Establishment and Evaluation of Mouse Model of Pregnancy Pain-depression Comorbidity Induced by Chronic Unpredictable Stress, Complete Freund's Adjuvant and Formalin

doi:10.12300/j.issn.1674-5817.2024.005

Abstract

Abstract:

Objective To establish a mouse model of pregnancy pain-depression comorbidity induced by chronic unpredictable stress (CUS), complete Freund's adjuvant (CFA), and formalin, and to systematically evaluate the associated phenotypes and preliminarily explore the pathological basis of the comorbidity. Methods Eight-week-old C57BL/6J female mice were randomly strarified divided into a control group (no intervention before pregnancy) and a CUS model group (CUS intervention before pregnancy) based on sucrose preference test (SPT) data. After completing the CUS treatment, female and male mice were paired and mated. Pain was induced by injecting 50% CFA and 5% formalin in the right hind foot during pregnancy to create a model of pregnancy pain-depression comorbidity. The experiment was divided into 8 subgroups: control-blank group, CUS-blank group, control-CFA group, CUS-CFA group, control-formalin group, CUS-formalin group, control-CFA+formalin group, and CUS-CFA+formalin group, with 10 mice in each group. The mice in each group were subject to behavioral tests, including the SPT, forced swimming test, tail suspension test, and open field test before and after CUS intervention, during pregnancy, and after delivery. Pain sensitivity changes were measured using mechanical allodynia and thermal hyperalgesia tests. Mice were then euthanized. Levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in hippocampus, as well as cortisol and adrenocorticotropic hormone (ACTH) in serum, were detected by enzyme-linked immunosorbent assay (ELISA). Results Compared with the control-blank group, the CUS-blank group showed a significant depression-like behavior with reduced pain threshold (P<0.001). The control-CFA+formalin group showed a decrease in pain threshold after both CFA injection and formalin injection (P<0.01). Compared with the control-blank and control-formalin groups, the pain threshold was significantly lower in the CUS-formalin group (P<0.01), with a sequential decrease among the three. Compared with the control-blank and control-CFA groups, the pain threshold was significantly lower in the CUS-CFA group (P<0.001), with a sequential decrease among the three. Compared with the control-blank and control-CFA+formalin groups, the mechanical pain threshold of mice in the CUS-CFA+formalin group was significantly lower (P<0.001) and the thermal radiation tolerance time was shorter (P<0.01), both with sequential decreases among the three. Compared with the control-CFA+formalin and the CUS-blank groups, the CUS-CFA+formalin group had a significantly lower percentage of sucrose preference (P<0.001), longer immobility time during the forced swimming test (P<0.001) and tail suspension test (P<0.001), reduced central exploration time in the open field test (P<0.001), reduced total exploration distance (P<0.001), and reduced percentage of distance traveled for central exploration (P<0.001). Compared with the control-CFA+formalin and CUS-blank groups, the serum cortisol and ACTH levels of the CUS-CFA+formalin group were significantly higher (P<0.01), and the levels of IL-6 and TNF-α in the hippocampus were higher (P<0.05). Conclusion The combination of CUS+CFA+formalin injections is an ideal method for establishing a C57BL/6J mouse model of pregnancy pain-depression comorbidity. The behavioral changes in model mice may be attributed to the regulation of inflammatory response in hippocampus and hormone levels in the hypothalamic-pituitary-adrenal (HPA) axis.

Key words: Pregnancy depression, Pain, Comorbidity, Inflammatory factors, Hypothalamic-pituitary-adrenal axis, C57BL/6J mice

CLC Number:

R-332

Yisu ZHANG,Xinru LIU,Ruojie WU,et al. Establishment and Evaluation of Mouse Model of Pregnancy Pain-depression Comorbidity Induced by Chronic Unpredictable Stress, Complete Freund's Adjuvant and Formalin[J]. Laboratory Animal and Comparative Medicine, 2024, 44(3): 259-269. DOI: 10.12300/j.issn.1674-5817.2024.005.

Figures/Tables 6

Figure 1 Experimental flowchart for the construction of a depression-pain comorbidity model in pregnant miceNote： CUS, chronic unpredictable stress; CFA, complete Freund's adjuvant; SPT, sucrose preference test; FST, forced swimming test; TST, tail suspension test; OFT, open field test; PTT, pain threshold test; T0, before CUS intervention; T4, after CUS intervention; T8, after pregnancy.

Figure 2 Depressive behavior evaluation and pain threshold detection in the CUS model of miceNote： A, Results of the sucrose preference test at the end of intervention periods before the start of the experiment (T0), one week after the experiment (T1), two weeks after the experiment (T2), three weeks after the experiment (T3), and four weeks after the experiment (T4) for the control group (n=40) and the chronic unpredictable stress (CUS) group (n=40); B, Forced swimming immobility time at the end of intervention periods T0 and T4 for each group; C, Tail suspension immobility time at the end of intervention periods T0 and T4 for each group; D, Percentage of time spent exploring the center of an open field for each group at the end of intervention periods T0 and T4; E, Right hind foot stabbing pain thresholds for each group at the end of intervention periods T0 and T4; F, Time to tolerate thermal radiation pain in the right hind foot for each group at the end of intervention T0 and T4. nsdifferences were not statistically significant, *P＜0.05, **P＜0.01, ***P＜0.001, n=40.

Figure 3 Depression affects the ability to perceive pain in pregnant miceNote： Control-blank group, i.e., without any treatment; Control-A group, i.e., without chronic unpredictable stress (CUS) but injected with formalin; CUS-A group, i.e., stimulated by CUS and injected with formalin; Control-C group, i.e., without CUS but injected with complete Freund's adjuvant (CFA); CUS-C group, i.e., stimulated by CUS and injected with CFA; Control-A+C group, i.e., without CUS but injected with formalin and CFA; CUS-blank group, i.e., stimulated by CUS but not injected with formalin and CFA; and CUS-A+C group, i.e., stimulated by CUS and injected with formalin and CFA. A, Pain threshold of the right hind foot in the control-blank, control-A, and CUS-A groups after delivery (T8); B, Pain threshold of the right hind foot at the end of gestation week 2 (T6) in the control-blank, control-C, and CUS-C groups; C, Pain threshold of the right hind foot at the end of gestation week 2 (T6) and after delivery (T8) in the control-C and CUS-C groups; D, Pain threshold of the right hind foot in control-blank, control-A+C, CUS-blank, and CUS-A+C mice before the CUS intervention (T0), after the CUS intervention (T4), at the end of the 2nd week of gestation (T6), and after delivery (T8); E, Pain threshold of the right hind foot after delivery (T8) of mice in the control-blank, control-A+C, and CUS-A+C groups; F, Plantar thermal radiation tolerance time of the right hind foot after delivery (T8) in control-blank, control-A+C, and CUS-A+C groups. nsdifferences were not statistically significant, **P＜0.01, ***P＜0.001, n=10.

Figure 4 Pain during pregnancy impacts depressive behaviors of miceNote： Control-A+C group, i.e., without chronic unpredictable stress (CUS) but injected with formalin and complete Freund's adjuvant (CFA); CUS-blank group, i.e., stimulated with CUS but not injected with formalin and CFA; CUS-A group, i.e., stimulated with CUS and injected with formalin; CUS-C group, i.e., stimulated with CUS and injected with CFA; CUS-A+C group, i.e., stimulated with CUS and injected with formalin and CFA. A-D, Preference for sugar water, swimming immobility time, tail suspension immobility time, and center exploration time in an open field as a percentage of time in each group after delivery (T8). **P＜0.01, ***P＜0.001, n=10.

Figure 5 Representative open field trajectory maps and heat maps for each groupNote： Control-blank group, i.e., without any treatment; control-A+C group, i.e., without chronic unpredictable stress (CUS) but injected with formalin and complete Freund's adjuvant (CFA); CUS-blank group, i.e., stimulated with CUS but not injected with formalin and CFA; and CUS-A+C group, i.e., stimulated with CUS and injected with formalin and CFA. A-D, Trajectory maps and heat maps of open field test in Control-blank, Control-A+C, CUS-blank, and CUS-A+C mice after delivery (T8); E-F, Ratio of central exploration distance to total distance after delivery (T8) in each group of mice. **P＜0.01, ***P＜0.001, n=10.

Figure 6 Higher hypothalamic-pituitary-adrenal axis （HPA axis） hormone levels and hippocampal inflammatory factor levels in mice with depression-pain comorbidity during pregnancyNote： Control-blank group, i.e., without any treatment; Control-A+C group, i.e., without chronic unpredictable stress (CUS) but injected with formalin and complete Freund's adjuvant (CFA); CUS-blank group, i.e., stimulated with CUS but not injected with formalin and CFA; and CUS-A+C group, i.e., stimulated with CUS and injected with formalin and CFA. A-D, Serum cortisol and ACTH levels, as well as hippocampal IL-6 and TNF-α levels, in mice from each group. **P＜0.01, ***P＜0.001, n=10.

References 37

1	DAGHER R K, BRUCKHEIM H E, COLPE L J, et al. Perinatal depression: challenges and opportunities. J Women's Health, 2021, 30(2):154-159. DOI: 10.1089/jwh.2020.8862 .
2	姚婷,余雨枫,李佳欣,等.中国女性产后抑郁发生率及影响因素meta分析[J].现代医药卫生, 2023, 39(20):3510-3515. DOI: 10.3969/j.issn.1009-5519.2023.20.020 .
	YAO T, YU Y F, LI J X, et al. Prevalence and influencing factors of postpartum depression in Chinese women: a meta-analysis [J]. J Mod Med Health, 2023, 39(20):3510-3515. DOI: 10. 3969/j.issn.1009-5519.2023.20.020 .
3	CHARLES R A, ABOUHASSAN S, MCFARLAND H, et al. Prescribing narcotics for pain: reconsider the fifth vital sign[J]. Ann Surg Open, 2022, 3(3): e186. DOI: 10.1097/AS9.0000000000000186 .
4	COLUZZI F, VALENSISE H, SACCO M, et al. Chronic pain management in pregnancy and lactation[J]. Minerva Anestesiol, 2014, 80(2):211-224.
5	HONG S S, CHEN L Y, ZHUANG H H, et al. Prevalence of lumbopelvic pain during pregnancy: a systematic review and meta-analysis of cross-sectional studies[J]. Acta Obstet Gynecol Scand, 2024, 103(2):225-240. DOI: 10.1111/aogs.14714 .
6	权紫微, 李铭麟, 苑莉莉, 等. 老年慢性疼痛与抑郁症的关系研究进展[J]. 实用老年医学, 2023, 37(3):223-225. DOI: 10.3969/j.issn.1003-9198.2023.03.003 .
	QUAN Z W, LI M L, YUAN L L, et al. Research progress on the relationship between chronic pain and depression in the elderly[J]. Pract Geriatr, 2023, 37(3):223-225. DOI: 10.3969/j.issn.1003-9198.2023.03.003 .
7	MA Y, XIANG Q, YAN C Y, et al. Relationship between chronic diseases and depression: the mediating effect of pain[J]. BMC Psychiatry, 2021, 21(1):436. DOI: 10.1186/s12888-021-03428-3 .
8	VIGNATO J, BECK C T, CONLEY V, et al. The lived experience of pain and depression symptoms during pregnancy[J]. MCN Am J Matern Child Nurs, 2021, 46(4):198-204. DOI: 10.1097/NMC.0000000000000724 .
9	徐龙进, 王可洲. 小鼠慢性应激性抑郁症易感品系筛选及其机制初步研究[J]. 实验动物与比较医学, 2016, 36(4):257-262. DOI: 10.3969/j.issn.1674-5817.2016.04.003 .
	XU L J, WANG K Z. Screening of mouse strain susceptive to chronic stress depression and preliminary research on its mechanism[J]. Lab Anim Comp Med, 2016, 36(4):257-262. DOI: 10.3969/j.issn.1674-5817.2016.04.003 .
10	WILLNER P, TOWELL A, SAMPSON D, et al. Reduction of sucrose preference by chronic unpredictable mild stress, and its restoration by a tricyclic antidepressant[J]. Psychopharmacology, 1987, 93(3):358-364. DOI: 10.1007/BF00187257 .
11	YOHN N L, BLENDY J A. Adolescent chronic unpredictable stress exposure is a sensitive window for long-term changes in adult behavior in mice[J]. Neuropsychopharmacology, 2017, 42(8):1670-1678. DOI: 10.1038/npp.2017.11 .
12	LOGAN R W, EDGAR N, GILLMAN A G, et al. Chronic stress induces brain region-specific alterations of molecular rhythms that correlate with depression-like behavior in mice[J]. Biol Psychiatry, 2015, 78(4):249-258. DOI: 10.1016/j.biopsych.2015.01.011 .
13	秦露, 唐静, 梁芯, 等. 跑步对CUS抑郁模型小鼠内侧前额叶皮质内PV⁺中间神经元的影响[J]. 陆军军医大学学报, 2023, 45(3):209-218. DOI: 10.16016/j.2097-0927.202208205 .
	QIN L, TANG J, LIANG X, et al. Running exercise shows anti-depressive effect in mice through PV⁺ interneurons in medial prefrontal cortex[J]. J Army Med Univ, 2023, 45(3):209-218. DOI: 10.16016/j.2097-0927.202208205 .
14	BUREK D J, MASSALY N, YOON H J, et al. Behavioral outcomes of complete Freund adjuvant-induced inflammatory pain in the rodent hind paw: a systematic review and meta-analysis[J]. Pain, 2022, 163(5):809-819. DOI: 10.1097/j.pain.0000000000002467 .
15	孙英舰, 张亚民, 白文举, 等. 完全弗氏佐剂诱导慢性炎性痛与焦虑共病的炎症反应特征[J]. 空军军医大学学报, 2022, 43(9):973-978. DOI: 10.13276/j.issn.2097-1656.2022.08.010 .
	SUN Y J, ZHANG Y M, BAI W J, et al. Inflammatory characteristics of chronic inflammatory pain and anxiety comorbidity induced by complete Freund's adjuvant[J]. J Air Force Med Univ, 2022, 43(9):973-978. DOI: 10.13276/j.issn.2097-1656.2022.08.010 .
16	林可可, 荆潇宽, 杨可, 等. 青藤碱对福尔马林诱导炎性疼痛的抑制作用[J]. 湖北科技学院学报(医学版), 2020, 34(3):202-204. DOI: 10.16751/j.cnki.2095-4646.2020.03.0202 .
	LIN K K, JING X K, YANG K, et al. Inhibitory effect of sinomenine on formalin-induced inflammatory pain[J]. J Hubei Univ Sci Technol Med Sci, 2020, 34(3):202-204. DOI: 10.16751/j.cnki.2095-4646.2020.03.0202 .
17	LI N, XIAO J, NIU J D, et al. Synergistic interaction between DAMGO-NH₂ and NOP01 in peripherally acting antinociception in two mouse models of formalin pain[J]. Peptides, 2023, 161:170943. DOI: 10.1016/j.peptides.2023.170943 .
18	徐平. 小鼠剖腹育仔若干问题探讨[J]. 上海实验动物科学, 1990, 10(4):222-223.
	XU P. Discussion on some problems of caesarean section in mice[J]. Shanghai Lab Anim Sci, 1990, 10(4):222-223.
19	CHIESA M, RABIEI H, RIFFAULT B, et al. Brain volumes in mice are smaller at birth after term or preterm cesarean section delivery[J]. Cereb Cortex, 2021, 31(8):3579-3591. DOI: 10.1093/cercor/bhab033 .
20	LIU M Y, YIN C Y, ZHU L J, et al. Sucrose preference test for measurement of stress-induced anhedonia in mice[J]. Nat Protoc, 2018, 13(7):1686-1698. DOI: 10.1038/s41596-018-0011-z .
21	HARRO J. Animal models of depression: pros and cons[J]. Cell Tissue Res, 2019, 377(1):5-20. DOI: 10.1007/s00441-018-2973-0 .
22	UENO H, TAKAHASHI Y, MURAKAMI S, et al. Effect of simultaneous testing of two mice in the tail suspension test and forced swim test[J]. Sci Rep, 2022, 12(1):9224. DOI: 10.1038/s41598-022-12986-9 .
23	KRAEUTER A K, GUEST P C, SARNYAI Z. The open field test for measuring locomotor activity and anxiety-like behavior[J]. Methods Mol Biol, 2019, 1916:99-103. DOI: 10.1007/978-1-4939-8994-2_9 .
24	何仁可, 鲁程, 陈薇, 等. 疼痛抑郁共病动物模型及评价方法研究进展[J]. 实验动物与比较医学, 2022, 42(1):68-73. DOI: 10.12300/j.issn.1674-5817.2021.097 .
	HE R K, LU C, CHEN W, et al. Advances in animal models and evaluation methods of pain and depression comorbidity[J]. Lab Anim Comp Med, 2022, 42(1):68-73. DOI: 10.12300/j.issn.1674-5817.2021.097 .
25	SONG J, KIM Y K. Animal models for the study of depressive disorder[J]. CNS Neurosci Ther, 2021, 27(6):633-642. DOI: 10.1111/cns.13622 .
26	YOU Z R, DING W H, DOHENY J T, et al. Methylphenidate and morphine combination therapy in a rat model of chronic pain[J]. Anesth Analg, 2020, 130(2):518-524. DOI: 10.1213/ANE. 0000000000004273 .
27	LÓPEZ-CANO M, FERNÁNDEZ-DUEÑAS V, LLEBARIA A, et al. Formalin murine model of pain[J]. Bio-protocol, 2017, 7(23): e2628. DOI: 10.21769/BioProtoc.2628 .
28	SHIGEMATSU-LOCATELLI M, KAWANO T, YASUMITSU-LOVELL K, et al. Maternal pain during pregnancy dose-dependently predicts postpartum depression: the Japan Environment and Children's Study[J]. J Affect Disord, 2022, 303:346-352. DOI: 10.1016/j.jad.2022.01.039 .
29	LIM G, LASORDA K R, FARRELL L M, et al. Obstetric pain correlates with postpartum depression symptoms: a pilot prospective observational study[J]. BMC Pregnancy Childbirth, 2020, 20(1):240. DOI: 10.1186/s12884-020-02943-7 .
30	MCHUGO M, TALATI P, ARMSTRONG K, et al. Hyperactivity and reduced activation of anterior hippocampus in early psychosis[J]. Am J Psychiatry, 2019, 176(12):1030-1038. DOI: 10.1176/appi.ajp.2019.19020151 .
31	KVICHANSKY A A, TRET'YAKOVA L V, VOLOBUEVA M N, et al. Neonatal proinflammatory stress and expression of neuroinflammation-associated genes in the rat hippocampus[J]. Biochemistry, 2021, 86(6):693-703. DOI: 10.1134/S0006297921060079 .
32	赵谦, 王安娜, 高雪松, 等. 慢性不可预知性温和应激致抑郁样大鼠模型的研究进展[J]. 实验动物与比较医学, 2020, 40(4):344-353. DOI: 10.3969/j.issn.1674-5817.2020.04.012 .
	ZHAO Q, WANG A N, GAO X S, et al. Research progress in depression-like rat model induced by chronic unpredictable mild stress[J]. Lab Anim Comp Med, 2020, 40(4):344-353. DOI: 10.3969/j.issn.1674-5817.2020.04.012 .
33	BELVEDERI MURRI M, PARIANTE C, MONDELLI V, et al. HPA axis and aging in depression: systematic review and meta-analysis[J]. Psychoneuroendocrinology, 2014, 41:46-62. DOI: 10.1016/j.psyneuen.2013.12.004 .
34	BERRIDGE K C. The debate over dopamine's role in reward: the case for incentive salience[J]. Psychopharmacology, 2007, 191(3):391-431. DOI: 10.1007/s00213-006-0578-x .
35	LAMMEL S, LIM B K, MALENKA R C. Reward and aversion in a heterogeneous midbrain dopamine system[J]. Neuropharmacology, 2014, 76 Pt B(0 0):351-359. DOI: 10.1016/j.neuropharm.2013.03.019 .
36	NESTLER E J, CARLEZON W A JR. The mesolimbic dopamine reward circuit in depression[J]. Biol Psychiatry, 2006, 59(12):1151-1159. DOI: 10.1016/j.biopsych.2005.09.018 .
37	WANG J, ZHAO Q H, ZHOU Y, et al. Subanesthetic dose of ketamine improved CFA-induced inflammatory pain and depression-like behaviors via caveolin-1 in mice[J]. J Neurosurg Anesthesiol, 2020, 32(4):359-366. DOI: 10.1097/ANA.0000000000000610 .

[1]	Zhengwen MA, Xiaying LI, Xiaoyu LIU, Yao LI, Jian WANG, Jin LU, Guoyuan CHEN, Xiao LU, Yu BAI, Xuancheng LU, Yonggang LIU, Wanyong PANG, Yufeng TAO. Interpretation and Elaboration for the ARRIVE Guidelines 2.0—Animal Research: Reporting In Vivo Experiments (V) [J]. Laboratory Animal and Comparative Medicine, 2024, 44(1): 105-114.
[2]	Min LIANG, Yang GUO, Jinjin WANG, Mengyan ZHU, Jun CHI, Yanjuan CHEN, Chengji WANG, Zhilan YU, Ruling SHEN. Construction of Dmd Gene Mutant Mice and Phenotype Verification in Muscle and Immune Systems [J]. Laboratory Animal and Comparative Medicine, 2024, 44(1): 42-51.
[3]	Han LI, Xiaorui ZHANG, Chengfang ZHANG. Mechanism of Intermittent Fasting in Improving Olanzapine-induced Metabolic Disorders in Mice [J]. Laboratory Animal and Comparative Medicine, 2023, 43(1): 3-10.
[4]	Sijia ZHAO, Xinyu HE, Quan JING, Lin MA, Chunlan GUO, Kuo WAN. Evaluation of Pain in Acute Pulpitis Hyperalgesia Model Rats [J]. Laboratory Animal and Comparative Medicine, 2022, 42(4): 333-341.
[5]	Changgeng PENG, Yan FU, Fengting ZHU, Ruilong XIA, Wei XIA. The Latest Research Progress of the Function of Spinal CD11c⁺ Microglia in Neuropathic Pain [J]. Laboratory Animal and Comparative Medicine, 2022, 42(3): 171-176.
[6]	Renke HE, Cheng LU, Wei CHEN, Mengya WANG, Aiping XU. Advances in Animal Models and Evaluation Methods of Pain and Depression Comorbidity [J]. Laboratory Animal and Comparative Medicine, 2022, 42(1): 68-73.
[7]	LI Zifa, ZHANG Hao, REN Meng, XU Kaiyong, HU Minghui, ZHOU Miaomiao, WANG Kezhou. Protective Effect of Quercetin on Lipid Metabolism Disorder in Mice Livers Caused by Cadmium [J]. Laboratory Animal and Comparative Medicine, 2021, 41(4): 305-312.
[8]	SONG Binbin, DONG Wenzhou, JIA Bingquan, ZHEN Ran, PENG Yu, YANG Xuan, YU Jia. Expression and Distribution of Molecules Regulating Protein Degradation Pathway in Nervous System of Mice [J]. Laboratory Animal and Comparative Medicine, 2020, 40(6): 463-469.
[9]	SUN Chengcheng, LIU Jiangang, LIU Meixia, LI Hao, LUO Zenggang. Neurological Dysfunction and Pathological Changes of Rats with [J]. Laboratory Animal and Comparative Medicine, 2020, 40(6): 470-476.
[10]	LEI Shan, LIU Qiang, HUANG Wei-jin, WANG You-chun. Influence of Strain, Gender and Hair Coat of Mice on Establishing Bioluminescent Imaging Pseudovirus Mouse Model [J]. Laboratory Animal and Comparative Medicine, 2019, 39(6): 423-428.
[11]	YANG Hua, ZHAO Ya-Juan, OU Qiang. Establishment of Nonalcoholic Fatty Liver Fibrosis Model and Expression of Inflammatory Factors in Mice [J]. Laboratory Animal and Comparative Medicine, 2017, 37(1): 20-24.
[12]	MIAO Jian-cheng, ZHAO Ying, CHEN Guo-qiang, ZHAO Li-ya, JIN Yi, LIU Da-hai, QIAO Wei-wei, ZHAO Xian-zhe. Evaluations on Rat Model of Tibia Cancer Pain with Bone Mineral Density [J]. Laboratory Animal and Comparative Medicine, 2012, 32(4): 290-294.
[13]	WANG Shou-li, ZHU-Jun, ZHU Yun-li, FU Pei-liang, WU Yu-li, WU Hai-shan, ZHAO Hui. The Perioperative Pain Control in Canine with Tibial Fracture and Internal Fixation [J]. Laboratory Animal and Comparative Medicine, 2012, 32(1): 56-59.
[14]	XIAO Nan1，ZHAO Wei2, BA Cai-feng2, SU Rong-jian1, SU Yu-hong1，3. Study on Gene Mutation and Muscles Subtypes’Expression in DMD Model Mice [J]. Laboratory Animal and Comparative Medicine, 2008, 28(6): 356-360.
[15]	XU Chang-Shui, LIANG Shang-Dong. Experimental Investigation of P2 Receptors and Pain [J]. Laboratory Animal and Comparative Medicine, 2004, 24(3): 167-171.