Article(id=1200023158267937344, tenantId=1146029695717560320, journalId=1189873630562394117, issueId=1200023152219746543, articleNumber=null, orderNo=null, doi=10.11855/j.issn.0577-7402.1942.2023.0817, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1662307200000, receivedDateStr=2022-09-05, revisedDate=null, revisedDateStr=null, acceptedDate=1682438400000, acceptedDateStr=2023-04-26, onlineDate=1764037416283, onlineDateStr=2025-11-25, pubDate=1698422400000, pubDateStr=2023-10-28, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1764037416283, onlineIssueDateStr=2025-11-25, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1764037416283, creator=13701087609, updateTime=1764037416283, updator=13701087609, issue=Issue{id=1200023152219746543, tenantId=1146029695717560320, journalId=1189873630562394117, year='2023', volume='48', issue='10', pageStart='1115', pageEnd='1236', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=0, createTime=1764037414841, creator=13701087609, updateTime=1764038706792, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1200028571126301693, tenantId=1146029695717560320, journalId=1189873630562394117, issueId=1200023152219746543, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1200028571126301694, tenantId=1146029695717560320, journalId=1189873630562394117, issueId=1200023152219746543, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=1129, endPage=1134, ext={EN=ArticleExt(id=1200023158641230418, articleId=1200023158267937344, tenantId=1146029695717560320, journalId=1189873630562394117, language=EN, title=Activation of glial cells in dorsal reticular nucleus of the medulla oblongata involved in the central mechanism of oral and maxillofacial pain sensitivity induced by occlusal interference in rats, columnId=1190310110212751762, journalTitle=Medical Journal of Chinese People’s Liberation Army, columnName=Basic Research, runingTitle=null, highlight=null, articleAbstract=

Objective To investigate the central mechanism of glial cells of the dorsal reticular nucleus (DRt) in the modulation of chronic orofacial pain after removal of experimental occlusal interference(EOI) in rats. Methods Twenty-four male SD rats (180-200 g) were randomly divided into three groups (8 in each group): sham group, EOI hyperalgesia maintaining group (occlusal interference appliance removed on day 8 after wearing 0.4 mm thick crowns), EOI hyperalgesia maintaining+DRt damage group (EOI hyperalgesia maintaining rats were injected with ibotenic acid to damage DRt). The non-reflexive behaviors of the three groups were evaluated by using orofacial operant test on 7, 10, 14 d after model establishment. Nine male SD rats were randomly divided into three groups (3 in each group): sham group, EOI hyperalgesia maintaining group (day 8 after wearing 0.4 mm thick crowns, before removal of EOI), EOI hyperalgesia maintaining group 6 d (6 days after EOI removed on day 8). DRt sections were obtained and processed for immunofluorescence staining for glai fibrillary acidic protein (GFAP) and OX-42. The levels of expression were hemi-quantitatively analyzed to evaluate the fluorescence area and fluorescence intensity of astrocytes and microglia. Results EOI hyperalgesia maintaining group and EOI hyperalgesia maintaining+DRt damage group exhibited lower feeding time in orofacial operant test, which implied hyperalgesia (P<0.05). The hyperalgesia in EOI hyperalgesia maintaining group persisted after the removal of EOI, and the difference was statistically significant at 10 d and 14 d compared with sham group (P<0.05), while the hyperalgesia in the EOI hyperalgesia maintaining+DRt damage group showed a rebound trend, and the difference was not statistically significant at 10 d and 14 d compared with sham group (P>0.05). The total feeding time at 14 d significant longer compared with the EOI hyperalgesia maintaining group (P<0.05). Semi-quantitative analysis of immunofluorescence staining showed that the fluorescence area and fluorescence intensity of GFAP and OX-42 in EOI hyperalgesia maintaining group did not show any increase compared with that of sham group (P>0.05), whereas the fluorescence area and fluorescence intensity of GFAP as well as the fluorescence area of OX-42 in EOI hyperalgesia maintaining group 6 d were significantly higher (P<0.05). The fluorescence area and fluorescence intensity of GFAP and OX-42 in EOI hyperalgesia maintaining group did not show any increase compared with that of EOI hyperalgesia maintaining group 6 d (P>0.05). Conclusion DRt was involved in the persistent maintenance of hyperalgesia in the EOI model after removal of occlusal interference, in which astrocyte and microglia activation in DRt were the central mechanisms for the maintenance of hyperalgesia.

, correspAuthors=Jian-Qiu Jin, authorNote=null, correspAuthorsNote=
E-mail:
, copyrightStatement=null, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, authorCompany=null, fund=null, authors=null, authorsList=Yong-Wei Xu, Ying-Ying Fan, Ye Cao, Zhi-Yue Lu, Jian-Qiu Jin), CN=ArticleExt(id=1200023162785202836, articleId=1200023158267937344, tenantId=1146029695717560320, journalId=1189873630562394117, language=CN, title=延髓背侧网状核胶质细胞活化参与大鼠咬合干扰致口颌面痛觉敏感的中枢机制, columnId=1190310110472798614, journalTitle=解放军医学杂志, columnName=基础研究, runingTitle=null, highlight=null, articleAbstract=

目的 探讨大鼠咬合干扰去除后痛觉敏感维持模型中延髓背侧网状核(DRt)胶质细胞活化参与口颌面痛觉敏感的中枢机制。方法 24只SD雄性大鼠(180~200 g)随机分为假手术组(于1%戊巴比妥钠全麻下保持开口5 min,但不粘固牙冠)、痛觉敏感维持组[实验性咬合干扰(EOI)8 d后去除干扰]、痛觉敏感维持(EOI 8 d后去除干扰)+DRt损毁组,每组8只,行为学实验动物建模后于7、10、14 d测定各组大鼠自我赏罚行为学表现。另取9只SD大鼠分为假手术组、痛觉敏感维持组(施加EOI 8 d去除EOI前)、痛觉敏感维持组6 d(施加EOI 8 d并去除EOI后6 d),每组3只,左心室灌流后取材进行DRt脑区胶质纤维酸性蛋白(GFAP)和大鼠小胶质细胞特异性标志物(OX-42)的免疫荧光染色,并进行半定量分析星形胶质细胞和小胶质细胞的荧光强度和荧光面积。结果 EOI 10 d时,与假手术组比较,痛觉敏感维持组总摄食时间明显缩短(P<0.05),痛觉敏感维持+DRt损毁组总摄食时间虽较假手术组缩短,但差异无统计学意义(P>0.05);EOI 14 d时,与假手术组比较,痛觉敏感维持组总摄食时间仍明显缩短(P<0.05),而痛觉敏感维持+DRt损毁组差异无统计学意义(P>0.05);与痛觉敏感维持组比较,痛觉敏感维持+DRt损毁组总摄食时间明显延长(P<0.05)。免疫荧光染色半定量分析显示,与假手术组比较,痛觉敏感维持组GFAP及OX-42的荧光面积、荧光强度差异均无统计学意义(P>0.05),而痛觉敏感维持组6 d的GFAP荧光面积、荧光强度及OX-42荧光面积均明显增高(P<0.05);与痛觉敏感维持组相比,痛觉敏感维持组6 d的GFAP及OX-42的荧光面积、荧光强度差异均无统计学意义(P>0.05)。结论 DRt参与了大鼠EOI模型在去除咬合干扰后痛觉敏感的维持,其中DRt的星形胶质细胞和小胶质细胞活化是痛觉敏感维持的中枢机制。

, correspAuthors=金建秋, authorNote=null, correspAuthorsNote=
金建秋,E-mail:
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许永伟,医学博士,主治医师,主要从事口腔修复和颌面部疼痛等方面的研究

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许永伟,医学博士,主治医师,主要从事口腔修复和颌面部疼痛等方面的研究

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许永伟,医学博士,主治医师,主要从事口腔修复和颌面部疼痛等方面的研究

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DRt. 延髓网状背侧亚核;EOI. 实验性咬合干扰

, figureFileSmall=Zs2NknfWrZo1KoTnSkWgww==, figureFileBig=OoWg5A+LujBG0Fd31e90cQ==, tableContent=null), ArticleFig(id=1200061230149894455, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1200023158267937344, language=EN, label=Fig.2, caption=Time course of body weight and orofacial operant test in different groups of rats, figureFileSmall=n880EbDEkEkVD/yWvqGw4Q==, figureFileBig=YhGia8bnWFyuvVEoAPGevw==, tableContent=null), ArticleFig(id=1200061230246363450, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1200023158267937344, language=CN, label=图2, caption=不同组实验大鼠体重情况与自我赏罚实验行为学表现

DRt. 延髓背侧网状核;A. 各组大鼠的体重变化情况;B. 各组大鼠总摄食时间比较;*P<0.05

, figureFileSmall=n880EbDEkEkVD/yWvqGw4Q==, figureFileBig=YhGia8bnWFyuvVEoAPGevw==, tableContent=null), ArticleFig(id=1200061230338638141, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1200023158267937344, language=EN, label=Fig.3, caption=Expression of GFAP immunofluorescence staining (A) and semiquantitative analysis (B) in the rats of each group, figureFileSmall=eNmeE88Mrzx75sBxKPLVAA==, figureFileBig=//lOO3u69IwYCEgBuVde2Q==, tableContent=null), ArticleFig(id=1200061230422524225, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1200023158267937344, language=CN, label=图3, caption=各组大鼠GFAP免疫荧光染色变化(A)及半定量分析(B)

GFAP. 胶质纤维酸性蛋白;*P<0.05

, figureFileSmall=eNmeE88Mrzx75sBxKPLVAA==, figureFileBig=//lOO3u69IwYCEgBuVde2Q==, tableContent=null), ArticleFig(id=1200061230527381828, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1200023158267937344, language=EN, label=Fig.4, caption=Expression of OX-42 immunofluorescence staining (A) and semiquantitative analysis (B) in the rats of each group, figureFileSmall=y84gwZJhrUt8ATW4h83TnA==, figureFileBig=2PATl8O7A5ypeI1DHghOXQ==, tableContent=null), ArticleFig(id=1200061230640628040, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1200023158267937344, language=CN, label=图4, caption=各组大鼠OX-42免疫荧光染色变化(A)以及半定量分析(B)

OX-42. 小胶质细胞特异性标志物;*P<0.05

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延髓背侧网状核胶质细胞活化参与大鼠咬合干扰致口颌面痛觉敏感的中枢机制
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许永伟 1 , 范莹莹 2 , 曹烨 3 , 陆支越 2 , 金建秋 2, *
解放军医学杂志 | 基础研究 2023,48(10): 1129-1134
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解放军医学杂志 | 基础研究 2023, 48(10): 1129-1134
延髓背侧网状核胶质细胞活化参与大鼠咬合干扰致口颌面痛觉敏感的中枢机制
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许永伟1, 范莹莹2, 曹烨3, 陆支越2, 金建秋2, *
作者信息
  • 1北京大学人民医院口腔科,北京 100044
  • 2北京医院口腔科/国家老年医学中心/中国医学科学院老年医学研究院,北京 100730
  • 3北京大学口腔医学院口腔医院修复科/口颌功能诊疗研究中心/国家口腔疾病临床医学研究中心/口腔数字化医疗技术和材料国家工程实验室/口腔数字医学北京市重点实验室,北京 100081
  • 许永伟,医学博士,主治医师,主要从事口腔修复和颌面部疼痛等方面的研究

通讯作者:

金建秋,E-mail:
Activation of glial cells in dorsal reticular nucleus of the medulla oblongata involved in the central mechanism of oral and maxillofacial pain sensitivity induced by occlusal interference in rats
Yong-Wei Xu1, Ying-Ying Fan2, Ye Cao3, Zhi-Yue Lu2, Jian-Qiu Jin2, *
Affiliations
  • 1Department of Stomatology, Peking University People's Hospital, Beijing 100044, China
  • 2Department of Stomatology, Beijing Hospital/National Center of Gerontology/Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing 100730, China
  • 3Department of Prosthodontics, Peking University School and Hospital of Stomatology/Center for Oral and Jaw Functional Diagnosis/National Clinical Research Center for Oral Diseases/National Engineering Laboratory for Digital and Material Technology of Stomatology/Beijing Key Laboratory of Digital Stomatology, Beijing 100081, China
出版时间: 2023-10-28 doi: 10.11855/j.issn.0577-7402.1942.2023.0817
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目的 探讨大鼠咬合干扰去除后痛觉敏感维持模型中延髓背侧网状核(DRt)胶质细胞活化参与口颌面痛觉敏感的中枢机制。方法 24只SD雄性大鼠(180~200 g)随机分为假手术组(于1%戊巴比妥钠全麻下保持开口5 min,但不粘固牙冠)、痛觉敏感维持组[实验性咬合干扰(EOI)8 d后去除干扰]、痛觉敏感维持(EOI 8 d后去除干扰)+DRt损毁组,每组8只,行为学实验动物建模后于7、10、14 d测定各组大鼠自我赏罚行为学表现。另取9只SD大鼠分为假手术组、痛觉敏感维持组(施加EOI 8 d去除EOI前)、痛觉敏感维持组6 d(施加EOI 8 d并去除EOI后6 d),每组3只,左心室灌流后取材进行DRt脑区胶质纤维酸性蛋白(GFAP)和大鼠小胶质细胞特异性标志物(OX-42)的免疫荧光染色,并进行半定量分析星形胶质细胞和小胶质细胞的荧光强度和荧光面积。结果 EOI 10 d时,与假手术组比较,痛觉敏感维持组总摄食时间明显缩短(P<0.05),痛觉敏感维持+DRt损毁组总摄食时间虽较假手术组缩短,但差异无统计学意义(P>0.05);EOI 14 d时,与假手术组比较,痛觉敏感维持组总摄食时间仍明显缩短(P<0.05),而痛觉敏感维持+DRt损毁组差异无统计学意义(P>0.05);与痛觉敏感维持组比较,痛觉敏感维持+DRt损毁组总摄食时间明显延长(P<0.05)。免疫荧光染色半定量分析显示,与假手术组比较,痛觉敏感维持组GFAP及OX-42的荧光面积、荧光强度差异均无统计学意义(P>0.05),而痛觉敏感维持组6 d的GFAP荧光面积、荧光强度及OX-42荧光面积均明显增高(P<0.05);与痛觉敏感维持组相比,痛觉敏感维持组6 d的GFAP及OX-42的荧光面积、荧光强度差异均无统计学意义(P>0.05)。结论 DRt参与了大鼠EOI模型在去除咬合干扰后痛觉敏感的维持,其中DRt的星形胶质细胞和小胶质细胞活化是痛觉敏感维持的中枢机制。

咬合干扰  /  口颌面痛觉敏感  /  延髓背侧网状核  /  胶质细胞

Objective To investigate the central mechanism of glial cells of the dorsal reticular nucleus (DRt) in the modulation of chronic orofacial pain after removal of experimental occlusal interference(EOI) in rats. Methods Twenty-four male SD rats (180-200 g) were randomly divided into three groups (8 in each group): sham group, EOI hyperalgesia maintaining group (occlusal interference appliance removed on day 8 after wearing 0.4 mm thick crowns), EOI hyperalgesia maintaining+DRt damage group (EOI hyperalgesia maintaining rats were injected with ibotenic acid to damage DRt). The non-reflexive behaviors of the three groups were evaluated by using orofacial operant test on 7, 10, 14 d after model establishment. Nine male SD rats were randomly divided into three groups (3 in each group): sham group, EOI hyperalgesia maintaining group (day 8 after wearing 0.4 mm thick crowns, before removal of EOI), EOI hyperalgesia maintaining group 6 d (6 days after EOI removed on day 8). DRt sections were obtained and processed for immunofluorescence staining for glai fibrillary acidic protein (GFAP) and OX-42. The levels of expression were hemi-quantitatively analyzed to evaluate the fluorescence area and fluorescence intensity of astrocytes and microglia. Results EOI hyperalgesia maintaining group and EOI hyperalgesia maintaining+DRt damage group exhibited lower feeding time in orofacial operant test, which implied hyperalgesia (P<0.05). The hyperalgesia in EOI hyperalgesia maintaining group persisted after the removal of EOI, and the difference was statistically significant at 10 d and 14 d compared with sham group (P<0.05), while the hyperalgesia in the EOI hyperalgesia maintaining+DRt damage group showed a rebound trend, and the difference was not statistically significant at 10 d and 14 d compared with sham group (P>0.05). The total feeding time at 14 d significant longer compared with the EOI hyperalgesia maintaining group (P<0.05). Semi-quantitative analysis of immunofluorescence staining showed that the fluorescence area and fluorescence intensity of GFAP and OX-42 in EOI hyperalgesia maintaining group did not show any increase compared with that of sham group (P>0.05), whereas the fluorescence area and fluorescence intensity of GFAP as well as the fluorescence area of OX-42 in EOI hyperalgesia maintaining group 6 d were significantly higher (P<0.05). The fluorescence area and fluorescence intensity of GFAP and OX-42 in EOI hyperalgesia maintaining group did not show any increase compared with that of EOI hyperalgesia maintaining group 6 d (P>0.05). Conclusion DRt was involved in the persistent maintenance of hyperalgesia in the EOI model after removal of occlusal interference, in which astrocyte and microglia activation in DRt were the central mechanisms for the maintenance of hyperalgesia.

occlusal interference  /  orofacial pain  /  subnucleus reticularis dorsalis  /  glia cells
许永伟, 范莹莹, 曹烨, 陆支越, 金建秋. 延髓背侧网状核胶质细胞活化参与大鼠咬合干扰致口颌面痛觉敏感的中枢机制. 解放军医学杂志, 2023 , 48 (10) : 1129 -1134 . DOI: 10.11855/j.issn.0577-7402.1942.2023.0817
Yong-Wei Xu, Ying-Ying Fan, Ye Cao, Zhi-Yue Lu, Jian-Qiu Jin. Activation of glial cells in dorsal reticular nucleus of the medulla oblongata involved in the central mechanism of oral and maxillofacial pain sensitivity induced by occlusal interference in rats[J]. Medical Journal of Chinese People’s Liberation Army, 2023 , 48 (10) : 1129 -1134 . DOI: 10.11855/j.issn.0577-7402.1942.2023.0817
机体疼痛下行调制(descending pain modulation)系统的下行易化(促进疼痛感受)和下行抑制(抑制疼痛感受)作用是协调工作的,当这种平衡被打破时,则出现下行调制的失衡:如果下行易化处于优势,则表现为疼痛感受加重;如果下行抑制占主导,则表现为疼痛感受迟钝或缺失[1-3]。疼痛下行调制系统的主要组成部分包括延髓头端腹内侧区(rostral ventromedial medulla,RVM)、延髓尾端腹外侧区(caudal ventrolateral medulla,VLM)和延髓网状背侧亚核(dorsal reticular nucleus,DRt)等[1-3]。RVM-VLM-DRt之间存在紧密的环路联系,在下行调制机制中具有重要作用。RVM的双向调节机制是下行调制的中继站,已有较多深入的研究[1-3]。而目前关于DRt的研究较少,少量研究倾向于认为DRt主要参与下行易化调制[4]。目前的研究主要针对DRt神经元机制,尚未见关于胶质细胞在DRt参与的下行调制中的作用。本研究将利用课题组成熟的对咬合干扰致口颌面痛觉敏感模型[5-6],对DRt胶质细胞参与咬合干扰所致口颌面疼痛的机制进行初步探讨,旨在为口颌面疼痛的治疗提供新的靶向脑区。
健康雄性SD大鼠33只,体重180~200 g,由北京维通利华实验动物技术有限公司提供。24 h昼夜交替,室温25 ℃。自我赏罚实验大鼠测试日预先禁食不禁水,其余时间自由饮食饮水;其余实验大鼠自由饮食饮水。所有动物实验均获北京大学医学部伦理委员会审批(LA2019353、LA2019354)。
将24只SD大鼠随机均分为假手术组、痛觉敏感维持组、痛觉敏感维持+DRt损毁组,每组8只。其中,假手术组大鼠于1%戊巴比妥钠全麻下保持开口5 min,但不粘固牙冠;痛觉敏感维持组参考课题组既往的实验方法[5]建立实验性咬合干扰(experimental occlusal interference,EOI)模型,EOI后8 d在1%戊巴比妥钠全麻下,用尖探针去除金属冠;痛觉敏感维持+DRt损毁组于DRt损毁后1周建立EOI模型8 d后去除金属冠,损毁药物使用1 g/μl鹅膏簟氨酸(ibotenic acid,IBO,MCE公司,中国)。实验大鼠在1%戊巴比妥钠麻醉下,将头部固定在脑立体定位仪上,根据大鼠脑图谱确定DRt坐标为前囟点后15 mm,左右旁开1.4 mm,从小脑表面进入,深度为6.5 mm。IBO用生理盐水稀释至1 μg/μl。使用前1 d配制。将1 μl的尖头微量注射器连接于微量注射泵上,双侧DRt给予0.3 μl IBO,持续5 min,注射后继续留针3 min以防止药物反溢。注射后缝合大鼠头部伤口,实验大鼠术后恢复1周后EOI,EOI后8 d去除咬合干扰。大鼠自我赏罚行为学实验采用Ugo Basile面部疼痛测试仪(Ugo Basile Orofacial Stimulation Test System,ComerioVA,意大利),具体测试方法参考文献[7]。实验流程见图1。采用盲法(测试者不知道动物分组情况)进行行为学测试,记录各组大鼠EOI,以及EOI 7、10、14 d的自我赏罚实验结果及体重。
将9只SD大鼠随机均分为假手术组(n=3,处理同1.2)、痛觉敏感维持组(n=3,EOI 8 d去除EOI前)及痛觉敏感维持组6 d(n=3,EOI 8 d去除EOI后6 d)。用1%戊巴比妥钠过量麻醉大鼠后行左心室灌注。先用250 ml温热生理盐水快速灌注,约300 ml 4%多聚甲醛灌流,然后取脑干及延髓以闩为中心头向4 mm,尾向1 mm组织块,放入4%多聚甲醛中固定、OCT包埋,冰冻切片,厚度为20 μm,每隔3片取出一片置于0.01 mol/L PBS溶液多孔皿中。取出4 ℃冰箱保存的组织漂片以0.01 mol/L PBS浸泡,10%驴血清封闭液室温封闭60 min,加入小胶质细胞特异性标志物(OX-42)(1∶200,Abcam,美国)或胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)(1∶400,CST,美国),4 ℃孵育过夜;0.01 mol/L PBS浸泡,驴抗小鼠IgG 1∶200室温孵育90 min;0.01 mol/L PBS浸泡、贴片、自然风干;防淬灭荧光封片剂封片,荧光显微镜下观察、拍片。荧光照片规格为512×512像素,保证相同增益及曝光条件下进行半定量分析。使用ImageJ软件对GFAP、OX-42的荧光面积及荧光强度进行半自动分析,每只大鼠选择5张脑切片进行分析。参考Colburn的胶质细胞活化分级标准[8]对大鼠DRt水平胶质细胞活化程度进行评价。基础染色:胶质细胞体积正常,均匀散在分布,伸出广泛细长的分支;轻度活化:胶质细胞染色微增强,呈分支状均匀分布;中度活化:胶质细胞胞体增大,染色增强,突起变短增粗,部分细胞之间有交叠;重度活化:胶质细胞胞体肥大,染色强阳性,突起短且粗,细胞相互之间重叠分布呈团块状。
采用SPSS 20.0软件进行统计分析。计量资料行正态性检验,呈正态分布者以$\bar{x}±s$表示,各组不同时间点的自我赏罚行为学结果采用重复测量的方差分析,进一步两两比较采用Bonferroni检验;各组相同时间点的机械刺激反应阈值与自我赏罚行为学结果采用多因素方差分析,进一步两两比较采用Bonferroni检验;对胶质细胞染色结果进行多因素方差分析,进一步两两比较采用Bonferroni检验。P<0.05为差异有统计学意义。
假手术组、痛觉敏感维持组及痛觉敏感维持+DRt损毁组大鼠在测试期间体重均出现缓慢增加,但各时间点差异无统计学意义(P>0.05,图2A)。
与假手术组比较,痛觉敏感维持组大鼠在8 d去除咬合干扰后总摄食时间缩短,在10、14 d时差异有统计学意义[10 d:(190.8±28.6) s vs. (293.3±31.4) s;14 d:(179.6±34.4) s vs. (309.2±19.2) s;P<0.05]。而痛觉敏感维持+DRt损毁组大鼠在8 d去除咬合干扰后,总摄食时间延长,在14 d时总摄食时间明显长于痛觉敏感维持组,差异有统计学意义[(286.1±17.1) s vs. (179.6±34.4) s,P<0.05];在10、14 d时与假手术组比较差异均无统计学意义[10 d:(241.6±20.4) s vs. (293.3±31.4) s;14 d:(286.1±17.1) s vs. (309.2±19.2) s;P<0.05,图2B]。
免疫染色结果显示,假手术组GFAP阳性染色位于星形胶质细胞的胞质和突起,从较小的胞体向周围发出细丝状分支,呈现为基础染色;痛觉敏感维持组星形胶质细胞与假手术组比较无明显变化;痛觉敏感维持组6 d星形胶质细胞胞体增大,分支变粗增多,染色增强,部分分支偶有交叠,呈现为中度活化(图3A)。半定量分析显示,与假手术组比较,痛觉敏感维持组星形胶质细胞的GFAP荧光强度及荧光面积变化差异均无统计学意义,而痛觉敏感维持组6 d星形胶质细胞的GFAP荧光强度及荧光面积均明显增高(P<0.05),且痛觉敏感维持组6 d与痛觉敏感维持组差异无统计学意义(P>0.05)(图3B)。
免疫染色结果显示,假手术组OX-42染色阳性表达于小胶质细胞的胞质和突起,从较小的胞体向周围发出爪状分支,呈现为基础染色;痛觉敏感维持组小胶质细胞与假手术组比较无明显变化;痛觉敏感维持组6 d小胶质细胞胞体增大,分支变粗增多,染色增强,部分聚集成团状,呈现为中度活化(图4A)。半定量分析显示,与假手术组比较,痛觉敏感维持组6 d小胶质细胞的OX-42荧光强度差异无统计学意义,但荧光面积较假手术组明显增大(P<0.05),而痛觉敏感维持组OX-42荧光强度及荧光面积与假手术组比较差异均无统计学意义,且痛觉敏感维持组6 d与痛觉敏感维持组差异无统计学意义(P>0.05)(图4B)。
慢性口颌面疼痛是临床治疗的困境,临床上可观察到咬合因素相关的口颌面疼痛患者在咬合因素去除后的转归不同,部分患者早期去除咬合因素后疼痛可缓解,而另一部分患者即使咬合因素去除后疼痛仍然存在。本课题组前期建立了一个接近临床、刺激可随时去除、可模拟不同痛觉敏感状态的咬合干扰相关的口颌面疼痛动物模型,证实在大鼠咬合干扰后8 d去除咬合干扰,大鼠的颌面部痛觉敏感仍持续存在,这与临床上患者长时间存在咬合相关颌面部疼痛时对因治疗(去除咬合)无效的情况相符[9-10]。因此,研究者对口颌面疼痛的下行调制机制进行了系列研究,揭示了不同痛觉敏感转归状态的下行调制机制动态变化[5-6],且发现了RVM内介导下行易化调制的ON-神经元和下行抑制调制的OFF-神经元的可塑性变化及二者的功能失衡在不同痛觉敏感转归状态下的作用,且RVM星形胶质细胞也参与了口颌面痛觉敏感维持的下行调制[5-6]。而DRt是参与下行易化机制的重要脑区,其能够接受来自RVM以及更高级中枢的调控信息,最终将下行调控信号投射至脊髓,这是本研究实验设计的重要基础[4]
本研究中,损毁DRt痛觉敏感维持模型去干扰前疼痛表现与未损毁时差异无统计学意义,而在去干扰后疼痛逆转,提示DRt在该模型去干扰后的痛觉敏感维持期起下行易化作用。在外界刺激存在时,DRt胶质细胞无明显活化,而当去除外界刺激后,疼痛上行传递通路作用减弱,但疼痛仍未恢复,此时胶质细胞活化,提示DRt的胶质细胞是痛觉敏感维持期下行易化的相关机制之一。
DRt中的胶质细胞参与下行易化的具体机制尚不明确,但下行易化的信号最终需要通过神经纤维传递,而神经元作为神经纤维疼痛信号传递的基础在疼痛的产生与传递中发挥着重要作用。有关上行传递通路的研究已发现胶质细胞与神经元细胞间存在着密切的相互作用。以脊髓为例,外周炎症或损伤后初级传入神经末梢会释放趋化因子、炎性介质等作用于胶质细胞受体,刺激胶质细胞活化。胶质细胞中丝裂原活化蛋白激酶磷酸化导致促炎细胞因子[如肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-6等]的产生及释放,这些促炎细胞因子一方面通过自分泌或旁分泌的方式形成自反馈作用,另一方面能够与神经元受体结合,增强神经元活性。有研究显示,IL-1β、TNF-α能够促进突触后N-甲基-D-天冬氨酸(NMDA)受体的突触传递并增加这类受体在神经元细胞膜上的表达;胶质细胞的活化与神经元细胞的敏化之间相互促进,形成了慢性疼痛机制中脊髓背角的一个正反馈循环[11-12]。在下行调制研究中,部分研究也指出下行调制系统中神经元与胶质细胞间可能存在类似的作用机制[13-14]。Wei等[13]发现,在三叉神经病理性疼痛大鼠中,RVM的小胶质细胞在建模后1~3 d出现早期活化,随后在RVM还出现了持续至少28 d的星形胶质细胞活化,并在14 d出现峰值;RVM注射胶质细胞抑制剂可明显减弱手术诱导的机械痛觉敏感。该研究进一步发现,RVM的星形胶质细胞通过细胞因子TNF-α及IL-1β作用于神经元的NMDA受体,使NMDA受体磷酸化,提示RVM星形胶质细胞活化分泌的TNF-α和IL-1β参与了维持神经病理性疼痛的脊髓上机制。Ni等[14]发现,在骨癌疼痛模型中趋化因子CXC配体-受体趋化因子CXC受体2信号通路参与了中脑导水管周围灰质的胶质细胞-神经元相互作用下的下行易化。一项关于脊髓注射5-羟色胺3(5-HT3)受体激动剂诱发机械痛觉敏感的研究发现,注射后出现神经元细胞敏化、细胞因子IL-18表达上调、胶质细胞活化,神经元-细胞因子-胶质细胞间的级联反应可能与5-HT3受体介导的下行易化相关[12]。上述研究提示,胶质细胞与神经元之间的相互作用可能是本研究中胶质细胞参与咬合干扰致口颌面痛觉敏感维持的具体机制。
本研究采用自我赏罚实验进行痛觉敏感测试。自我赏罚实验允许动物在接受奖赏或逃避伤害性刺激间做出选择,与反射行为测试方法相比更客观,更能反映动物高级中枢对伤害性刺激加工后的表现[15-16]。临床上亦可观察到咬合干扰相关口颌面疼痛患者,其焦虑、抑郁等评分较高[17-18]。前期研究还发现,海马、前扣带回等均参与了咬合干扰致口颌面痛觉敏感的中枢机制[19-20],提示该咬合干扰模型的疼痛特点与情绪认知高级中枢功能活动等相关。因此,使用自我赏罚实验方法结合反射行为实验结果可更全面地反映该模型的行为学特点。该方法能够反映高级中枢对疼痛的整合,且在实验过程中操作方便,实验大鼠适应期训练结束后,实验人员每次测试仅需安装操作仪器,并不需要对实验大鼠反复操作,可减轻实验动物的恐惧、紧张等心理,实验结果稳定,大鼠摄食水平稳定。但该方法也存在一些缺点:如实验周期长,适应期训练即需要2~3周,实验动物存在训练失败的可能;实验前还需对大鼠禁食。综上,自我赏罚实验是颌面部痛觉敏感测定的一种可靠、易行的行为学方法。
综上所述,本研究探索了大鼠咬合干扰去除后痛觉敏感维持模型中DRt胶质细胞活化参与口颌面痛觉敏感的中枢机制,对临床问题有一定提示意义。但本研究也存在一些局限:虽然发现咬合干扰后痛觉敏感维持与DRt胶质细胞活化相关,且胶质细胞可能起下行易化作用,但并未进一步探索胶质细胞下行易化的深入机制。因此,未来可进一步关注下行调制机制中胶质细胞的具体作用机制、神经元的作用及神经元-胶质细胞的相互作用等,以研究更高级中枢在颌面部疼痛中的复杂调控机制,助力于减少颌面部慢性疼痛的发生,缓解疼痛患者的症状,促进其身心健康。
  • 中央高水平医院临床科研业务费(BJ-2022-177)
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2023年第48卷第10期
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doi: 10.11855/j.issn.0577-7402.1942.2023.0817
  • 接收时间:2022-09-05
  • 首发时间:2025-11-25
  • 出版时间:2023-10-28
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  • 收稿日期:2022-09-05
  • 录用日期:2023-04-26
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National High Level Hospital Clinical Research Funding(BJ-2022-177)
中央高水平医院临床科研业务费(BJ-2022-177)
作者信息
    1北京大学人民医院口腔科,北京 100044
    2北京医院口腔科/国家老年医学中心/中国医学科学院老年医学研究院,北京 100730
    3北京大学口腔医学院口腔医院修复科/口颌功能诊疗研究中心/国家口腔疾病临床医学研究中心/口腔数字化医疗技术和材料国家工程实验室/口腔数字医学北京市重点实验室,北京 100081

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2种不同金属材料的力学参数

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genus
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Percentage of
total species (%)

Genus
种数
Number of
species
占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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