2008). The Mincle/Syk pathway plays a role in traumatic brain injury (de Rivero Vaccari et al., 2015), subarachnoid hemorrhage, and ischemic stroke (Suzuki et al., 2013; He et al., 2015; Xie et al.,2017). However, the regulatory mechanism of the Mincle/Syk pathway in cerebral hemorrhage remains unclear. Fur‐thermore, it is also not known whether acupuncture can treat cerebral hemorrhage by regulating the Mincle/Syk pathway.

In this study, we first used an autohemic blood infusion method to establish a rat model of cerebral hemorrhage for studying the effectiveness of acupuncture throughBaihuitoQubin. We then determined the therapeutic mechanism of acupuncture through the Mincle/Syk pathway.

Materials and Methods

Animals

A total of 312 male speci fic pathogen‐free Sprague‐Dawley rats, aged 8 weeks and weighing 280—320 g, were purchased from the Laboratory Animal Center, Heilongjiang University of Chinese Medicine, China (license No. SYXK (Hei) 2016‐015). All experiments were approved by the Animal Care and Use Committee of Heilongjiang University of Chinese Medi‐cine of China (approval No. 2016‐09‐02‐01). Animal care and experimental procedures were performed in accordance with the United State National Institutes of Health Guide for the Care and Use of Laboratory Animals (publication No. 85–23,revised 1985). All surgeries were performed under pentobar‐bital anesthesia, and all efforts were made to minimize suffer‐ing. Rats were housed under a 12‐hour light/dark cycle at 21± 2°C and 50 ± 5% relative humidity, with unlimited access to standard food and water. Rats were randomly divided into four groups: sham (n= 72), intracerebral hemorrhage (ICH)(n= 72), piceatannol (a Syk inhibitor) (n= 72), and acupunc‐ture (n= 72). All groups were evenly divided into four sub‐groups (n= 18 each), which were assessed at 6, 12, 24, and 72 hours after treatment.

Establishment of a cerebral hemorrhage rat model

Rats were anesthetized with pentobarbital (60 mg/kg; in‐traperitoneally) (Royalton, Dalian, China), and fixed on a stereotactic frame (STW‐3X; Chengdu Instrument Factory,Chengdu, China). Bregma and the coronal sulcus were ex‐posed by cutting the scalp midline. A dental drill was used at the interchange of 0.2 mm behind and 3.5 mm right from bregma. Drilling was performed to a diameter of 1 mm until the meninges was reached. Autologous tail tip blood was in‐jected into the caudate nucleus (20 μL/min) (anteroposterior:?0.24 mm, lateral: 3.5 mm, dorsal: 6 mm) (right side is the in‐jury side) (Paxinos and Watson, 2007). The microsyringe was exited from the caudate—putamen after 5 minutes. The wound was filled using dental cement, and subsequently stitched,bandaged, and sterilized (Liu et al., 2017). According to Berd‐erson’s scale (Bederson et al., 1986), the model was successful if the score was 1 to 3 at 2 hours after modeling.

Operations for sham models were identical except for blood injection.

Treatment

After successful model establishment, in the acupuncture group,Baihui(DU20) (speci fic location: head between the middle ears) andQubin(GB7) (specific location: leading edge of ear root) points were identi fied (Li, 2007). Hair was cut around theBaihui(DU20) point, and a 0.30 × 25 mm acupuncture needle (Hua Tuo Brand; Suzhou Medical Ap‐pliance, Suzhou, China) used to pierce from the epicranial aponeurosis to theQubin(GB7) point in the bottom right direction. The needle pierced to a depth of 15 mm, and was then rotated at 200 r/min for 5 minutes. For each 30‐minute session, needling at 200 r/min was performed for three ses‐sion, each lasting for 5 minutes.

In the sham and ICH groups, rats were fixed on the frame for 30 minutes without any operation.

In the piceatannol group, rats were intraperitoneally in‐jected with piceatannol (34.62 mg/kg) (license No. 10083‐24‐6; Selleck, Shanghai, China) at 1 hour after model estab‐lishment.

Modi fied neurological severity score (mNSS)

The mNSS was analyzed at 6, 12, 24, 72, and 168 hours after model establishment (Lei et al., 2015). Higher scores indicate poorer neurological function. More detailed information is provided in Additional file 1.

Brain water content (BWC)

Brains were obtained by decapitation at 6, 12, 24, and 72 hours after treatment in each subgroup. Left and right hemispheres were separated. Wet weight was weighed. Dry weight was weighed after a 72‐hour incubation in a drying cabinet (Binder, Germany) at 105°C. BWC was calculated by: wet weight — dry weight) / wet weight × 100%.

Histopathological assay

Brain tissue of rats in each group was collected at 72 hours after surgery, perfused with 4% paraformaldehyde, and cut into 4‐μm slices for routine hematoxylin and eosin staining to observe organizational structure. Images were captured using an optical microscope (Leica, Germany) at 200×magni fication.

Immunohistochemistry

Rats were treated with pentobarbital (60 mg/kg) by intra‐peritoneal injection, followed by perfusion fixation with 4%paraformaldehyde. Brain tissue was obtained after decapita‐tion, and fixed in paraformaldehyde following dehydration.After paraffin embedding and histological sectioning (5 μm),sections were incubated with primary antibodies, namely rab‐bit anti‐Mincle (1:500; Bioss, Beijing, China), rabbit anti‐Syk(1:500; Bioss), and rabbit anti‐caspase recruitment domain family member 9 (CARD9) (1:500; Abcam, Cambridge, UK)overnight at 4°C. Sections were then incubated with goat anti‐rabbit IgG (1:2000; Abcam) as a secondary antibody at 37°C for 30 minutes. Images were captured using a micro‐scope imaging system (Moticam 3000; Motic, Hong Kong Special Administrative Region, China). Each sample was ex‐amined in captured images from five non‐overlapping visual fields under a 400× magni fication. Average number of posi‐tive cells was calculated (Xue et al., 2014).

Western blot assay

The right hemisphere of rats at 6, 12, 24, and 72 hours after ICH was lysed, with protein samples isolated and collected.Fifty micrograms (50 μg) of sample was loaded onto a so‐dium dodecyl polyacrylamide gel and transferred to a poly‐vinylidene difluoride membrane. The membrane was then blocked using 5% non‐fat dry milk, and subsequently incu‐bated with primary antibody overnight at 4°C, followed by secondary antibody at room temperature for 2 hours. Rabbit anti‐Mincle (1:1000; Bioss), rabbit anti‐Syk (1:1000; Bioss),rabbit anti‐CARD9 (1:1000; Abcam), and rabbit anti‐β‐ac‐tin (1:1000; Bioss) were used as primary antibodies. Goat anti‐rabbit IgG (1:2000; Abcam) was used as the secondary

antibody. Protein signal was marked using a commercially available 3, 3′‐diaminobenzidine (DAB) electrochemilumi‐nescence system (Beyotime, Jiangsu, China), and images captured (Duris et al., 2011). Optical density values for speci fic bands were measured using ImageJ software (NIH,Bethesda, MD, USA).

Real-time polymerase chain reaction (PCR)

RNA was extracted from the brain using a commercially available kit (RNAiso Plus; Takara Bio Inc., Dalian, China),according to the manufacturer’s instructions. Complemen‐tary DNA (cDNA) was reverse transcribed using the Prime Script II 1st Strand cDNA Synthesis Kit (Takara Bio Inc.).The reverse transcription conditions were: 42°C for 60 min‐utes, 95°C for 5 minutes, and then storage on ice. The prim‐ers used were: β‐actin‐forward, 5′‐AAC ACC CCA GCC ATG TAC GTA‐3′; β‐actin‐reverse, 5′‐TGG CCA TCT CTT GCT CGA A‐3′; IL‐1β‐forward, 5′‐AAG GGG ACA TTA GGC AGC AC‐3′; and IL‐1β‐reverse, 5′‐ATG AAA GAC CTC AGT GCG GG‐3′. Real‐time PCR was performed us‐ing the SYBR Premix Ex Taq kit (Takara Bio Inc.). The PCR conditions were: 95°C for 30 seconds, 95°C for 5 seconds,and 60°C for 34 seconds for 40 cycles, with reactions held at 4°C. IL‐1β mRNA was calculated by the 2‐ΔΔCtmethod (Xie et al., 2017).

Statistical analysis

Data are represented as mean ± SD for mNSS, western blot‐ting, and as median (interquartile range) for BWC, immu‐nohistochemistry, and real‐time PCR. All analyses were per‐formed using Graph Pad Prism 6 software (GraphPad, San Diego, CA, USA).Pvalues for mNSS and western blotting were determined by one‐way analysis of variance followed by Tukey’spost hoctest. BWC, immunohistochemistry and real‐time PCR were examined using the Kruskal‐Wallis test followed by Mann‐WhitneyUtest for pairwise comparisons.A value ofP＜ 0.05 was considered statistically signi ficant.

Results

Effect of acupuncture on neural function after ICH

No signi ficant differences in mNSS were detectable among subgroups (6, 12, 24, 72, and 168 hours) in the sham group(P＞ 0.05). Compared with the sham group, mNSS score was signi ficantly increased in the ICH group (P＜ 0.01). mNSS was signi ficantly lower in the acupuncture and piceatannol groups compared with the ICH group (P＜ 0.01). No signif‐icant differences were observed in mNSS score between the acupuncture and piceatannol groups (P＞ 0.05; Figure 1).

Effect of acupuncture on BWC after ICH

BWC was relatively minor in the sham group (Figure 2).However, compared with the sham group, BWC was signi fi‐cantly increased in the ICH group (P＜ 0.05). BWC was sig‐ni ficantly lower in the acupuncture and piceatannol groups compared with the ICH group (P＜ 0.05). No signi ficant dif‐ference in BWC was observed between the acupuncture and piceatannol groups (P＞ 0.05; Figure 2).

Figure 1 Effect of acupuncture through Baihui (DU20) to Qubin(GB7) on mNSS in a rat model of intracerebral hemorrhage.

Figure 2 Effect of acupuncture through Baihui (DU20) to Qubin (GB7)on brain water content in a rat model of intracerebral hemorrhage.

Figure 3 Effect of acupuncture through Baihui (DU20) to Qubin (GB7) on brain histopathology of rats after intracerebral hemorrhage(hematoxylin and eosin staining, original magni fication, 200×).

Effect of acupuncture on brain histopathology after ICH

Hematoxylin and eosin staining was performed to examine histological changes 72 hours after ICH (Figure 3). Rats re‐ceiving sham surgery had no blood injection into the brain and showed normal microscopic features, with no inflam‐matory infiltration from the semi‐dark area. However, the ICH group showed intracranial blood injection, tissue ede‐ma, partial nerve cell necrosis, glial cell swelling, intracellular vacuoles, and inflammatory cell infiltration. Furthermore,in rats receiving acupuncture or piceatannol treatment after blood injection, the pathological features were less severe.

Effect of acupuncture on the Mincle/Syk pathway in rats with ICH

Immunopositivity of Mincle, Syk, and CARD9 in the brain as detected by immunohistochemistry

Immunopositivity of Syk and CARD9 were signi ficantly re‐duced in the acupuncture and piceatannol groups compared with the ICH group (P＜ 0.05). Immunopositivity of Syk and CARD9 showed no statistical differences between the acu‐puncture and piceatannol groups (P＞ 0.05). Compared with the ICH group, Mincle immunopositivity was decreased in the acupuncture group (P＜ 0.05). However, Mincle immu‐nopositivity was not signi ficantly different between the ICH and piceatannol groups (P＞ 0.05; Figure 4).

Protein expression of Mincle, Syk, and CARD9 in the brain as detected by western blot assay

Expression levels of Syk and CARD9 were signi ficantly re‐duced in the acupuncture and piceatannol groups compared with the ICH group (P＜ 0.01). There were no statistically signi ficant differences in Syk and CARD9 expression levels between the acupuncture and piceatannol groups (P＞ 0.05).Compared with the ICH group, Mincle expression levels were signi ficantly decreased in the acupuncture group (P＜0.01). However, Mincle expression levels were not signifi‐cantly different between the ICH and piceatannol groups (P＞ 0.05; Figure 5.

IL-1β mRNA expression in the brain as detected by real-time PCR

IL‐1β mRNA levels were signi ficantly increased in the ICH group compared with the sham group (P＜ 0.01). Compared with the ICH group, expression levels of IL‐1β mRNA were significantly decreased in the acupuncture and piceatan‐nol groups (P＜ 0.01). However, expression levels of IL‐1β mRNA were not different between the acupuncture and piceatannol groups (P＞ 0.05; Figure 6).

Figure 5 Effect of acupuncture through Baihui (DU20) to Qubin (GB7) on protein expression of CARD9, Syk, and Mincle in the brain of rats at 6,12, 24, and 72 hours after intracerebral hemorrhage (western blot assay).

Figure 6 Effect of acupuncture through Baihui (DU20) to Qubin(GB7) on mRNA expression of IL-1β at 6, 12, 24, and 72 hours in the brain of rats after intracerebral hemorrhage.

Discussion

Peripheral tissues are exposed to a massive amount of blood after ICH, which in turn provokes a variety of cellular and molecular effects. Activation of cells and in flammation can trigger infiltration of polymorphonuclear leukocytes and monocytes, activation of microglia, damage to the blood‐brain barrier, and development of brain edema, which contributes to secondary brain injury (Aronowski and Hall,2005; Wang and Dore, 2007). Meanwhile, previous studies have shown that acupuncture can reduce neural in flamma‐tion (Liu et al., 2016), suppress cell apoptosis, and alleviate nerve dysfunction (Ma et al., 2016) after stroke, but it re‐mains unclear whether acupuncture is effective for treating cerebral hemorrhage. Consequently, in this study, we first examined the protective effects of acupuncture throughBaihui(DU20) toQubin(GB7) in a cerebral hemorrhage animal model. We then further investigated the effect of acupuncture on in flammation and its protective mechanism.Our results indicate that acupuncture could be an excellent tool for ICH therapy.

With the advent7 of college, I was brought to grips with the problem of earning a living. Part-time teaching of piano and harmony and, upon graduation, occasional concerts and lectures, proved only partial sources of livelihood8. In terms of time and effort involved, the financial remuneration（,） was disheartening. This induced within me searing self-doubt and dark moods of despondency. Adding to my dismal9 sense of inadequacy10 was the repeated experience of seeing my sisters and friends go off to exciting dates. How grateful I was for my piano, where—through Chopin, Brahms, and Beethoven -- I could mingle11 my longing12 and seething13 energy with theirs. And where I could dissolve my frustration14 in the beauty and grandeur15（,） of their conceptions.

As already described, inflammation plays an important role in ICH (Tang et al., 2017). Inflammation leads to cell swelling and damage, which in turn induces brain edema.In flammation, clotting, and erythrocyte lysis caused by pri‐mary injury can result in brain edema, which is associated with poor recovery from injury and may lead to more severe and prolonged brain injury (Zheng et al., 2016). Thus, de‐creasing brain edema is critical for protecting neurovascular structures. Acupuncture is one of the most important com‐ponents of traditional Chinese medicine, and has greatly contributed to reducing the rate of stroke‐induced disability and improving recovery of neural function. Our results show that acupuncture throughBaihui(DU20) toQubin(GB7) can decrease mNSS and reduce BWC in a cerebral hemorrhage animal model. Moreover, hematoxylin‐eosin staining showed that acupuncture throughBaihui(DU20)toQubin(GB7) also decreased capillary permeability and leakage. Our data are consistent with other similar studies(Liu et al., 2017).

The innate immune system is critical in development of neurovascular injury and a major reason for excessive pa‐renchymatous in flammation. Fang et al., (2013) found that both innate immunity and in flammation participate in the pathological process of ICH. Necrosis of leukocytes can in‐duce microglial cells and macrophages to release proin flam‐matory cytokines, thereby leading to further brain injury(Wang and Dore, 2007). Animal experiments have shown that factors inhibiting microglia/macrophages can remark‐ably reduce brain injury and improve neural function in rodents (Zhao et al., 2011).

Mincle is a pattern‐recognition receptor primarily ex‐pressed in myeloid cells, especially antigen‐presenting cells. It is also expressed on the surface of B cells, microglia,neurons, and mast cells (Flornes et al., 2004; McKimmie et al., 2006; Ribbing et al., 2011; Kawata et al., 2012; He et al., 2015). Mincle expression is low under normal circum‐stances. During infection and tissue damage, Mincle is upregulated and binds to endogenous antigens, leading to recruitment and activation of Syk. Syk can activate protein kinase C‐δ, which phosphorylates downstream CARD9.This activates the nuclear factor‐κB pathway via CARD9/B‐cell lymphoma/leukemia 10 (BCL 10), which ultimately generates biologically active IL‐1β (Takizawa et al., 2001;Strasser et al., 2012; Yasukawa et al., 2014). As a mediator of the immune response and activation point of in flammation,IL‐1β expression levels directly re flect grades of in flamma‐tion after cerebral hemorrhage (Gross et al., 2009). Previous studies have shown that the Mincle/Syk signaling pathway is involved in many innate immune responses including isch‐emic stroke, traumatic brain injury, and subarachnoid hem‐orrhage (Suzuki et al., 2013; de Rivero Vaccari et al., 2015;He et al., 2015; Arumugam et al., 2017; Xie et al., 2017). Our study found that acupuncture markedly suppressed protein expression in the Mincle/Syk pathway and decreased IL‐1β expression in rat brain tissue following hemorrhage. These results suggest that acupuncture throughBaihui(DU20) toQubin(GB7) may mitigate cerebral hemorrhage injury neu‐ritis and improve neural function by suppressing the Min‐cle/Syk pathway, which further downregulates expression levels of downstream in flammatory cytokines.

Piceatannol, a Syk inhibitor, suppresses Syk/CARD9 ex‐pression during ischemic stroke and subarachnoid hemor‐rhage, reduces IL‐1β levels, improves neural function, and facilitates brain edema therapy (Suzuki et al., 2013; He et al., 2015; Xie et al., 2017). Our study found no significant differences in the effects of therapy in rats treated with acu‐puncture compared with the piceatannol group. This further suggests that acupuncture throughBaihui(DU20) toQubin(GB7) can in fact affect the Mincle/Syk pathway, mitigate inflammatory brain injury, improve neural function, and alleviate brain edema.

Our study has a few limitations. First, we have searched relevant literature within and outside China, but reports on the Mincle/Syk signaling pathway are still limited. The mechanism by which blocking Mincle improves ICH out‐come needs further investigation. Second, we did not ex‐amine changes of Mincle/Syk between different time points after ICH as the purpose of our study was to determine whether the therapeutic effect of acupuncture was signi ficant compared with the other experimental groups at the same time point. Third, connections between Mincle and other signaling pathways after ICH requires further studies.

In summary, acupuncture throughBaihui(DU20) toQubin(GB7) may suppress the Mincle/Syk pathway and reduce the release of proinflammatory cytokines, thereby mitigating neurological damage after cerebral hemorrhage.

Author contributions:XYL and XHD participated in study conception and design. XYL, WZ, and XPY wrote this paper. WT, YW, WWY, and HHM were responsible for data analysis. QXC, PL, RQG, and SSD were responsible for tissue processing. All authors approved the final version of the paper.

Financial support:This study was supported by the National Natural Science Foundation of China, No. 81473764, No. 81273824; the Key Project of Natural Science Foundation of Heilongjiang Province of China, No. ZD201204; the Doctoral Fund Program of Ministry of Education of China, No. 20102327110003. All authors declare that financial support does not affect the opinion of the article and the objective statistical analysis and report of the research results in this study.

Institutional review board statement:All experiments were approved by the Animal Care and Use Committee of Heilongjiang University of Chinese Medi cine of China (approval No. 2016-09-02-01). Animal care and experimental procedures were performed in accordance with the United State National Institutes of Health Guide for the Care and Use of Laboratory Animals (publication No. 85-23, revised 1985).

Copyright license agreement:The Copyright License Agreement has been signed by all authors before publication.

Data sharing statement:Datasets analyzed during the current study are available from the corresponding author on reasonable request.

Plagiarism check:Checked twice by iThenticate.

Peer review:Externally peer reviewed.

Open access statement:This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution-Non-Commercial-ShareAlike 4.0 License, which allows others to remix, tweak,and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms.

Open peer reviewer:Clarissa Cavarsan, Universidade Federal do Paraná, Brazil.

Additional files:

Additional file 1:Modi fied neurological severity score points.

Additional file 2:The primary data of Figure 2.