Observation of Digestive Tract Tissue Morphology in Mice Using Probe-Based Confocal Laser Endomicroscopy

doi:10.12300/j.issn.1674-5817.2025.035

Abstract

Abstract:

Objective To explore the application value of probe-based confocal laser endomicroscopy (pCLE) in rapidly detecting and evaluating the morphological characteristics of digestive tract tissues in mice. Methods Twelve male SPF Kunming mice aged 6 weeks were randomly divided into two groups. Six mice were subjected to gastric gavage with 52% Red Star Erguotou to establish the model, and six were given saline by gastric gavage as a control. After 28 days of modeling, 3 mice were randomly selected from each group. After deep anesthesia induced by inhalation of 3% isoflurane, the mice were sacrificed by cervical dislocation. The stomach, duodenum, jejunum, and rectum tissues were excised and immersed in 1% fluorescein sodium solution for staining. The microstructure of the mucosal surface of each tissue was observed using pCLE. The remaining mice in the model group and the control group were deeply anesthetized by inhaling 3% isoflurane, then cardiac perfusion was performed successively with saline and 4% paraformaldehyde. The stomach, duodenum, jejunum, and rectum tissues were excised for dehydration, section and hematoxylin-eosin (HE) staining, and the morphological changes of the tissues were observed under a microscope. Results Under pCLE imaging, fluorescence staining on the surface of the gastrointestinal mucosa was uniform in the control group; the morphology of gastric pits, intestinal villi, and intestinal crypts was intact, arranged compactly, and had distinct boundaries. In the model group, the gastrointestinal mucosa exhibited mucosal swelling and deformation, with uneven fluorescence staining and fluorescein leakage. Furthermore, some tissues showed defects or cell shedding, and the boundaries between adjacent characteristic structures (e.g., gastric pits, intestinal crypts) were blurred. HE staining showed that the gastrointestinal tissue structure of the control group mice was normal and well-organized, with no structural defects. Moreover, submucosal glands were uniform in size, with no hyperplasia observed, and no obvious inflammatory cell infiltration. In the model group, some gastrointestinal mucosal structures were defective and sparsely arranged; submucosal glands showed atrophy, accompanied by obvious inflammatory cell infiltration. The histological characteristics detected by pCLE were consistent with those of HE staining. Conclusion pCLE can be used to obtain rapid, real-time, large-scale, and high-resolution microscopic imaging of the gastrointestinal mucosa, realistically and comprehensively displaying its physiological and microstructural characteristics. It shows promising prospects and practical utility in the histological evaluation of digestive system injuries in small animals.

Key words: Chronic alcohol exposure injury, Probe-based confocal laser endomicroscopy, Digestive tracts, Mice

CLC Number:

Q95-33

LIU Yueqin,XUE Weiguo,WANG Shuyou,et al. Observation of Digestive Tract Tissue Morphology in Mice Using Probe-Based Confocal Laser Endomicroscopy[J]. Laboratory Animal and Comparative Medicine, 2025, 45(4): 457-465. DOI: 10.12300/j.issn.1674-5817.2025.035.

Figures/Tables 5

Figure 1 HE staining results of liver tissue （×40） and serum ALT and AST levels in miceNote： A shows HE staining images of liver tissue in control and model group mice， the blue arrows indicate the hepatocytes with macrovesicular fat， and the black arrow indicates the nuclei pushed to the edge. B shows the alanine aminotransferase（ALT） and aspartate aminotransferase（AST） levels in the two groups detected by ultraviolet microplate method. n = 6；Compared with the control group，??P＜0.01.

Figure 2 pCLE images of gastric mucosa surface （×1 000） and HE staining images of gastric tissue （×20） in miceNote： In probe-based confocal laser endomicroscopy（pCLE） images of the gastric mucosa surface， red dashed circle indicates gastric foveae and blue arrows indicate the border of the gastric foveae in the control group， yellow dashed circle shows swollen and deformed gastric foveae and the red arrows are detached tissue cell fragments on the gastric mucosa surface in the model group. In HE staining images of gastric tissues， black arrows indicate neatly arranged glandular ducts in the control group and the red asterisks indicate edematous areas of gastric tissues in the model group.

Figure 3 pCLE images of duodenal mucosa surface （×1 000） and HE staining images of duodenal tissue （×20） in miceNote： In probe-based confocal laser endomicroscopy（pCLE）images of the duodenal mucosa surface in mice， blue arrow indicates a clear duodenal mucosal boundary， red arrows indicate detached tissue cell fragments on the duodenal mucosa surface， yellow asterisks indicate bright areas of fluorescein leakage. In HE staining images of mouse duodenal tissues， black arrows indicate inflammatory cell infiltration， red asterisk indicates intestinal villi edema and black asterisk indicates intestinal villi necrosis and shedding.

Figure 4 pCLE images of jejunum mucosa surface （×1 000） and HE staining images of jejunum tissue （×20） in miceNote： In probe-based confocal laser endomicroscopy（pCLE）images of the jejunum mucosa surface in the model group， red arrows indicate focal necrosis area of surface tissue cells in the jejunum mucosa， and yellow asterisks show local bright area of fluorescein leakage. In HE staining images of jejunal tissues in the model group， black arrows indicate the intestinal villi with destroyed morphological structure， exhibiting apical rupture and shedding， and red dashed circle indicates crowded glands.

Figure 5 pCLE images of rectal mucosa surface （×1 000） and HE staining images of rectal tissue （×10） in miceNote： In probe-based confocal laser endomicroscopy（pCLE）images of the rectal mucosa surface， red dashed circle indicates rectal crypts and blue arrow indicates clear rectal crypts boundary in the control group， yellow dashed circle shows fusion of adjacent crypts， and red arrows indicate tissue cells exfoliation on the rectal mucosa surface in the model group. In HE staining images of rectal tissues in the model group， black arrows indicate disordered arrangement of glandular ducts and red asterisks indicate that the intestinal epithelial structure is incomplete.

References 21

[1]	曾锦树, 汤晓琼, 阮炜炜, 等. 前列腺肿瘤组织的超声弹性与原子力显微成像[J]. 福建师范大学学报(自然科学版), 2021, 37(2):51-56. DOI: 10.12046/j.issn.1000-5277.2021.02.008 .
	ZENG J S, TANG X Q, RUAN W W, et al. Ultrasound elastography and atomic force microscopy of prostate tumor tissue[J]. J Fujian Norm Univ Nat Sci Ed, 2021, 37(2):51-56. DOI: 10.12046/j.issn.1000-5277.2021.02.008 .
[2]	梁忠泉, 刘畅, 杨志娜, 等. 脂肪组织石蜡切片制作方法探讨[J]. 中国组织化学与细胞化学杂志, 2019, 28(2): 170-173. DOI: 10.16705/j.cnki.1004-1850.2019.02.012 .
	LIANG Z Q, LIU C, YANG Z N, et al. Discussion on preparation method for paraffin section of adipose tissues [J]. Chin J Histochem Cytochem, 2019, 28 (2): 170-173. DOI: 10.16705/j.cnki.1004-1850.2019.02.012 .
[3]	王贵龙, 王爱瑶. 共聚焦激光显微内镜与普通结肠镜对溃疡性结肠炎患者炎症活动度及黏膜屏障功能改变的判定价值比较[J]. 上海医药, 2021, 42(7):60-63, 71. DOI: 10.3969/j.issn.1006-1533.2021.07.017 .
	WANG G L, WANG A Y. The comparison of confocal laser microscopy and common colonoscopy to determine changes in inflammatory activity and mucosal barrier function in ulcerative colitis patients[J]. Shanghai Med Pharm J, 2021, 42(7):60-63, 71. DOI: 10.3969/j.issn.1006-1533.2021.07.017 .
[4]	薛培婷. 共聚焦激光显微内镜下溃疡性结肠炎炎症分级的人工智能辅助诊断模型建立与验证 [D]. 济南: 山东大学, 2023. DOI: 10.27272/d.cnki.gshdu.2023.002239 .
	XUE P T. Artificial intelligence-assisted confocal laser endomicroscopy for grading the inflammation degree of ulcerative colitis [D]. Jinan: Shandong University, 2023. DOI: 10.27272/d.cnki.gshdu.2023.002239 .
[5]	刘志美, 张静. 共聚焦激光显微内镜在早期胃癌诊断中的应用进展[J]. 中国微创外科杂志, 2024, 24(9):623-627.DOI:10.3969/j.issn.1009-6604.2024.09.006 .
	LIU Z M, ZHANG J. Application progress of confocal laser microscopic endoscopy in the diagnosis of early gastric cancer[J]. Chin J Minim Invasive Surg, 2024, 24(9):623-627.DOI:10.3969/j.issn.1009-6604.2024.09.006 .
[6]	王羽宸. 原位实时光学活检改善低位直肠癌肛门功能的对照研究[D]. 广州: 南方医科大学, 2024. DOI: 10.27003/d.cnki.gojyu.2024.000947 .
	WANG Y C. In situ real-time optical biopsy improves anal function in low rectal cancer: a controlled study[D]. Gangzhou: Southern Medical University, 2024. DOI: 10.27003/d.cnki.gojyu.2024.000947 .
[7]	丁慧, 陈慧敏, 李晓波, 等. 十二指肠镜联合探头式共聚焦激光显微内镜对十二指肠疾病的观察研究[J]. 临床内科杂志, 2024, 41(7): 446-450. DOI: 10.3969/j.issn.1001-9057.2024.07.003 .
	DING H, CHEN H M, LI X B, et al. Observation study of small bowel diseases by enteroscopy combined with probe-based confocal laser endomicroscopy[J]. J Clin Intern Med, 2024, 41(7): 446-450. DOI: 10.3969/j.issn.1001-9057.2024.07.003 .
[8]	刘凯. 激光共聚焦显微内镜在上消化道早癌和癌前病变中的应用进展[J]. 胃肠病学, 2024, 29(3):186-192. DOI: 10.3969/j.issn.1008-7125.2024.03.006 .
	LIU K. Progress of confocal laser endomicroscopy in early cancer and precancerous lesions of upper gastrointestinal tract[J]. Chin J Gastroenterol, 2024, 29(3):186-192. DOI: 10.3969/j.issn.1008-7125.2024.03.006 .
[9]	左秀丽, 许树长, 王林恒, 等. 中国显微内镜消化系统疾病临床应用共识意见[J]. 胃肠病学, 2023, 28(2):91-106. DOI: 10.3969/j.issn.1008-7125.2023.02.005 .
	ZUO X L, XU S C, WANG L H, et al. Chinese expert consensus on clinical application of endomicroscopy in digestive diseases[J]. Chin J Gastroenterol, 2023, 28(2):91-106. DOI: 10.3969/j.issn.1008-7125.2023.02.005 .
[10]	SONG X J, WANG S Y, ZHAO C, et al. Visual method for evaluating liver function: targeted in vivo fluorescence imaging of the asialoglycoprotein receptor[J]. Biomed Opt Express, 2019, 10(10):5015-5024. DOI: 10.1364/BOE.10.005015 .
[11]	李丽. 探头式共聚焦激光显微内镜对早期胃癌及癌前病变诊断价值的研究[D]. 武汉:华中科技大学, 2020. DOI: 10.27157/d.cnki.ghzku.2020.006230 .
	LI L. The study on the diagnostic value of probe-based confocal laser endomicroscopy in early gastric cancer and precancerous lesions[D]. Wuhan: Huazhong University of Science and Technology, 2020. DOI: 10.27157/d.cnki.ghzku.2020.006230 .
[12]	杨静. 探头式共聚焦激光显微内镜在胆管良恶性狭窄鉴别诊断中的研究[D]. 济南: 山东大学, 2016. DOI: 10.7666/d.Y3032821 .
	YANG J. Research on the differential diagnosis of benign and malignant bile duct stenosis by probe-type confocal laser endoscopy[D]. Jinan: Shandong University, 2016. DOI: 10.7666/d.Y3032821 .
[13]	张燕萍. 探头式共聚焦激光显微内镜对大肠息肉的诊断价值[D]. 北京: 首都医科大学, 2015.
	ZHANG Y P. Diagnostic value of probe-type confocal laser endomicroscopy for colorectal polyps[D]. Beijing: Capital Medical University, 2015.
[14]	杨雪芳, 刘哲晰, 王璞. 共聚焦内窥显微成像技术及其应用[J]. 中国激光, 2022, 49(19):1907002. DOI: 10.3788/CJL202249.1907002 .
	YANG X F, LIU Z X, WANG P. Confocal endoscopic microscopy and its applications[J]. Chin J Lasers, 2022, 49(19):1907002. DOI: 10.3788/CJL202249.1907002 .
[15]	MOUSSATA D, GOETZ M, GLOECKNER A, et al. Confocal laser endomicroscopy is a new imaging modality for recognition of intramucosal bacteria in inflammatory bowel disease in vivo [J]. Gut, 2011, 60(1):26-33. DOI: 10.1136/gut.2010.213264 .
[16]	RASMUSSEN D N, KARSTENSEN J G, RIIS L B, et al. Confocal laser endomicroscopy in inflammatory bowel disease: a systematic review[J]. J Crohns Colitis, 2015, 9(12):1152-1159. DOI: 10.1093/ecco-jcc/jjv131 .
[17]	BUCHNER A M. Confocal laser endomicroscopy in the evaluation of inflammatory bowel disease[J]. Inflamm Bowel Dis, 2019, 25(8):1302-1312. DOI: 10.1093/ibd/izz021 .
[18]	IMAEDA A. Confocal laser endomicroscopy for the detection of atrophic gastritis: a new application for confocal endomicroscopy?[J]. J Clin Gastroenterol, 2015, 49(5):355-357. DOI:10.1097/MCG.0000000000000309 .
[19]	宋晓晶, 熊枫, 贾术永, 等. 大鼠腹内壁中线间质通道微观结构的活体激光共聚焦成像观察[J]. 激光生物学报, 2021, 30(5):435. DOI: 10.3969/j.issn.1007-7146.2021.05.008 .
	SONG X J, XIONG F, JIA S Y, et al. Observation of microstructure of midline interstitial channels of the inner abdominal wall in rat for in vivo confocal laser imaging[J]. Acta Laser Biol Sin, 2021, 30(5):435. DOI: 10.3969/j.issn.1007-7146.2021.05.008 .
[20]	刘婷婷, 杨宁江. 消化道内镜活检标本石蜡切片制作分析[J]. 中国继续医学教育, 2019, 11(31):99-101. DOI: 10.3969/j.issn.1674-9308.2019.31.041 .
	LIU T T, YANG N J. Analysis in making paraffin sections from gastrointestinal endoscopic biopsy specimens[J]. China Continuing Med Educ, 2019, 11(31):99-101. DOI: 10.3969/j.issn.1674-9308.2019.31.041 .
[21]	王梓义, 陈静, 周学谦, 等. 探头式共聚焦激光显微内镜对胃底腺息肉的诊断价值[J]. 陆军军医大学学报, 2024, 46(10):1150-1157. DOI: 10.16016/j.2097-0927.202310082 .
	WANG Z Y, CHEN J, ZHOU X Q, et al. Diagnostic value of probe-based confocal laser microendoscopy in differential diagnosis of fundic gland polyps[J]. J Army Med Univ, 2024, 46(10):1150-1157. DOI: 10.16016/j.2097-0927.202310082 .