小鼠耳蜗血管纹血-迷路屏障免疫组织化学研究
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摘要
目的利用免疫组织化学、免疫荧光染色及激光共聚焦显微镜技术研究小鼠血管纹组织形态、血-迷路屏障的组成,为耳蜗血管纹血-迷路屏障病理生理学研究提供基础。方法快速分离出完整的小鼠血管纹组织,应用免疫组织化学、免疫荧光染色等方法对血管纹上各层组织、细胞进行标记,利用激光共聚焦显微镜详细观察其形态结构特点,观察血-迷路屏障的组成及形态特点。结果完整解剖出小鼠的血管纹组织呈自然弯曲状态,颜色为半透明淡红色,免疫荧光染色观察三层细胞,边缘细胞呈近圆形的多边形,致密排列,其紧密连接蛋白呈线性分布;基底细胞呈扁长形连成一片;毛细血管网夹于边缘细胞与基底细胞之间贯穿血管纹全程,周细胞伴行血管并包裹毛细血管;中间细胞的血管周巨噬细胞呈树枝样,其足突伸出与微血管壁紧密接触。结论边缘细胞、中间细胞、基底细胞及毛细血管网构成耳蜗血管纹的基本结构,毛细血管内皮细胞、基底膜、周细胞、血管周巨噬细胞通过细胞间的紧密连接共同组成血-迷路屏障。
Objective To study morphology of the Stria vascularis and composition of the blood-labyrinth barrier(BLB) in mice by immunohistochemistry, immunofluorescence staining and confocal laser scanning microscopy, to facilitate the study of its pathophysiology. Methods Intact stria vascularis tissue was rapidly isolated, and then labeled by immunohistochemistry and immunofluorescence. Morphological and structural characteristics were observed by laser confocal microscopy. Results The intact stria vascularis in mice was naturally curved and translucent reddish in color. Three layers of cells were observed by immunofluorescence staining. The marginal cell was essentially hexagon in shape with tight junction proteins linearly distributed. The basal cells were elongated and flat. The stria capillary network was sandwiched between the marginal and basal cells, accompanied and wrapped by pericytes. Perivascular-resident macrophages in the intermediate cells were dendritic, with their processes in close contact with microvessels. Conclusion Marginal cells, intermediate cells, basal cells and the capillary network constitute the basic structures of the cochlear stria vascularis. Endothelial cells, basilar membrane, pericytes and Perivascular-resident macrophages form a blood-to-labyrinth barrier through tight junctions between cells.
Objective To study morphology of the Stria vascularis and composition of the blood-labyrinth barrier(BLB) in mice by immunohistochemistry, immunofluorescence staining and confocal laser scanning microscopy, to facilitate the study of its pathophysiology. Methods Intact stria vascularis tissue was rapidly isolated, and then labeled by immunohistochemistry and immunofluorescence. Morphological and structural characteristics were observed by laser confocal microscopy. Results The intact stria vascularis in mice was naturally curved and translucent reddish in color. Three layers of cells were observed by immunofluorescence staining. The marginal cell was essentially hexagon in shape with tight junction proteins linearly distributed. The basal cells were elongated and flat. The stria capillary network was sandwiched between the marginal and basal cells, accompanied and wrapped by pericytes. Perivascular-resident macrophages in the intermediate cells were dendritic, with their processes in close contact with microvessels. Conclusion Marginal cells, intermediate cells, basal cells and the capillary network constitute the basic structures of the cochlear stria vascularis. Endothelial cells, basilar membrane, pericytes and Perivascular-resident macrophages form a blood-to-labyrinth barrier through tight junctions between cells.
引文
1 Wu J,Han W,Chen XR,et al.MMP-2 and MMP-9 Contribute to Functional Integrity and Noise-induced Damage to Blood-Labyrinth Barrier[J].Molecular Medicine Reports.2017,16:1731-1738.
2张桐,韩维举.耳蜗血管纹血-迷路屏障病理生理学研究进展[J].中华耳科学杂志.2017,15(2):257-262.Zhang T,Han WJ.Advances in Pathophysiological Studies of Cochlear Blood-Labyrinth Barrier[J].Chinese Journal of Otology.2017,15(2):257-262.
3 Bibas A,Liang J,Michaels L,et al.The Development of the Stria Vascularis in the Human Foetus[J].Clin Otolaryngol Allied Sci.2000,25(2):126-129.
4 Kikuchi K,Hilding DA.The Development of the Stria Vascularis in the Mouse[J].Acta Otolaryngol.1966,62(4):277-291.
5 Ando M,Takeuchi S.Postnatal Vascular Development in the Lateral Wall of the Cochlear Duct of Gerbils:Quantitative Analysis by Electron Microscopy and Confocal Laser Microscopy[J].Hear Res.1998,123:48-156.
6林琳,孙建和,黄德亮.胎儿耳蜗血管纹的扫描电镜观察[J].临床耳鼻咽喉科杂志.2005,4:157-159.Lin L,Sun JH,Huang DL.Scanning Electron Microscopic Observation of the Fetal Stria Vasculari[J].J Clin Otorhinolarygol(China).2005,4:157-159.
7 Wu YX.Noise Alters Guinea Pig's Blood-labyrinth Barrier Ultrastructure and Permeability along with a Decrease of Cochlear Claudin-5 and Occludin[J].BMC Neurosci.2014,15:136.
8 Juhn SK,Hunter BA,Odland RM.Blood-labyrinth Barrier and Fluid Dynamics of the Inner Ear[J].Int Tinnitus J.2001,7(2):72-83.
9 Zhang W,Dai M,Fridberger A,et al.Perivascular-resident Macrophage-like Melanocytes in the Inner Ear are Essential for the Integrity of the Intrastrial Fluid-blood Barrier[J].Proc Natl Acad Sci U.S.A.2012,109(26):10388-93.
10张纪帅,陈星睿,吴军,等.白噪声暴露前后豚鼠听觉功能检测[J].中华耳科学杂志.2015,13(1):141-144.Zhang JS,Wu J,Chen XR,et al.Detection of Guinea Pigs’Hearing Functions before and after White Noise Exposure[J].Chinese Journal of Otology.2015,13(1):141-144.
11 Davis H.Biophysics and Physiology of the Inner Ear[J].Physiol Rev.1957,37(1):1-49.
12 Liu H,Li Y,Chen L,et al.Organ of Corti and Stria Vascularis:Is there an Interdependence for Survival?[J].PLo S One.2016.11(12):1-21.
13 Chen L,Guo W,Ren L,et al.A de novo Silencer Causes Elimination of MITF-M Expression and Profound Hearing Loss in Pigs[J].BMC Biol.2016,14:52.
14 Armulik A,GenovéG,Betsholtz C.Pericytes:Developmental,Physiological,and Pathological Perspectives,Problems,and Promises[J].Dev Cel.2011,21(1):193-215.
15 Armulik A,GenovéG,M?e M,et al.Pericytes Regulate the Blood-brain Barrier[J].Nature,2010.468(7323):557-61.
16 Bell RD,Winkler EA,Sagare AP,et al.Pericytes Control Key Neurovascular Functions and Neuronal Phenotype in the Adult Brain and during Brain Aging[J].Neuron.2010,68(3):409-27.
17 Shi X,Han WJ,Yamamoto H,et al.The Cochlear Pericytes[J].Microcirculation.2008,15(6):515-29.
18 Kniesel U,Wolburg H.Tight Junctions of the Blood-brain Barrier[J].Cell Mol Neurobiol.2000,20(1):57-76.
19 Hawkins BT,Davis TP.The Blood-brain Barrier/Neurovascular Unit in Health and Disease[J].Pharmacol Rev.2005,57(2):173-185.
20 Lee HS,Namkoong K,Kim DH.Hydrogen Peroxide-induced Alterations of Tight Junction Proteins in Bovine Brain Microvascular Endothelial Cells[J].Microvasc Res.2004,68(3):231-238.
21 Hu BH,Cai QF,Hu ZH.Metalloproteinases and their Associated Genes Contribute to the Functional Integrity and Noise-induced Damage in the Cochlear Sensory Epithelium[J].J Neurosci.2012,32(43):14927-14941.
22吴军,韩维举.耳蜗血-迷路屏障与噪声性听力损伤[J].听力学及言语疾病杂志.2015,23(4):427-430.Wu J,Han WJ.Damage of Blood-Labyrinth Barrier and Noise-induced Hearing Loss[J].Journal of Audiology and Speech Pathology.2015,23(4):427-430.
23 Inamura N,Salt AN.Permeability Changes of the Blood-labyrinth Barrie Measured in vivo during Experimental Treatments[J].Hear Res.1992,61:12-18.
24 Neuwelt EA,Baker DE,Pagel MA.Cerebrovascular Permeability and Delivery of Gentamicin to Normal Brain and Experimental Brain Abscess in Rats[J].Neurosurg.1984,61:430-439.
25 Juhn SK,Rybak LP,Prado S.Nature of Blood-labyrinth Barrier in Experimental Conditions[J].Ann Otol Rhinol Laryngol.1981,90:135-141.
2张桐,韩维举.耳蜗血管纹血-迷路屏障病理生理学研究进展[J].中华耳科学杂志.2017,15(2):257-262.Zhang T,Han WJ.Advances in Pathophysiological Studies of Cochlear Blood-Labyrinth Barrier[J].Chinese Journal of Otology.2017,15(2):257-262.
3 Bibas A,Liang J,Michaels L,et al.The Development of the Stria Vascularis in the Human Foetus[J].Clin Otolaryngol Allied Sci.2000,25(2):126-129.
4 Kikuchi K,Hilding DA.The Development of the Stria Vascularis in the Mouse[J].Acta Otolaryngol.1966,62(4):277-291.
5 Ando M,Takeuchi S.Postnatal Vascular Development in the Lateral Wall of the Cochlear Duct of Gerbils:Quantitative Analysis by Electron Microscopy and Confocal Laser Microscopy[J].Hear Res.1998,123:48-156.
6林琳,孙建和,黄德亮.胎儿耳蜗血管纹的扫描电镜观察[J].临床耳鼻咽喉科杂志.2005,4:157-159.Lin L,Sun JH,Huang DL.Scanning Electron Microscopic Observation of the Fetal Stria Vasculari[J].J Clin Otorhinolarygol(China).2005,4:157-159.
7 Wu YX.Noise Alters Guinea Pig's Blood-labyrinth Barrier Ultrastructure and Permeability along with a Decrease of Cochlear Claudin-5 and Occludin[J].BMC Neurosci.2014,15:136.
8 Juhn SK,Hunter BA,Odland RM.Blood-labyrinth Barrier and Fluid Dynamics of the Inner Ear[J].Int Tinnitus J.2001,7(2):72-83.
9 Zhang W,Dai M,Fridberger A,et al.Perivascular-resident Macrophage-like Melanocytes in the Inner Ear are Essential for the Integrity of the Intrastrial Fluid-blood Barrier[J].Proc Natl Acad Sci U.S.A.2012,109(26):10388-93.
10张纪帅,陈星睿,吴军,等.白噪声暴露前后豚鼠听觉功能检测[J].中华耳科学杂志.2015,13(1):141-144.Zhang JS,Wu J,Chen XR,et al.Detection of Guinea Pigs’Hearing Functions before and after White Noise Exposure[J].Chinese Journal of Otology.2015,13(1):141-144.
11 Davis H.Biophysics and Physiology of the Inner Ear[J].Physiol Rev.1957,37(1):1-49.
12 Liu H,Li Y,Chen L,et al.Organ of Corti and Stria Vascularis:Is there an Interdependence for Survival?[J].PLo S One.2016.11(12):1-21.
13 Chen L,Guo W,Ren L,et al.A de novo Silencer Causes Elimination of MITF-M Expression and Profound Hearing Loss in Pigs[J].BMC Biol.2016,14:52.
14 Armulik A,GenovéG,Betsholtz C.Pericytes:Developmental,Physiological,and Pathological Perspectives,Problems,and Promises[J].Dev Cel.2011,21(1):193-215.
15 Armulik A,GenovéG,M?e M,et al.Pericytes Regulate the Blood-brain Barrier[J].Nature,2010.468(7323):557-61.
16 Bell RD,Winkler EA,Sagare AP,et al.Pericytes Control Key Neurovascular Functions and Neuronal Phenotype in the Adult Brain and during Brain Aging[J].Neuron.2010,68(3):409-27.
17 Shi X,Han WJ,Yamamoto H,et al.The Cochlear Pericytes[J].Microcirculation.2008,15(6):515-29.
18 Kniesel U,Wolburg H.Tight Junctions of the Blood-brain Barrier[J].Cell Mol Neurobiol.2000,20(1):57-76.
19 Hawkins BT,Davis TP.The Blood-brain Barrier/Neurovascular Unit in Health and Disease[J].Pharmacol Rev.2005,57(2):173-185.
20 Lee HS,Namkoong K,Kim DH.Hydrogen Peroxide-induced Alterations of Tight Junction Proteins in Bovine Brain Microvascular Endothelial Cells[J].Microvasc Res.2004,68(3):231-238.
21 Hu BH,Cai QF,Hu ZH.Metalloproteinases and their Associated Genes Contribute to the Functional Integrity and Noise-induced Damage in the Cochlear Sensory Epithelium[J].J Neurosci.2012,32(43):14927-14941.
22吴军,韩维举.耳蜗血-迷路屏障与噪声性听力损伤[J].听力学及言语疾病杂志.2015,23(4):427-430.Wu J,Han WJ.Damage of Blood-Labyrinth Barrier and Noise-induced Hearing Loss[J].Journal of Audiology and Speech Pathology.2015,23(4):427-430.
23 Inamura N,Salt AN.Permeability Changes of the Blood-labyrinth Barrie Measured in vivo during Experimental Treatments[J].Hear Res.1992,61:12-18.
24 Neuwelt EA,Baker DE,Pagel MA.Cerebrovascular Permeability and Delivery of Gentamicin to Normal Brain and Experimental Brain Abscess in Rats[J].Neurosurg.1984,61:430-439.
25 Juhn SK,Rybak LP,Prado S.Nature of Blood-labyrinth Barrier in Experimental Conditions[J].Ann Otol Rhinol Laryngol.1981,90:135-141.