新疆阿魏干预神经源性疼痛模型大鼠痛阈及脊髓Fos蛋白和星形胶质细胞的表达

doi:10.3969/j.issn.2095-4344.2015.40.017

中国组织工程研究 ›› 2015, Vol. 19 ›› Issue (40): 6485-6491.doi: 10.3969/j.issn.2095-4344.2015.40.017

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新疆阿魏干预神经源性疼痛模型大鼠痛阈及脊髓Fos蛋白和星形胶质细胞的表达

黄异飞^1，2，胡炜¹，李磊³，刘岩路¹

1新疆维吾尔自治区中医医院脊柱二科，新疆维吾尔自治区乌鲁木齐市 830000； 2新疆医科大学附属中医医院博士后流动站，新疆维吾尔自治区乌鲁木齐市 830000； 3新疆医科大学研究生学院，新疆维吾尔自治区乌鲁木齐市 830000

出版日期:2015-09-30 发布日期:2015-09-30
通讯作者: 胡炜，博士，副主任医师，新疆维吾尔自治区中医医院脊柱二科，新疆维吾尔自治区乌鲁木齐市 830000
作者简介:黄异飞，男，1969年生，广东省人，汉族，2003年北京大学医学部毕业，博士，主任医师，教授，主要从事脊柱相关疾病的中西医结合治疗。
基金资助:
新疆维吾尔自治区自然科学基金项目(201233146-17)

Effect of Ferula sinkiangensis K.M. Shen on pain threshold and Fos protein expression and astrocyte activation in the spinal cord of neuropathic pain rats

Huang Yi-fei^{1, 2}, Hu Wei¹, Li Lei³, Liu Yan-lu¹

1 Second Department of Spine Surgery, Traditional Chinese Medicine Hospital of Xinjiang Uygur Antonomous Region, Urumqi 830000, Xinjiang Uygur Antonomous Region, China 2 Postdoctoral Research Station, Traditional Chinese Medicine Hospital of Xinjiang Uygur Antonomous Region, Urumqi 830000, Xinjiang Uygur Antonomous Region, China 3 Postgraduate School of Traditional Chinese Medice Hospital of Xinjiang Uygur Antonomous Region, Urumqi 830000, Xinjiang Uygur Antonomous Region, China

Online:2015-09-30 Published:2015-09-30
Contact: Hu Wei, M.D., Associate chief physician, Second Department of Spine Surgery, Traditional Chinese Medicine Hospital of Xinjiang Uygur Antonomous Region, Urumqi 830000, Xinjiang Uygur Antonomous Region, China
About author:Huang Yi-fei, M.D., Professor, Chief physician, Second Department of Spine Surgery, Traditional Chinese Medicine Hospital of Xinjiang Uygur Antonomous Region, Urumqi 830000, Xinjiang Uygur Antonomous Region, China; Postdoctoral Research Station, Traditional Chinese Medicine Hospital of Xinjiang Uygur Antonomous Region, Urumqi 830000, Xinjiang Uygur Antonomous Region, China
Supported by:
the Natural Science Foundation of Xinjiang Uygur Antonomous Region of China, No. 201233146-17

摘要/Abstract

摘要：

背景：新疆阿魏主要由挥发油、树脂和树胶组成，具有抗炎、抗过敏及解痉镇痛作用。但其镇痛作用机制尚不明确，
目的：观察新疆阿魏对神经病理性疼痛大鼠热痛及机械痛及脊髓Fos蛋白和星形胶质细胞表达的影响。
方法：成年SD大鼠80只制备慢性坐骨神经松结扎大鼠模型后随机分为5组：分别灌胃低、中、高剂量阿魏0.075，0.15，0.30 g/kg，赛来昔布和生理盐水。各组分别在术前1 d及术后1，2，3，5，7，14 d 进行热痛及机械痛测定，并取S4-5脊髓组织，并采用免疫组织化学染色的方法观察脊髓Fos蛋白和星形胶质细胞表达变化。
结果与结论：给药后1，5 d，低、中、高剂量阿魏组均高于生理盐水组(P < 0.01)，中剂量阿魏组热痛阈值下降幅度最小(P < 0.05)，高剂量阿魏组机械痛阈值减小幅度最小(P < 0.01)。术后各时间点低、中、高剂量阿魏组及赛来昔布组在的Fos蛋白阳性细胞数均小于生理盐水组(P < 0.05)，中、高剂量阿魏组各时间点Fos蛋白阳性细胞数均高于赛来昔布组(P < 0.05)。高剂量阿魏组及赛来昔布组在术后各个时间点的脊髓组织星形胶质细胞阳性细胞数均明显少于生理盐水组(P < 0.05)；中、高剂量阿魏组与赛来昔布组各时间点的星形胶质细胞阳性细胞数比较差异有显著性意义 (P < 0.05)。结果证实，新疆阿魏可以有效缓解大鼠神经病理性疼痛，其机制可能与脊髓Fos蛋白和星形胶质细胞的激活有关。

中国组织工程研究杂志出版内容重点：肾移植；肝移植；移植；心脏移植；组织移植；皮肤移植；皮瓣移植；血管移植；器官移植；组织工程

关键词: 实验动物, 脑及脊髓损伤动物模型, 新疆阿魏, 神经源性疼痛, 坐骨神经性疼痛, 大鼠, 镇痛, 痛阈, 热痛, 机械痛, Fos蛋白, 星形胶质细胞, 新疆维吾尔自治区自然科学基金

Abstract:

BACKGROUND: Ferula sinkiangensis K.M. Shen is composed of volatile oil, resin and gum that have the anti-inflammatory, anti-allergic, antispasmodic and analgesic effects. But its analgesic mechanism is unclear.

OBJECTIVE: To observe the effect of Ferula sinkiangensis K.M. Shen on heat pain, mechanical pain, Fos protein expression and astrocyte activation in spinal cord of rats with neuropathic pain.

METHODS: Eighty adult Sprague-Dawley rat models of chronic sciatic nerve injury were randomly divided into five groups and then intragastically administered Ferula sinkiangensis K.M. Shen at low, moderate and high doses (0.075, 0.15, 0.30 g/kg), celecoxib or physiological saline. Heat pain and mechanical pain were measured at 1 day before operation and at 1, 2, 3, 5, 7, 14 days after operation. The spinal cord tissue at S₄_-₅ segments was harvested and Fos protein expression and astrocyte activation in the spinal cord of rats were observed by immunohistochemical staining method.

RESULTS AND CONCLUSION: After 1 and 5 days of medication, behavioral pain scores of rats in the low-, moderate-, and high-dose Ferula sinkiangensis K.M. Shen groups were significantly higher than that in the physiological saline group (P < 0.01). The largest reduction in heat pain threshold was measured in the moderate-dose Ferula sinkiangensis K.M. Shen group compared to the other groups (P < 0.01). The most significant reduction in rat mechanical pain threshold was measured in the high-dose Ferula sinkiangensis K.M. Shen group than in the other groups (P < 0.01). At each time point post-operation, the number of Fos protein-positive cells in the low-, moderate- and high-dose Ferula sinkiangensis K.M. Shen and celecoxib groups was significantly lower than that in the physiological saline group (P < 0.05); the number of Fos protein-positive cells in the moderate- and high-dose Ferula sinkiangensis K.M Shen groups was significantly higher than that in the celecoxib group (P < 0.05). At each time point post-operation, the number of astrocytes in the spinal cord tissue of rats in the high-dose Ferula sinkiangensis K.M. Shen and celecoxib groups was significantly lower than that in the physiological saline group (P < 0.05). There was significant difference in the number of astrocytes between the moderate- and high-dose Ferula sinkiangensis K.M shen groups and celecoxib group (P < 0.05). These results confirm that Ferula sinkiangensis K.M. Shen may effectively alleviate the neuropathic pain of rats, and the mechanism of which may be related to the activation of Fos protein and astrocytes in the spinal cord.

中国组织工程研究杂志出版内容重点：肾移植；肝移植；移植；心脏移植；组织移植；皮肤移植；皮瓣移植；血管移植；器官移植；组织工程

Key words: Tissue Engineering, Model, Animal, Pain, Sciatic Nerve

中图分类号:

R318

黄异飞，胡炜，李磊，刘岩路. 新疆阿魏干预神经源性疼痛模型大鼠痛阈及脊髓Fos蛋白和星形胶质细胞的表达[J]. 中国组织工程研究, 2015, 19(40): 6485-6491.

Huang Yi-fei, Hu Wei, Li Lei, Liu Yan-lu. Effect of Ferula sinkiangensis K.M. Shen on pain threshold and Fos protein expression and astrocyte activation in the spinal cord of neuropathic pain rats[J]. Chinese Journal of Tissue Engineering Research, 2015, 19(40): 6485-6491.

图/表（结果） 3

The number of animals and process of modeling

Eighty rats were included in the final analysis without any loss.

Effects of Ferula sinkiangensis K.M. Shen on behavioral pain scores

Spontaneous pain scores were significantly higher in all groups at 1, 2, 3, 5, 7 and 14 days after CCI compared to those before CCI, with the highest scores observed at 7 days after injury (P < 0.01). Scores were significantly lower in the CCI+A2, CCI+A3 and CCI+S groups compared to the CCI+NS group at 2, 3, 5 and 7 days following drug administration (P < 0.05), suggesting that Ferula sinkiangensis K.M. Shen acts as an analgesic. Pain scores were significantly higher in the CCI+A1 and CCI+A2 groups compared to the CCI+S group at 3, 5, 7 and 14 days after drug administration (P < 0.01), indicating less analgesic efficacy compared to the COX-2 inhibitor celecoxib. In addition, higher scores were observed in the CCI+A3 group compared to the CCI+S group at 5, 7 and 14 days after drug administration (P < 0.05; Table 1). TWL changes

There were no significant differences in TWL among groups before CCI (P=0.148). After injury, however, all groups exhibited a decrease in TWL which was apparent at 1 day before treatment (P=0.033 compared to pre-CCI). The largest reduction after CCI was measured in the CCI+ NS group. In this group, TWL was slowly increased at the beginning at about 5 days before treatment. In the three Ferula sinkiangensis K.M. Shen groups, however, TWLs were significantly higher than those in the CCI+NS group at days 1 and 5 before treatment (P=0.008), and significantly higher in the CCI+A2 group compared to the CCI+NS group at each time point (P=0.034). The TWLs were significantly lower in the CCI+A1 and CCI+A3 groups compared to CCI+A2 at 2, 5 and 14 days before treatment (P=0.033), suggesting that the moderate dose provided optimal thermal analgesia. Although TWLs did not recover to the values before treatment in any group at 14 days before treatment. TWLs in the CCI+A1 and CCI+A2 groups were still significantly higher than that in the CCI+NS group (P=0.001; Table 2).

WMT changes There was no significant difference in WMT among the five groups before CCI (P=0.752). The largest reduction of WMT after treatment was observed in the CCI+NS group, with the most significant reduction at 1 day (P=0.03). The WMT reached a minimal value at 5 day before treatment, and then gradually recovered. There was no significant difference in the WMT between the CCI+A1 group and the CCI+NS group(P=0.107). However, significantly higher WMTs were measured in the CCI+A2 group compared to the CCI+NS group at 2, 5, 7 and 14 days before treatment (P=0.000). The WMTs in the CCI+A3 group were also ignificantly higher than those in the CCI+NS group at 1, 2, 3, 5, 7 and 14 days after CCI (P=0.000) and significantly higher than those in the CCI+A1 group at 1, 2, 3, 5, 7 and 14 days after CCI (P=0.000). Furthermore, WMTs were significantly higher in the CCI+A3 group compared to the CCI+A2 group at 3 and 5 days after treatment (P=0.003), suggesting that high dose Ferula sinkiangensis K.M. Shen provided moderately superior mechanical analgesia. All WMTs recovered to the pre-treatment values in the three Ferula sinkiangensis K.M. Shen groups at 14 days after CCI (Table 3).

Fos protein expression in rat spinal cord

Most of the Fos-like immunoreactive cells in the rat spinal cord appeared spindle-shaped or oval, and staining was restricted to cell nuclei. Significant increases in the number of Fos-like immunoreactive cells were observed in all groups after CCI treatment, but the numbers were significantly lower in the CCI+A1, CCI+A2, CCI+A3 and CCI+S groups compared to the CCI+NS group at 1, 2, 3, 5, 7 and 14 days after CCI (P < 0.05 or 0.01), indicating that analgesia after CCI reduced spinal Fos expression. The number of Fos-like immunoreactive cells was slightly greater in the CCI+A3 group compared to the CCI+S group at 1, 2, 3, 5, 7 and 14 days after CCI, but the difference was not significant (P > 0.05). Fos-like immunoreactivity was significantly higher in the CCI+A1 and CCI+A2 groups than that in the CCI+S group at 1, 2, 3, 5, 7 and 14 days after treatment (P < 0.05; Table 4, Figure 1).

Astroglial GFAP expression in rat spinal cord

The largest number of GFAP-positive cells was observed at 7 days after CCI for all treatment groups. There were no significant differences in the number of GFAP-positive cells among the CCI+A1, CCI+A2 and CCI+NS groups at 1, 2, 3, 5, 7 and 14 days after CCI (P > 0.05). Larger numbers of GFAP-positive cells were significantly observed in the CCI+A3 and CCI+S groups compared to the CCI+NS group at 1, 2, 3, 5, 7 and 14 days after CCI (P < 0.05). In addition, the numbers of GFAP-positive cells in the CCI+A1 and CCI+A2 groups were significantly different from the CCI+S group at 1, 2, 3, 5, 7 and 14 days after CCI (P < 0.05), while there was no significant difference in the number of GFAP-positive cells between the CCI+A3 group and CCI+S group (P > 0.05). Therefore, the number of GFAP-positive cells was temporally correlated with hyperalgesia after CCI (Figure 2).

参考文献

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引言

Neuropathic pain can be induced by lesion or dysfunction in the peripheral or central nervous systems, such as nerve injury, infection, metabolic diseases or nerve compression. It is defined as the pain caused by a primary lesion or dysfunction in the nervous system. At present, opioid alkaloids is the main alagesic drug acting on the central nervous system, has analgesic, sedative, antitussive and emetic effects, inhibits respiration, and narrows the pupil. The analgesic drug is highly effective, selective and specific, and is in accordance with the characteristics of drug binding to receptors. Strong analgesic effect is valid for various types of pain, but drug addiction limits its use. Non-central analgesic drugs, i.e., non-opioid analgesic drugs, are mainly antipyretic analgesic and anti-inflammatory and non-steroidal drugs, mainly exert their effects by suppressing the secretion of inflammatory mediators (such as leukotrienes and arachidonic acid), which mainly contain non-steroidal anti-inflammatory drugs. Highly selective cyclooxygenase-2 inhibitors such as celecoxib have the advantage of few side effects. Steroids apparently relieve symptoms, but hormones have many side effects and impact therapeutic efficacy. Therefore, the mechanisms of neuropathic pain and the research and development of analgesic drugs have attracted widespread attention. Ferula sinkiangensis K.M. Shen is composed of volatile oils, resins and gums, has anti-inflammatory, anti-allergic, anti-bacterial, anti-ulcer and antispasmodic analgesic effects^[1-2]. Subcutaneous injection or oral administration of volatile oil emulsions can evidently inhibit carrageenan-induced rat paw edema, inflammatory mediator histamine- and 5-hydroxytryptamine (5-HT)-induced increase of vascular permeability. Ferula sinkiangensis K.M. Shen has a noticeable effect on acute inflammation, which is possibly associated with its effect on anti-inflammatory mediators. Ferula sinkiangensis K.M. Shen has been shown to suppress Freund’s adjuvant- induced immune inflammatory responses, and to inhibit delayed hypersensitivity^[3-4]. The analgesic effect of Ferula sinkiangensis K.M. Shen has been verified in the clinic and research^[5-6]. Volatile oil of Ferula sinkiangensis K.M. Shen elevated the pain threshold value of withdrawal syndrome and precipitated withdrawal in morphine- dependent rats, had analgesic effect^[5-6], but its analgesic effect and the underlying mechanisms have not been confirmed by a rigorous experiment. 中国组织工程研究杂志出版内容重点：肾移植；肝移植；移植；心脏移植；组织移植；皮肤移植；皮瓣移植；血管移植；器官移植；组织工程

A rat model of chronic constriction injury (CCI) is a commonly used model for neuropathic pain, and can simulate peripheral nerve injury-induced neuropathic pain. This study sought to observe the effects of gastric administration of Ferula sinkiangensis K.M. Shen on heat pain, mechanical pain, spinal cord Fos protein and astrocyte activation in a rat model of neuropathic pain after CCI, and then to explore its possible mechanisms in analgesia.

材料方法

Animals

Eighty Sprague-Dawley rats (male:female ratio of 1:1, weight 270±20 g) were purchased from Xinjiang Experimental Animal Research Center (Xinjiang Uygur Antonomous Region, Urumqi, China; Certificate No. SCXK2011-0003).

Methods

Preparation of rat models of chronic sciatic nerve injury

Rats were intraperitoneally injected with 10% chloral hydrate (3 mg/kg) for induction of surgical anesthesia. The hair on the lower back and thigh was shaved and the skin was sterilized. The rats were secured tightly, and neuropathic pain was induced by CCI as described previously^[7]. Briefly, an approximately 2 cm incision in length was cut parallel to the femur in the rear middle thigh, gluteus maximus, and biceps flexor cruris directly dissociated to expose the sciatic nerve in the spatium intermusculare. The surrounding tissues were gently separated to expose the sciatic nerve, and three ligatures (silk 4-0) were placed around the nerve proximal to the trifurcation, with approximately 1 mm between each ligature. The ligatures were slowly tightened until a brief flick of the ipsilateral hind limb was observed. After nerve ligation, muscular and skin layers were immediately sutured with thread. Penicillin sodium was injected intramuscularly at 3 × 10⁴ U per rat for 3 successive days after surgery to prevent infection. All surgical procedures were performed under normal sterile conditions by the same experimenter.

Standards for the success of sciatic nerve injury

The main parts of sciatic nerve were subjected to a loose ligation with the first line before bifurcation. Three ligations were performed respectively with 1 mm spacing. Taking a mild calf muscles quiver as a symbol, sciatic nerve and arterial mild compression on nerve surface were visible under dissecting microscope, without blood flow interruption.

Grouping and drug administration

Eighty adult Sprague-Dawley CCI rats were randomly divided into five groups using a random number generator: low-dose Ferula. sinkiangensis K.M. Shen (CCI+A1), moderate-dose Ferula. sinkiangensis K.M. Shen (CCI+A2), high-dose Ferula. sinkiangensis K.M. Shen (CCI+A3), physiological saline (CCI+NS), and celecoxib groups (CCI+S).Ferula sinkiangensis K.M. Shen (Xinjiang Institute of Materia Medica, Urumqi, China) was mixed with distilled water and ground evenly in a mortar. Rats were intragastically administered Ferula sinkiangensis K.M. Shen at 0.075, 0.15 and 0.3 g/kg in the CCI+A1, CCI+A2 and CCI+A3 groups respectively, while physiological saline was intragastically administered (CCI+NS group) at 3 mL/100 g. The drug doses used were extrapolated from human studies. The moderate drug dose (1.5/60×6.5 g/kg) was calculated by conversion from a human study using the body surface area (BSA)^[8]. The low dose was one half of the moderate dose, and the high dose was double of the moderate dose.

Behavioral pain scores

Behavioral pain assessment was performed at 1 day before operation and at 1, 2, 3, 5, 7, 14 days after operation. Spontaneous pain was assessed based on the position of the paw on the surgical side according to Attal et al ^[9]. Score 0, normal paw position; score 1, the paw is positioned slightly on the cage bottom and appears slightly flexed; score 2, only the internal edge of the paw touches the cage bottom; score 3, only the foot heel touches the cage bottom, with paw inversion observed; score 4, paw suspension; score 5, rat licks the paw on the surgical side.

Thermal withdrawal latency measurement (TWL)

The TWL of the bottom foot of rat was measured with a PL-200 thermal pain stimulator (Chengdu Taimeng Technology Co., Ltd., Sichuan Province, China)^[10]. At 1 day before CCI and at 1, 2, 3, 5, 7 and 14 days after CCI, rats were placed gently in a Plexiglass box on the upper glass plate of the thermal pain stimulator for 10 minutes. Until the rats were habituated to the environment, the sole of the posterior limb was exposed to thermal light at an intensity of 60%, and rat paw TWL was recorded. The duration of exposure to light was limited to 25 seconds and the interval between two measurements was 15 minutes. The measurement was repeated for three times to yield the mean TWL response.

Rat mechanical allodynia latency measurement

The 50% paw mechanical allodynia latency in rats (WMT) was assessed using von Frey filaments (Stoelting; Wood Dale, IL, USA) and the up-and-down method at 1 day before treatment and at 1, 2, 3, 5, 7 and 14 days after treatment^[11]. Rats were placed in a Plexiglass box on a metal grid for 15 minutes. Then, the bottom of medial foot of the posterior limb was stimulated successively with 2, 4, 6, 8, 10 and 15 g von Frey filaments for less than 6 seconds. A positive reaction was defined as lifting or licking the stimulated foot. If stimulating with a lower force did not induce positive response, a higher force was used. If a positive response was present, stimulation with a lower force was applied to a neighboring point on the bottom of foot. The stimulation was applied 10 times with 30 seconds interval between each of stimulation. Mechanical hyperalgesia was defined as the presence of ≥ 5 positive responses in 10 times of stimulation, and the percentage of positive response occurring in 10 times of stimulation was defined as the positive rate of maximum folding force. If < 5 positive responses occurred in 10 times of stimulation, the positive rate of the minimal folding force that induced the positive response was estimated, and such a folding force indicated the minimal folding force. The maximum intensity was 15 g, and intensity more than 15 g was also recorded as 15 g. The folding force that induces a positive response was calculated using the following formula^[12]: folding force to induce positive response = the maximum folding force-(maximum folding force-minimal folding force)/(positive rate of maximum folding force-positive rate of minimal folding force) × (positive rate of maximum folding force-50%), with 50% of this folding force used as the WMT.

Determination of Fos protein expression in spinal cord

At 3, 7 and 14 days after treatment, three rats were randomly selected from each group and were sacrificed. The chest was rapidly opened and a catheter was inserted into the ascending aorta. The remaining blood was flushed with 100 mL NaCl (9 g/L), followed by perfusion of 500 mL phosphate buffered saline (PBS; 0.1 mol/L) containing 40 g/L paraformaldehyde for 1 to 2 hours. The fixed spinal cord was excised and embedded in paraffin for slice preparation. Spinal cord samples were cut into cross sections, and Fos protein expression was determined by immunohistochemistry method. Briefly, slices were dewaxed, hydrated, washed three times with 0.01 mol/L PBS (pH < 7.4) (5 minutes/wash) and incubated in H₂O₂ for 15 minutes to quench endogenous peroxidase activity. Specimens were washed three times with PBS (5 minutes/wash) and blocked in normal goat serum for 5-10 minutes. The excess serum was removed and slices incubated in rabbit anti-Fos antibody (1:8 000) at 4 ℃ for 48 hours, followed by three PBS washes (5 minutes/wash). Immunolabeled slices were then incubated in biotin-labeled goat anti-rabbit intravenous gamma-globulin at room temperature for 30 minutes and wash three times with PBS (5 minutes/wash). Immunolabeling was visualized by 3,3'-Diaminobenzidine (DAB) staining (10 minutes). Labeled slices were flushed with running water, counterstained with hematoxylin and eosin (H&E) for approximately 30 seconds, dried, dehydrated, made transparent, and sealed under coverslips. Cells stained positive for Fos were counted under an Olympus BH2 microscope (Olympus, Tokyo, Japan) at 400× magnification. Six sections were sampled from the spinal cord of each rat and the average number of neurons with Fos-like immunoreactivity (FLI) was reported.

Determination of astrocyte activation in the spinal cord

The spinal cord samples were cut into sections and stained for glial fibrillary acidic protein (GFAP). Briefly, paraffin-embedded spinal cord sections were dewaxed, made transparent in dimethylbenzene, hydrated in a graded alcohol series, and treated with citric acid for antigen retrieval. The sections were washed three times with PBS (3 minutes/wash), and blocked in (animal) serum in a humidity box at 37 ℃ for 30 hours. The blocking solution was removed, and slices incubated in mouse anti-GFAP (1:400; Sigma) at 4 ℃ overnight. Immunolabeled sections were washed three times with PBS (3 minutes/wash), and incubated in fluorophore-conjugated secondary antibody (1:100; Sigma) in a humidity box at room temperature for 1 hour. The sections were washed three times with PBS (10 minutes/wash), preserved with fluorescent mounting medium, and visualized under a confocal microscope. The spinal cord gray matter was demarcated into 4 Rexed layers: the superficial layer (Rexed layers I-II), laminae propria (Rexed layers III-IV), neck of the dorsal horn (Rexed layers V-VI), and ventral horn (Rexed layers VII-X). Six sections were sampled from the spinal cord of each rat, and the number of immunopositive astrocytes on each section was counted. The mean number of GFAP-positive astrocytes was calculated as an estimate of astroglial reactivity (activation). The staining intensity in individual cells was not taken into account for this estimation.

Main outcome measures

Behavior pain scores; TWL measurement results; WMT measurement results; Fos protein expression in rat spinal cord; astroglial GFAP expression in rat spinal cord.

Statistical analysis

All measurement data are expressed as the mean ± SD, and all statistical analyses were performed using the Statistic Package for Social Science 13.0 software (SPSS, Chicago, IL, USA). Repeated-measures data were tested for statistical significance by analysis of variance, and treatment groups were compared by two independent samples t-tests. Categorical values were expressed as proportions and compared by Chi-square test. A P-value < 0.05 was considered statistically significant.

讨论

The CCI of rat develops behavioral signs of spontaneous pain and hyperalgesia for both thermal and mechanical stimulation. In the current study, both the thermal and mechanical pain thresholds were significantly reduced in the control (physiological saline-treated) CCI group at 2 days after treatment, reached minima at 7 days (indication of greatest hyperalgesia), and did not recover to the pre-treatment value until 14 days. Conversely, gavage feeding of the traditional medicinal plant Ferula sinkiangensis K.M. Shen reduced spontaneous pain and both mechanical and thermal hyperalgesia, and this analgesic effect was associated with the reduced spinal Fos expression. These results suggest that Ferula sinkiangensis mitigated the hyperalgesic effects of CCI by altering patterns of gene expression after CCI. Peak hyperalgesia was temporally correlated with peak spinal astroglial activation. Both TWL and WMT started to decline in all Ferula sinkiangensis dose groups and the celecoxib group as early as at 1 day after CCI. In general, the analgesic effect was strongest in the moderate-dose group (CCI+A2), indicating that Ferula. sinkiangensis K.M. Shen at an appropriate dose can alleviate neuropathic pain in rats.

c-Fos is an immediate early gene, a variety of noxious stimuli can induce c-Fos expression in the dorsal horn of the spinal cord^[13], and C-Fos is much more expressed in the spinal dorsal horn I, II, V layers, so it can be used as a sign of neuropathic pain stimuli^[14]. Results from this study showed that the number of Fos-like immunoreactive cells apparently increased in each group after the surgery. The number of Fos-like immunoreactive cells was evidently lower in the CCI+A1, CCI+A2, CCI+A3 and CCI+S groups than that in the CCI+NS group at each time point, suggesting that analgesia reduced Fos expression after CCI. The number of Fos-like immunoreactive cells was higher in the CCI+A3 group than that in the CCI+S group at each time point. Significant differences in number of Fos-like immunoreactive cells were detected between the CCI+A1, CCI+A2 groups and CCI+S group. These findings indicate that Resina Ferulae inhibited c-Fos protein expression in the spinal cord, and alleviate neuropathic pain.

Glial cells (mainly microglia and astrocytes) are widely present in the dorsal horn of the spinal cord of animals and are involved in the occurrence and maintenance of neuropathic pain^[15-17]; when subjected to painful stimuli, glial cells, especially activated astrocytes, produce reactions which lead to significantly increased GFAP marker^[18].

Studies confirmed that microglia and astrocytes are involved in the occurrence of chronic neuropathic pain^[15]. Especially in pathological pain stimuli, astrocytes release neuroactive substances such as reactive oxygen species, nitric oxide, leukotrienes, arachidonic acid and nerve growth factor. These active substances act on the spinal cord dorsal horn nociceptive afferent neurons and they are hypersensitive. In addition, it releases pro-inflammatory cytokines, such as interleukin-1, interleukin-6 and tumor necrosis factor-α. The receptors of these cytokines are expressed in the spinal cord glial cells and neurons, which themselves also have a strong pain-inducing effect. Because the large surface of astrocytes can express GFAP, noxious stimuli may directly activate astrocytes to cause neuropathic pain, therefore, drugs which can reduce astrocyte activation can inhibit neuropathic pain.

Our results demonstrated that the number of GFAP- mmunoreactive cells reached the highest level at approximately 7 days after surgery in each group. The number of fluorescence positive cells was significantly less in the CCI+A3 and CCI+S groups compared with the CCI+NS group at each time point. Significant differences in the number of fluorescence positive cells were detectable between the CCI+A1 and CCI+A2 groups and CCI+S group at each time point. No significant difference in the number of fluorescence positive cells was seen between the CCI+A3 group and the CCI+S group at different time points. Results from this study also demonstrated that Ferula sinkiangensis K.M. Shen may alleviate neuropathic pain through inhibiting the expression of astrocytes through alleviating neuropathic pain.

In conclusion, Ferula sinkiangensis K.M. Shen can effectively alleviate rat sciatica, and c-Fos protein and GFAP may be involved in the underlying mechanism of analgesic action 中国组织工程研究杂志出版内容重点：肾移植；肝移植；移植；心脏移植；组织移植；皮肤移植；皮瓣移植；血管移植；器官移植；组织工程

新疆阿魏干预神经源性疼痛模型大鼠痛阈及脊髓Fos蛋白和星形胶质细胞的表达

Effect of Ferula sinkiangensis K.M. Shen on pain threshold and Fos protein expression and astrocyte activation in the spinal cord of neuropathic pain rats

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