Article(id=1190669164684652763, tenantId=1146029695717560320, journalId=1189873630562394117, issueId=1190669163988398295, articleNumber=null, orderNo=null, doi=10.11855/j.issn.0577-7402.1430.2024.1217, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1726588800000, receivedDateStr=2024-09-18, revisedDate=null, revisedDateStr=null, acceptedDate=1731513600000, acceptedDateStr=2024-11-14, onlineDate=1761807250424, onlineDateStr=2025-10-30, pubDate=1745769600000, pubDateStr=2025-04-28, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1761807250424, onlineIssueDateStr=2025-10-30, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1761807250424, creator=13701087609, updateTime=1761807250424, updator=13701087609, issue=Issue{id=1190669163988398295, tenantId=1146029695717560320, journalId=1189873630562394117, year='2025', volume='50', issue='4', pageStart='367', pageEnd='503', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=0, createTime=1761807250258, creator=13701087609, updateTime=1761807667423, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1190670913772339410, tenantId=1146029695717560320, journalId=1189873630562394117, issueId=1190669163988398295, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1190670913772339411, tenantId=1146029695717560320, journalId=1189873630562394117, issueId=1190669163988398295, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=452, endPage=457, ext={EN=ArticleExt(id=1190669164881785054, articleId=1190669164684652763, tenantId=1146029695717560320, journalId=1189873630562394117, language=EN, title=Three-dimensional light sheet microscopy imaging for evaluating intraplaque neovascularization in arterial plaques and the efficacy of interventions, columnId=1190310110212751762, journalTitle=Medical Journal of Chinese People’s Liberation Army, columnName=Basic Research, runingTitle=null, highlight=null, articleAbstract=

Objective To investigate application value of three-dimensional light sheet microscopy imaging for evaluating intraplaque neovascularization in arterial plaques and the efficacy of intervention, and to assess the effect of melatonin (MLT) on neovascularization by these means. Methods Thirty-six ApoE-/- model mice were randomly divided into three groups (n=12): vehicle group, MLT group, and MLT+GW9662 intervention group (MLT+GW). The mice were treated with vehicle, MLT alone, or MLT combined with peroxisome proliferator activated receptor‑γ (PPARγ) inhibitor GW9662, respectively. The carotid arteries of the models were three-dimensionally imaged using a light sheet microscopy, and the length, volume and other indicators of neovascularization were quantitatively analyzed using Imaris software. Subsequently, CD31 immunohistochemical staining was performed for verification. Results The light sheet microscopy preliminarily achieved the three-dimensional visualization of intraplaque neovascularization, and its structure was observed to be three-dimensionally reticular and scattered. The results of Imaris quantitative analysis showed that, compared with vehicle group, the total intraplaque neovas cularization length in the MLT group was shortened [(15.79±12.90) mm vs. (33.42±11.16) mm, P<0.05], the total volume was reduced [(1.34±1.47)×10-3 mm3 vs. (13.44±7.35)×10-3 mm3, P<0.05], and the volume ratio was decreased (0.44%±0.47% vs. 3.76%±1.74%, P<0.05). The above indicators in MLT+GW group were significantly increased compared with those in MLT group [total length: (35.31±4.69) mm, total volume: (8.87±3.46)×10-3 mm3, volume ratio: 2.89%±0.38%; P<0.05]. The CD31 immunohistochemical staining also supported the above findings (P<0.05). Conclusions Based on the light sheet microscopy imaging technology, the three-dimensional visualization and quantitative analysis of intraplaque neovascularization were preliminarily realized. It was found that MLT could reduce the overall burden of intraplaque neovascularization, and PPARγ might be involved in its regulatory process.

, correspAuthors=Yun-Dai Chen, authorNote=null, correspAuthorsNote=
E-mail:
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目的 探索三维光片显微成像在动脉斑块内新生血管可视化及定量分析中的应用价值,并借此评估褪黑素(MLT)对新生血管的影响。方法 36只ApoE-/-模型小鼠随机分为3组(n=12):溶剂组、MLT组及MLT联合GW9662干预(MLT+GW)组,分别接受Vehicle、MLT单独干预及MLT联合过氧化物酶体增殖物激活受体γ(PPARγ)抑制剂GW9662干预。采用光片显微镜对模型进行颈动脉三维成像,并借助Imaris软件对新生血管的长度、体积等指标进行定量分析,随后通过CD31免疫组化染色进行验证。结果 光片显微镜初步实现了斑块内新生血管的三维可视化,并观察到其结构呈三维网状且散在分布。Imaris定量分析结果显示,与溶剂组比较,MLT组斑块内新生血管的总长度缩短[(15.79±12.90) mm vs. (33.42±11.16) mm,P<0.05),总体积缩小[(1.34±1.47)×10-3 mm3 vs. (13.44±7.35)×10-3 mm3P<0.05],且体积分数降低(0.44%±0.47% vs. 3.76%±1.74%,P<0.05),MLT+GW组上述指标则较MLT组明显上调[总长度:(35.31±4.69) mm;总体积:(8.87±3.46)×10-3 mm3;体积分数:2.89%±0.38%;P<0.05]。CD31免疫组化染色也佐证了上述发现(P<0.05)。结论 基于光片显微成像技术初步实现了斑块内新生血管的三维可视化及定量分析,并发现MLT可减轻斑块内新生血管的整体负荷,且PPARγ可能参与了其调控过程。

, correspAuthors=陈韵岱, authorNote=null, correspAuthorsNote=
陈韵岱,E-mail:
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江雨凡,硕士研究生,主要从事动脉粥样硬化病理生理机制方面的研究

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江雨凡,硕士研究生,主要从事动脉粥样硬化病理生理机制方面的研究

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TS. 串联狭窄;A. 动物实验流程;B. 颈动脉TS手术示意图

, figureFileSmall=KQkvlafOD9xL987WgGjHKQ==, figureFileBig=cqP8QTlhE4P3IC4RPe1/XQ==, tableContent=null), ArticleFig(id=1190669353826792231, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1190669164684652763, language=EN, label=Fig.2, caption=Three-dimensional images of plaque and intraplaque neovascularization acquired by light sheet microscopy, figureFileSmall=lHpSWDneTAZhez/AB6HL5A==, figureFileBig=h1v4hidAx7WGxKYeiKiAMg==, tableContent=null), ArticleFig(id=1190669353889706792, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1190669164684652763, language=CN, label=图2, caption=光片显微镜获取的颈动脉斑块及斑块内新生血管三维图像

Isolectin. 内皮细胞荧光染料;TO-PRO. 细胞核荧光染料;A. 颈动脉原始荧光信号的三维视图;B. 颈动脉原始荧光信号的截面视图(bar=100 μm)

, figureFileSmall=lHpSWDneTAZhez/AB6HL5A==, figureFileBig=h1v4hidAx7WGxKYeiKiAMg==, tableContent=null), ArticleFig(id=1190669353952621353, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1190669164684652763, language=EN, label=Fig.3, caption=Tracking of fluorescence signal of intraplaque neovasculari-zation by Imaris software, figureFileSmall=97esXZ3ygpQ0O+OsJtICZA==, figureFileBig=YTkjFjR8rChs9DdpjB8isQ==, tableContent=null), ArticleFig(id=1190669354040701738, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1190669164684652763, language=CN, label=图3, caption=Imaris三维图像分析软件对斑块内新生血管荧光信号的拟合和追踪

Isolectin. 内皮细胞荧光染料;A、B. Imaris拟合的斑块内新生血管与原始荧光信号的对照;C、D. Imaris拟合的斑块内新生血管三维重建模型(bar=100 μm)

, figureFileSmall=97esXZ3ygpQ0O+OsJtICZA==, figureFileBig=YTkjFjR8rChs9DdpjB8isQ==, tableContent=null), ArticleFig(id=1190669355022168875, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1190669164684652763, language=EN, label=Fig.‍4, caption=Effect of melatonin (MLT) on overall burden of intraplaque neovascularization, figureFileSmall=L8bmCc2vV7hcqLquTyG6dQ==, figureFileBig=IGPKmEjFaspfGPtamxm2ug==, tableContent=null), ArticleFig(id=1190669355089277741, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1190669164684652763, language=CN, label=图4, caption=MLT对斑块内新生血管整体负荷的影响

MLT. 褪黑素;GW. GW9662,为PPARγ抑制剂;A. I段颈动脉斑块内新生血管的三维重建(bar=200 μm);B. 斑块内新生血管整体负荷对比(n=6);*P<0.05

, figureFileSmall=L8bmCc2vV7hcqLquTyG6dQ==, figureFileBig=IGPKmEjFaspfGPtamxm2ug==, tableContent=null), ArticleFig(id=1190669355147997998, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1190669164684652763, language=EN, label=Fig.5, caption=Effect of melatonin (MLT) on intraplaque neovascularization (CD31 immunohistochemical staining), figureFileSmall=ftvInH4elVJ+etS/TNYkew==, figureFileBig=PCnK9eD1L7eWG/ENeVhmzg==, tableContent=null), ArticleFig(id=1190669355210912559, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1190669164684652763, language=CN, label=图5, caption=MLT对斑块内新生血管的影响(CD31免疫组化染色)

MLT. 褪黑素;GW. GW9662,为PPARγ抑制剂;A. 斑块内新生血管的CD31免疫组化染色(SP法,bar=100 μm,仅孵育免疫组化二抗作为阴性对照);B. 各组斑块内新生血管密度对比(n=6);*P<0.05

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三维光片显微成像在动脉斑块内新生血管可视化及其干预中的应用研究
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江雨凡 1, 2 , 马强 2 , 佟伟 2 , 李越洋 2 , 陈韵岱 1, 2, *
解放军医学杂志 | 基础研究 2025,50(4): 452-457
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解放军医学杂志 | 基础研究 2025, 50(4): 452-457
三维光片显微成像在动脉斑块内新生血管可视化及其干预中的应用研究
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江雨凡1, 2, 马强2, 佟伟2, 李越洋2, 陈韵岱1, 2, *
作者信息
  • 1南开大学医学院,天津 300071
  • 2解放军总医院第六医学中心心血管医学部,北京 100048
  • 江雨凡,硕士研究生,主要从事动脉粥样硬化病理生理机制方面的研究

通讯作者:

陈韵岱,E-mail:
Three-dimensional light sheet microscopy imaging for evaluating intraplaque neovascularization in arterial plaques and the efficacy of interventions
Yu-Fan Jiang1, 2, Qiang Ma2, Wei Tong2, Yue-Yang Li2, Yun-Dai Chen1, 2, *
Affiliations
  • 1School of Medicine, Nankai University, Tianjin 300071, China
  • 2Department of Cardiology, the Sixth Medical Center of Chinese PLA General Hospital, Beijing 100048, China
出版时间: 2025-04-28 doi: 10.11855/j.issn.0577-7402.1430.2024.1217
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目的 探索三维光片显微成像在动脉斑块内新生血管可视化及定量分析中的应用价值,并借此评估褪黑素(MLT)对新生血管的影响。方法 36只ApoE-/-模型小鼠随机分为3组(n=12):溶剂组、MLT组及MLT联合GW9662干预(MLT+GW)组,分别接受Vehicle、MLT单独干预及MLT联合过氧化物酶体增殖物激活受体γ(PPARγ)抑制剂GW9662干预。采用光片显微镜对模型进行颈动脉三维成像,并借助Imaris软件对新生血管的长度、体积等指标进行定量分析,随后通过CD31免疫组化染色进行验证。结果 光片显微镜初步实现了斑块内新生血管的三维可视化,并观察到其结构呈三维网状且散在分布。Imaris定量分析结果显示,与溶剂组比较,MLT组斑块内新生血管的总长度缩短[(15.79±12.90) mm vs. (33.42±11.16) mm,P<0.05),总体积缩小[(1.34±1.47)×10-3 mm3 vs. (13.44±7.35)×10-3 mm3P<0.05],且体积分数降低(0.44%±0.47% vs. 3.76%±1.74%,P<0.05),MLT+GW组上述指标则较MLT组明显上调[总长度:(35.31±4.69) mm;总体积:(8.87±3.46)×10-3 mm3;体积分数:2.89%±0.38%;P<0.05]。CD31免疫组化染色也佐证了上述发现(P<0.05)。结论 基于光片显微成像技术初步实现了斑块内新生血管的三维可视化及定量分析,并发现MLT可减轻斑块内新生血管的整体负荷,且PPARγ可能参与了其调控过程。

三维成像  /  动脉粥样硬化  /  斑块内新生血管

Objective To investigate application value of three-dimensional light sheet microscopy imaging for evaluating intraplaque neovascularization in arterial plaques and the efficacy of intervention, and to assess the effect of melatonin (MLT) on neovascularization by these means. Methods Thirty-six ApoE-/- model mice were randomly divided into three groups (n=12): vehicle group, MLT group, and MLT+GW9662 intervention group (MLT+GW). The mice were treated with vehicle, MLT alone, or MLT combined with peroxisome proliferator activated receptor‑γ (PPARγ) inhibitor GW9662, respectively. The carotid arteries of the models were three-dimensionally imaged using a light sheet microscopy, and the length, volume and other indicators of neovascularization were quantitatively analyzed using Imaris software. Subsequently, CD31 immunohistochemical staining was performed for verification. Results The light sheet microscopy preliminarily achieved the three-dimensional visualization of intraplaque neovascularization, and its structure was observed to be three-dimensionally reticular and scattered. The results of Imaris quantitative analysis showed that, compared with vehicle group, the total intraplaque neovas cularization length in the MLT group was shortened [(15.79±12.90) mm vs. (33.42±11.16) mm, P<0.05], the total volume was reduced [(1.34±1.47)×10-3 mm3 vs. (13.44±7.35)×10-3 mm3, P<0.05], and the volume ratio was decreased (0.44%±0.47% vs. 3.76%±1.74%, P<0.05). The above indicators in MLT+GW group were significantly increased compared with those in MLT group [total length: (35.31±4.69) mm, total volume: (8.87±3.46)×10-3 mm3, volume ratio: 2.89%±0.38%; P<0.05]. The CD31 immunohistochemical staining also supported the above findings (P<0.05). Conclusions Based on the light sheet microscopy imaging technology, the three-dimensional visualization and quantitative analysis of intraplaque neovascularization were preliminarily realized. It was found that MLT could reduce the overall burden of intraplaque neovascularization, and PPARγ might be involved in its regulatory process.

three-dimensional imaging  /  atherosclerosis  /  intraplaque neovascularization
江雨凡, 马强, 佟伟, 李越洋, 陈韵岱. 三维光片显微成像在动脉斑块内新生血管可视化及其干预中的应用研究. 解放军医学杂志, 2025 , 50 (4) : 452 -457 . DOI: 10.11855/j.issn.0577-7402.1430.2024.1217
Yu-Fan Jiang, Qiang Ma, Wei Tong, Yue-Yang Li, Yun-Dai Chen. Three-dimensional light sheet microscopy imaging for evaluating intraplaque neovascularization in arterial plaques and the efficacy of interventions[J]. Medical Journal of Chinese People’s Liberation Army, 2025 , 50 (4) : 452 -457 . DOI: 10.11855/j.issn.0577-7402.1430.2024.1217
斑块内新生血管能够加速动脉斑块的进展,增加急性心血管事件的发生风险[1-3]。本课题组前期研究发现,褪黑素(melatonin,MLT)调控过氧化物酶体增殖物激活受体γ(peroxisome proliferator-activated receptor-γ,PPARγ)可影响斑块内新生血管的形成,且斑块内新生血管呈不均匀分布的三维连续结构[4]。然而,传统病理观察仅能展现某几个解剖层面的二维组织学特征,一定程度限制了其在此类组织中的应用[4-10]。2004年,Huisken等[11]发明了光片荧光显微镜(简称光片显微镜),该技术具有低光漂白性及高效的三维成像能力[12]。随着组织透明化技术的进步,光片显微镜已应用于大样本如鼠脑以及难以透明化的样本如骨、心脏的整体成像[6]。在动脉粥样硬化研究中,Becher等[5]和Buglak等[6]使用光片显微镜成功展示了动脉的管腔内皮细胞覆盖率及斑块空间分布等信息。但由于样本处理和动物模型的限制,目前尚未实现对整段血管中斑块内新生血管的三维可视化及定量分析。本研究使用光片显微镜对透明化的模型颈动脉进行三维成像,并借助Imaris三维图像分析软件对新生血管进行定量分析,旨在为斑块内新生血管及其干预效果提供有效的评估手段,并为全面研究斑块形成的病理生理机制及促进该领域的临床转化提供有力支持。
36只6周龄雄性ApoE-/- C57BL/6J小鼠(SPF级)购自北京维通利华实验动物技术有限公司[实验动物许可证号:SCXK(京)2021-0006],饲养于温度(22±3) ℃、湿度45%~60%的环境下,自由摄食饮水。将小鼠随机分为3组(n=12):溶剂组、MLT组及MLT联合GW9662干预(MLT+GW)组,分别接受vehicle、MLT单独干预及MLT联合PPARγ抑制剂GW9662干预。使用高脂饮食联合颈动脉串联狭窄(tandem stenosis,TS)手术构建斑块内新生血管模型:自小鼠6周龄起给予高脂饮食(含15%脂肪和1.25%胆固醇),并按分组给予MLT[10 mg/(kg.d)][13-14]或GW9662[3 mg/(kg.d)][15-16]灌胃干预10周。于小鼠12周龄时行TS手术,依据文献[17]的方法在小鼠右颈动脉创建管腔直径150 μm的串联狭窄(图1A、B)。最终于术后4周取Ⅰ段颈动脉为观察样本(图1B)。本研究获北京希诺因生物科技动物管理委员会批准(XNY-20230814001),且遵循《中华人民共和国实验动物管理条例》的相关规定。
Isolectin荧光染料已被既往研究用于标记心肌、肾和肺等组织的微血管结构[10,18]。因此,本研究参考文献[10],于TS术后4周给予小鼠尾静脉注射100 μl Isolectin GS-IB4(0.5 μg/μl,德国Thermo Fischer公司)用于颈动脉微血管结构的荧光标记,30 min后使用生理盐水和4%多聚甲醛溶液灌流并固定。随后使用TO-PRO荧光染料(1:1000,德国Thermo Fischer公司)标记细胞核,再将样本包埋入低熔点琼脂糖中。根据iDISCO透明化方法[19],将样本浸入四氢呋喃水溶液中进行梯度脱水,随后置入二氯甲烷中脱水、脱脂直至组织沉降,最后浸入二苄醚(dibenzyl ether,DBE)中直至组织呈透明状态。
使用ZEISS Lightsheet 7光片显微镜(A23000102,德国ZEISS公司)对透明化的颈动脉样本进行三维成像。首先将样本粘贴于载物架上,注入DBE作为折射率匹配的成像介质,物镜选择20×/1.0(RI=1.53),使用488 nm(TO-PRO)和647 nm(Isolectin)荧光通道,曝光时长设定为30 ms,激光能量调整为30%。成像完成后,将图像导入Imaris 10.1三维图像分析软件(Bitplane software)进行后续分析。采用Surface分析模块对颈动脉血管壁和斑块内新生血管的荧光信号进行拟合及渲染,随后采用Filament分析模块对提取的斑块内新生血管荧光信号进行拟合及追踪。将上述分析流程保存为模板,用于对所有样本的标准化、流程化分析。最后,在“Statistics”工具栏中提取实验相关数据。
TS术后4周,使用生理盐水和4%多聚甲醛溶液对模型动物进行灌流并固定,取其颈动脉组织样本竖直包埋入石蜡中,取石蜡切片行CD31(1:2000,ab182981,英国Abcam公司)免疫组化染色,取相邻切片作为阴性对照(HRP标记山羊抗兔二抗,PN0046,1:500,武汉皮诺飞生物科技有限公司),随后使用显微镜成像,并采用ImageJ软件对斑块内新生血管进行定量分析。
采用GraphPad Prism 9.0软件进行统计分析。所有数据均为计量资料且符合正态分布,以x±s表示,多组间比较采用单因素方差分析(one-way ANOVA),进一步两两比较采用Tukey法。P<0.05为差异有统计学意义。
本研究借助细胞核荧光信号(TO-PRO)在三维视图(图2A)上展示了一段颈动脉样本,其中可见条索状交错分布的内皮细胞荧光信号(Isolectin);而在截面视图(图2B)上进一步观察到了不同程度增厚的斑块及狭窄的管腔,且Isolectin展示了管腔内皮细胞及斑块内的新生血管。
借助Imaris三维图像分析软件进一步对斑块内的新生血管进行可视化及定量分析,图3A展示了内皮细胞荧光通道,并使用该原始荧光信号对斑块内新生血管进行拟合(图3B、C);在三维视图中可观察到斑块内新生血管多数呈三维网状结构(图3C、D)。在此基础上通过软件提取相关数据以用于后续的统计分析。
进一步分析MLT对模型小鼠颈动脉(图1B,I段颈动脉)斑块内新生血管三维整体负荷的影响显示,各组均存在散在的斑块内新生血管,且分布不均(图4A)。与溶剂组比较,MLT组斑块内新生血管的总长度缩短[(15.79±12.90) mm vs. (33.42±11.16) mm,P<0.05],总体积缩小[(1.34±1.47)×10-3 mm3 vs. (13.44±7.35)×10-3 mm3P<0.05],且体积分数降低(0.44%±0.47% vs. 3.76%±1.74%,P<0.05);与MLT组比较,MLT+GW组斑块内新生血管的整体负荷明显上调[总长度:(35.31±4.69) mm;总体积:(8.87±3.46)×10-3 mm3;体积分数:2.89%±0.38%],差异均有统计学意义(P<0.05),且MLT+GW组与溶剂组比较差异无统计学意义(P>0.05)(图4B)。
CD31免疫组化染色结果显示,溶剂组小鼠的颈动脉管腔狭窄,存在斑块和斑块内新生血管(图5A)。与溶剂组相比,MLT组斑块内新生血管数量减少,表现为CD31阳性染色范围缩小,斑块内新生血管密度降低(11.66%±3.88% vs. 3.44%±2.69%,P<0.05),而同时给予GW9662(PPARγ抑制剂)后CD31阳性染色范围扩大,斑块内新生血管密度增高(8.69%±3.62%,P<0.05)(图5B)。
三维成像使研究者能够从多维度解析模型样本,有利于全面评估样本的组织学特征,进而推进对疾病病理生理机制的研究[6,20]。Jansen等[21]采用病理三维重构技术分析肿瘤组织的侵袭程度,发现三维重构能够展示肿瘤组织不规则的立体轮廓,从而更准确地对肿瘤进行分期、分型。但该技术需要对样本连续切片并制片,过程复杂耗时,且破坏了原有的拓扑结构,导致组织层面不连续,不利于高效获取三维数据。共聚焦显微镜也能够对一定厚度(4~50 μm)的样本进行三维成像,但无法实现对较大组织的整体成像,且成像时间较长、光漂白和光毒性较大[10],一定程度上限制了其应用。近年来,光片显微镜已逐渐应用于大样本的整体成像[6]。Gorelashvili等[7]利用光片显微镜分析了骨髓细胞在骨髓腔内的分布及位移,发现巨核细胞可影响造血干细胞向血管的趋化运动,进而影响造血干细胞动员。Epah等[10]则利用光片显微镜观察了心肌、肾组织样本的微血管三维结构,为这些疾病模型提供了多维度的分析手段。在动脉粥样硬化方面,Becher等[5]利用该技术分析了斑块表面内皮细胞的覆盖情况,为斑块侵蚀模型的评估提供了更加全面可靠的研究手段。而本研究实现了对整段斑块内新生血管的可视化及定量分析,初步观察到斑块内新生血管呈密度不均的三维网状分布。此外,本研究还发现,构建高脂饮食联合颈动脉TS手术模型4周后,其斑块内新生血管总长度为(33.42±11.16) mm,体积约为(13.44±7.35)×10-3 mm3,占颈动脉的体积分数约为3.76%±1.74%,为斑块内新生血管的研究提供了一种多维度的分析方法,并为该领域的三维数据分析提供了参考。
MLT对动脉粥样硬化的影响已被多项基础研究证实[22-24],但目前临床上仅用于治疗睡眠障碍,因此,需更全面且丰富的研究结果来为MLT在动脉粥样硬化中的临床应用奠定基础。由于斑块内新生血管缺乏完整的基底膜,常导致斑块内出血并加速斑块的进展[2,25-26]。有研究发现,MLT干预后斑块内出血的发生率降低[13,27];同时MLT可抑制肿瘤或眼底疾病中的病理性血管新生过程[28],但MLT对斑块内新生血管的影响及其潜在机制仍需进一步探讨。近年来,多项研究发现肿瘤与动脉粥样硬化可能存在共通的病理生理机制[29-32],动脉斑块内低氧及新生血管的微环境也与肿瘤组织存在相似性,而PPARγ能够抑制肿瘤组织中的病理性血管新生过程[33]。笔者前期研究发现,MLT可上调斑块中PPARγ的表达,减少斑块CD31免疫组化的阳性染色区域,而抑制PPARγ可增加其阳性染色范围[4]。但由于斑块内新生血管呈三维连续结构且分布不均,而传统病理观察仅能展示某些解剖层面的二维组织学特征,难以反映样本的完整信息[4,9]。因此,本研究通过斑块内新生血管的三维成像及分析,完整揭示了MLT及PPARγ对其体积及空间分布的影响,发现MLT能够减轻模型小鼠颈动脉斑块内新生血管的整体负荷,表现为其总长度缩短约17.63 mm,总体积缩小约12.1×10-3 mm3,且体积分数降低约3.32%,而在此基础上抑制PPARγ将导致新生血管的总长度延长约19.52 mm,总体积增大约7.53×10-3 mm3,体积分数提高约2.45%。此外,本研究发现MLT组斑块内新生血管的总长度约为溶剂组的47%,但其体积约为溶剂组的10%,抑制PPARγ能够使其总长度增加1倍,但其体积增长6倍,提示MLT在缩短斑块内新生血管长度的同时,也缩小了其管腔面积,且PPARγ可能参与了这一过程的调控。该发现为MLT在动脉粥样硬化干预治疗中的应用提供了潜在的研究方向。此外,作为经典的物质代谢调控分子,PPARγ可参与多种细胞糖代谢的调控[34-36],而异常升高的糖酵解反应是内皮细胞形成新生血管的代谢特点之一[37],但PPARγ是否通过调控内皮细胞糖代谢进而影响斑块内新生血管的形成仍有待进一步研究。
光片显微镜三维成像根植于组织透明化技术的革新,其成像质量直接受制于组织透明化处理的效果[38]。目前的组织透明化技术应用的试剂主要分为有机试剂和亲水试剂两类[38]。本研究选用的iDISCO有机试剂透明化方法,可在保留组织原有荧光信号的同时,增加外源性荧光信号的标记效率,进而提升样本的成像质量[38]。然而,相对于亲水试剂,大部分有机试剂透明化方法虽高效且透明度高,但常伴随着组织皱缩的问题[38],目前对于该问题暂无有效的解决方案[39],本研究中也发现了类似现象。然而,也有研究发现,亲水试剂透明化方法Ce3D在小鼠小肠壁组织中展现出了高透明化、低形变的优势[39],但由于不同组织的成分相差较大,同一种透明化方法对不同组织的处理效果不尽相同,Ce3D对动脉斑块等组织的适用性尚待验证。因此,在追求高透明度、良好荧光保存与渗透的同时减少组织形变,可能仍是透明化技术未来的发展方向。
综上所述,基于光片显微成像技术,本研究初步实现了斑块内新生血管的三维可视化及定量分析,为深化病理生理机制研究及推动相关成果的临床转化提供了可能的发展方向;此外,本研究还发现MLT干预能够减轻斑块内新生血管的整体负荷,且PPARγ可能参与了这一过程的调控。然而,本研究仍存在一定的局限性,如未深入探讨PPARγ影响斑块内新生血管形成的具体机制。近年来,异常糖代谢被视为影响斑块进展的重要因素[29,40-43],因此,PPARγ作为糖代谢的调控分子,是否通过糖代谢影响斑块内新生血管的形成仍有待进一步探索。
  • 国家自然科学基金(U23A6011)
  • 国家自然科学基金(82400060)
  • 国家自然科学基金(82300568)
  • 解放军总医院青年自主创新科学基金(22QNFC102)
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doi: 10.11855/j.issn.0577-7402.1430.2024.1217
  • 接收时间:2024-09-18
  • 首发时间:2025-10-30
  • 出版时间:2025-04-28
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  • 收稿日期:2024-09-18
  • 录用日期:2024-11-14
基金
National Natural Science Foundation of China(U23A6011)
国家自然科学基金(U23A6011)
National Natural Science Foundation of China(82400060)
国家自然科学基金(82400060)
National Natural Science Foundation of China(82300568)
国家自然科学基金(82300568)
Youth Innovation Science Foundation of Chinese PLA General Hospital(22QNFC102)
解放军总医院青年自主创新科学基金(22QNFC102)
作者信息
    1南开大学医学院,天津 300071
    2解放军总医院第六医学中心心血管医学部,北京 100048

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2种不同金属材料的力学参数

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total species (%)

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种数
Number of
species
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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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