经耳后乳突部皮下注射途径靶向内耳给药的初步研究
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摘要
血-迷路屏障( blood labyrinth barrier, BLB)是近年来耳科方面研究的热点。由于BLB的存在,内耳(inner ear,IE)结构得以和血液相隔,从而保持了IE各种液体成分的稳定,也保证了IE少受各种毒性药物、微生物、自身系统免疫性疾病的侵袭和破坏。但同时也因为BLB的存在,分子结构较大的有效的治疗药物难以进入IE甚至被完全阻隔,即便某些小分子的药物也因其所带电荷的不合宜而无法通过。因此理论上跨BLB局部内耳用药比全身用药更有效、更安全。但由于IE结构、位置的特殊性,目前临床上采用的跨BLB局部内耳用药方法都存在着各自的不足。如何让药物更安全、持续、高效的进入IE,这一问题目前仍未得到有效的解决。
伊文思蓝(Evans blue,EB)是一种四钠重氮染料,能与血清白蛋白彻底、稳定地结合形成不能透过BLB的荧光颗粒,该荧光颗粒不能通过BLB进入内耳。本实验利用EB此特点,将其作为示踪剂,探讨耳后乳突部皮下微淋巴管是否与内耳的内淋巴或外淋巴之间存在着直接的淋巴通路,这条通路可以使注射到耳后皮下的药物能够绕过BLB的阻拦,直接运输至IE内。
目的
验证经耳后乳突部皮下组织注射给药后输送药物到IE的可行性。通过实验验证耳后皮下组织与IE之间是否存在直接的通路,循此通路可使经耳后皮下途径注射的药物大部分直接进入内耳,而不需要进入血液循环、通过BLB。若存在该通路的话,分析并阐述该通路的性质及特点。
方法
1、将白色红目鼠随机分为静脉注射给药组、耳后皮下给药组。分别采用颈内静脉注射及耳后皮下注射方法为两组豚鼠注射EB(40mg/kg体重),观察豚鼠皮肤、粘膜的蓝染情况,用二甲基甲酰胺(dimethylformamide,DMF)摄取进入耳蜗内的EB,用荧光分光光度计(Spectrofluorophotometer,SPF)对DMF进行EB定量。
2、取豚鼠血液,分离血清,制备EB血清溶液并做涂片,在荧光显微镜下观察其荧光性。将豚鼠随机分为静脉注射给药组、耳后皮下给药组。分别采用颈内静脉注射及耳后皮下给药方法为两组豚鼠注射EB血清溶液,抽取耳蜗外淋巴液并在荧光显微镜下寻找EB白蛋白荧光颗粒,做耳蜗超薄冰冻切片,在荧光显微镜下观察EB白蛋白荧光颗粒在内耳的分布情况。
结果
1、静脉给药组豚鼠出现躯体、四肢、眼部、耳部皮肤及口唇粘膜的蓝染要比耳后皮下给药组迅速,而且蓝染程度亦重。SPF测得的摄取了进入耳蜗内的EB的DMF的荧光值极低,无实际意义,未能检测到两组豚鼠的耳蜗外淋巴和内淋巴含有明确的EB。
2、可见EB血清溶液的涂片内有片状红色荧光,但荧光强度较弱,其间有发出明亮鲜红色荧光的颗粒,较明显,而EB生理盐水溶液涂片内无红色荧光的颗粒,而是呈现均匀的发出极弱红色荧光的斑片,难以观察或拍照。两组豚鼠耳蜗的外淋巴液中均并未见到发出红色荧光的EB白蛋白颗粒。在耳蜗切片中,未见到膜蜗管内前庭膜、基底膜、柯蒂氏器等部位存在发出红色荧光的EB白蛋白颗粒。
结论
经静脉途径及耳后皮下途径注射EB生理盐水溶液后2h后,通过荧光分光测定法,两组豚鼠耳蜗外淋巴及内淋巴内均未检测到确实存在的EB含量。EB可与血清中的白蛋白结合形成荧光性明显增强的EB白蛋白荧光颗粒。经静脉途径及耳后皮下途径注射EB血清溶液,通过荧光显微镜下观察耳蜗外淋巴及耳蜗超薄冰冻切片,未能观察到外淋巴及膜蜗管内存在EB白蛋白荧光颗粒。综上所述:本实验的两种方法均未能证实豚鼠耳后皮下组织与IE间存在着绕过BLB而直接相通的淋巴通道。
Blood labyrinth barrier is a focus in auristics research in recent years. The tissue of inner ear is separated from blood by BLB, which could maintain the stability of composition of fluid in inner ear. Not simply, BLB keeps IE away from invasion and destruction by various toxic substances、microorganisms、autoimmune disease as far as possible. Curatives consisting of large molecules could not get into inner ear easily because of the baffle of BLB, besides, some curatives consisting of small molecules could not overcome the obstacle either for they possess incorrect electric charge. So theoretically local application is safer and more effective than systemic administration because the former could get round BLB. However, due to the special structure and anatomical position of IE, there are respective shortages in all the local application methods. It is urgent to find a new administration route in order to make medicine get into IE more safely、effectively and persistently.
Objective:
To verify the feasibility of inner ear targeted drug delivery by postauricular hypodermic injection. To find out if there is a direct lymph-channel between the inner ear and hypodermia of postauricular mastoidea, through which the curatives could bypass BLB and get into endolymph or perilymph. To analyze and expound its characteristics if there is indeed such a channel.
Methods:
(1) The guinea pigs were randomly allocated into two groups: EB-vein shot group, EB-postauricular hypodermic injection group. EB-Saline solution was injected through vein and postauricular hypodermia respectively. The level of blue-stain in skin and mucous membrane were observed. The EB in endolymph and endolymph was absorbed by using dimethylformamide( DMF ). Amount of EB in DMF was detected by spectrofluorophotometer.
(2) Serum was isolated from guinea pig’s blood. The fluorescence of EB-serum solution was observed by the method of smear. The guinea pigs were randomly allocated into two groups: EB-vein shot group, EB-postauricular hypodermic injection group. EB-serum solution was injected through vein and postauricular hypodermia respectively. Perilymph was collected from cochlea and observed carefully with fluorescent microscope if there were EB-albumin fluorescent particles in it. Distribution of EB-albumin fluorescent particles in frozen cochlea section was observed.
Results:
(1) The level of blue-stain of skin and mucous membrane in EB-vein shot group was higher than which in EB-postauricular hypodermic injection group. The amount of EB in inner ear was not detected in the two groups reliably.
(2) There were fluorescent particles emitting bright red flourescence in EB-serum solution smear. However, there were not fluorescent particles in EB-saline solution smear. EB-serum fluorescent particles were not detected in perilymph、membrana vestibularis、basal membrane、Corti's organ or other parts in the two groups.
Conclusions:
The amount of EB in perilymph was not detected in the two groups. Abundant EB molecules could gather into fluorescent particles complexed with albumin. And the fluorescence intensity will enhance remarkably accordingly, which could be easily observed with fluorescence microscope. EB-serum fluorescent particles were not detected in inner ears of two groups. To sum up: there is no evidence to prove that there is a direct lymph-channel between the inner ear and hypodermia of postauricular mastoidea .
伊文思蓝(Evans blue,EB)是一种四钠重氮染料,能与血清白蛋白彻底、稳定地结合形成不能透过BLB的荧光颗粒,该荧光颗粒不能通过BLB进入内耳。本实验利用EB此特点,将其作为示踪剂,探讨耳后乳突部皮下微淋巴管是否与内耳的内淋巴或外淋巴之间存在着直接的淋巴通路,这条通路可以使注射到耳后皮下的药物能够绕过BLB的阻拦,直接运输至IE内。
目的
验证经耳后乳突部皮下组织注射给药后输送药物到IE的可行性。通过实验验证耳后皮下组织与IE之间是否存在直接的通路,循此通路可使经耳后皮下途径注射的药物大部分直接进入内耳,而不需要进入血液循环、通过BLB。若存在该通路的话,分析并阐述该通路的性质及特点。
方法
1、将白色红目鼠随机分为静脉注射给药组、耳后皮下给药组。分别采用颈内静脉注射及耳后皮下注射方法为两组豚鼠注射EB(40mg/kg体重),观察豚鼠皮肤、粘膜的蓝染情况,用二甲基甲酰胺(dimethylformamide,DMF)摄取进入耳蜗内的EB,用荧光分光光度计(Spectrofluorophotometer,SPF)对DMF进行EB定量。
2、取豚鼠血液,分离血清,制备EB血清溶液并做涂片,在荧光显微镜下观察其荧光性。将豚鼠随机分为静脉注射给药组、耳后皮下给药组。分别采用颈内静脉注射及耳后皮下给药方法为两组豚鼠注射EB血清溶液,抽取耳蜗外淋巴液并在荧光显微镜下寻找EB白蛋白荧光颗粒,做耳蜗超薄冰冻切片,在荧光显微镜下观察EB白蛋白荧光颗粒在内耳的分布情况。
结果
1、静脉给药组豚鼠出现躯体、四肢、眼部、耳部皮肤及口唇粘膜的蓝染要比耳后皮下给药组迅速,而且蓝染程度亦重。SPF测得的摄取了进入耳蜗内的EB的DMF的荧光值极低,无实际意义,未能检测到两组豚鼠的耳蜗外淋巴和内淋巴含有明确的EB。
2、可见EB血清溶液的涂片内有片状红色荧光,但荧光强度较弱,其间有发出明亮鲜红色荧光的颗粒,较明显,而EB生理盐水溶液涂片内无红色荧光的颗粒,而是呈现均匀的发出极弱红色荧光的斑片,难以观察或拍照。两组豚鼠耳蜗的外淋巴液中均并未见到发出红色荧光的EB白蛋白颗粒。在耳蜗切片中,未见到膜蜗管内前庭膜、基底膜、柯蒂氏器等部位存在发出红色荧光的EB白蛋白颗粒。
结论
经静脉途径及耳后皮下途径注射EB生理盐水溶液后2h后,通过荧光分光测定法,两组豚鼠耳蜗外淋巴及内淋巴内均未检测到确实存在的EB含量。EB可与血清中的白蛋白结合形成荧光性明显增强的EB白蛋白荧光颗粒。经静脉途径及耳后皮下途径注射EB血清溶液,通过荧光显微镜下观察耳蜗外淋巴及耳蜗超薄冰冻切片,未能观察到外淋巴及膜蜗管内存在EB白蛋白荧光颗粒。综上所述:本实验的两种方法均未能证实豚鼠耳后皮下组织与IE间存在着绕过BLB而直接相通的淋巴通道。
Blood labyrinth barrier is a focus in auristics research in recent years. The tissue of inner ear is separated from blood by BLB, which could maintain the stability of composition of fluid in inner ear. Not simply, BLB keeps IE away from invasion and destruction by various toxic substances、microorganisms、autoimmune disease as far as possible. Curatives consisting of large molecules could not get into inner ear easily because of the baffle of BLB, besides, some curatives consisting of small molecules could not overcome the obstacle either for they possess incorrect electric charge. So theoretically local application is safer and more effective than systemic administration because the former could get round BLB. However, due to the special structure and anatomical position of IE, there are respective shortages in all the local application methods. It is urgent to find a new administration route in order to make medicine get into IE more safely、effectively and persistently.
Objective:
To verify the feasibility of inner ear targeted drug delivery by postauricular hypodermic injection. To find out if there is a direct lymph-channel between the inner ear and hypodermia of postauricular mastoidea, through which the curatives could bypass BLB and get into endolymph or perilymph. To analyze and expound its characteristics if there is indeed such a channel.
Methods:
(1) The guinea pigs were randomly allocated into two groups: EB-vein shot group, EB-postauricular hypodermic injection group. EB-Saline solution was injected through vein and postauricular hypodermia respectively. The level of blue-stain in skin and mucous membrane were observed. The EB in endolymph and endolymph was absorbed by using dimethylformamide( DMF ). Amount of EB in DMF was detected by spectrofluorophotometer.
(2) Serum was isolated from guinea pig’s blood. The fluorescence of EB-serum solution was observed by the method of smear. The guinea pigs were randomly allocated into two groups: EB-vein shot group, EB-postauricular hypodermic injection group. EB-serum solution was injected through vein and postauricular hypodermia respectively. Perilymph was collected from cochlea and observed carefully with fluorescent microscope if there were EB-albumin fluorescent particles in it. Distribution of EB-albumin fluorescent particles in frozen cochlea section was observed.
Results:
(1) The level of blue-stain of skin and mucous membrane in EB-vein shot group was higher than which in EB-postauricular hypodermic injection group. The amount of EB in inner ear was not detected in the two groups reliably.
(2) There were fluorescent particles emitting bright red flourescence in EB-serum solution smear. However, there were not fluorescent particles in EB-saline solution smear. EB-serum fluorescent particles were not detected in perilymph、membrana vestibularis、basal membrane、Corti's organ or other parts in the two groups.
Conclusions:
The amount of EB in perilymph was not detected in the two groups. Abundant EB molecules could gather into fluorescent particles complexed with albumin. And the fluorescence intensity will enhance remarkably accordingly, which could be easily observed with fluorescence microscope. EB-serum fluorescent particles were not detected in inner ears of two groups. To sum up: there is no evidence to prove that there is a direct lymph-channel between the inner ear and hypodermia of postauricular mastoidea .
引文
1. 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.
2. Juhn SK, Rybak LP. Labyrinthine barriers and cochlear homeostasis[J]. Acta Otolaryngol. 1981, 91(5-6): 529-534.
3. Juhn SK, Rybak LP, Prado S. Nature of blood labyrinth barrier in experimental conditions[J]. Ann Otol Rhinol Laryngol. 1981, 90(2): 135-141.
4. Suzuki M, Kaga K. Effect of cisplatin on the negative charge barrier in strial vessels of the guinea pig. A transmission electromicroscopic study using polyethyleneimine molecules[J]. Arch Otorhinolaryngol. 1996, 253(6): 351-355.
5. Auerbach R, Alby L, Morrissey LW, et al. Expression of organ-specific antigens on capillary endothelial cells[J]. Microvasc Res. 1985, 29(3): 401-411.
6. Sakagami M, Matsunaga T, Hashimito PH, et al. Fine structure and permeability of capillaries in the stria vascular and spiral ligament of the inner ear of the guinea pig[J]. Cell Tissue Res. 1982, 226(3): 5112-5221.
7. Kroll RA, Neuwelt EA. Outwitting the blood-brain barrier for therapeutic purposes: osmotic opening and othermeans[J]. Neurosurgery. 1998, 42(5): 10832-11001.
8. Sakagami M, Sano M, Matsunaga T. Ultrastructural study of the effect of acute hypertension on the stria vascularis and spiral ligament[J]. Acta Otolaryngol. 1984, 97(122): 532-611.
9. Zhang ZJ, Saito T, Kimura Y, et al. Disruption of mdr1ap-glycoprotein generesults in dysfunction of blood-inner ear barrier in mice[J]. Brain Res. 2000, 852(1): 1162-1261.
10. Lenhardt E, Plotzliche Horstomngen. Aufbeiden Seiten gleichzeitig oder nacheinander aufgetreten[J]. Z Laryngol Rhinol Otol. 1958, 37: 1-16.
11. McCabe BF. Autoimmune sensorineural hearing loss[J]. Ann Otol Rhinol Laryngol. 1979, 88(5 pt 1): 585 - 589.
12. Harris JP, Sharp PA. Inner ear autoantibodies in patients with rapidly progressive sensorineural hearing loss[J]. Laryngoscope. 1990, 100(5): 516- 524.
13. Harris JP, Ryan AF. Fundamental immune mechanisms of the brain and inner ear[J]. Otolaryngol Head Neck Surg. 1995, 112(6): 639 - 653.
14. Tomiyama S, Harris JP. The role of the endolymphatic sac in inner ear immunity[J ]. Acta Otolaryngol. 1987, 103(3-4): 182 - 188.
15. Revel JP. Hexagonal array of subunits in intercellular junctions of the mouse heart and liver[J]. J Cell Biol. 1967, 33(3): 7-12.
16. Banyard SH, Stammers DK, Harrison PM. Electron density map of apoferritin at 2.8-A resolution[J]. Nature. 1978, 271(5642): 282-284.
17.葛贤锡,沈云珍,刘建中.铁蛋白示踪观察甘油开放血-迷路屏障的作用[J].中华耳鼻咽喉科杂志. 1989, 21(2): 70-71.
18. Saria A, Lundberg JM. Evans blue fluorescence: quantitative and morphological evaluation of vascular permeability in animal tissues[J]. J Neu-rosci Mothods. 1983, 8(1): 41-49.
19.张学渊,汪吉宝.豚鼠内耳微循环障碍对血迷路屏障通透性的影响[J].中华耳鼻咽喉科杂志. 2000, 35(5): 339-341.
20. Gianoli GJ, Li JC. Transtympanic steroid for treatment of sudden hearing loss[J]. Otolaryngol Head Neck Surg. 2001, 125(3): 142-146.
21. Nordang L, Laurent C, Mollnes TE. Complement Activation in Sudden Deafness[J]. Arch Otolaryngol Head neck surgery. 1998, 124(6): 633-636.
22. Lopez IA, Ishiyama G, Lee M, et al. Immunohistochemical localization of aquaporins in the human inner ear[J]. Cell Tissue Res. 2007, 328(3): 453-460.
23. Fukushima M, Kitahara T, Fuse Y, et al. Changes in aquaporin expression in the inner ear of the rat after i.p. injection of steroids[J]. Acta Otolaryngol Suppl. 2004, 553: 13-18.
24. Beitz E, Zenner HP, Schultz JE. Aquaporin-mediated fluid regulation in the inner ear[J]. Cell Mol Neurobiol. 2003, 23(3): 315-329.
25. Trune DR, Kempton JB, et al. Mineralocorticoid receptor mediates glucocorticoid treatment effects in the autoimmune mouse ear[J]. Hear Res. 2006, 212((1-2): 22-32. .
26. Lee JH, Marcus DC. Nongenomic effects of corticosteroids on ion transport by stria vascularis[J]. Audiol Neurootol. 2002, 7(2): 100-106.
27. Trune DR, Kempton JB. Aldosterone and prednisolone control of cochlear function in MRL/MpJ-Fas(lpr) autoimmune mice[J]. Hear Res. 2001, 155(1-2): 9-20.
28. Nagashima R, Ogita K. Enhanced biosynthesis of glutathione in the spiral ganglion of the cochlea after in vivo treatment with dexamethasone in mice[J]. Brain Res. 2006, 1117(1): 101-108.
29. Stone JH, Francis HW. Immune-mediated inner ear disease[J]. Current Curr Opin Rheumatol. 2000, 12(1): 32-40.
30.Rahman MU, Poe DS, Choi HK. Autoimmune vestibulo-cochlear disorders[J]. Curr Opin Rheumatol. 2001, 13(3): 184-189.
31. Chandrasekhar, Sujans S. Intratympanic dexamethasone for sudden sensorineural hearing loss: Clinical and laboratory evaluation[J].Otolaryngology and Neurotology. 2001, 22(1): 18-23.
32. Scherer EQ, Herzog M, Wangemann P. Endothelin-1-induced vasospasms of spiral modiolar artery are mediated by rho-kinase-induced Ca(2+) sensitization of contractile apparatus and reversed by calcitonin gene-related Peptide[J]. Stroke. 2002, 33(12): 2965-2971.
33. Schuknecht HF. Ablation therapy for the relief of Meniere’s disease[J]. Laryngoscpe. 1956, 66(7): 859- 870.
34. Chandrasekhar SS, Rubinstein RY, Kwartler JA, et al. Dexamethasone pharmacokinetics in the inner ear : comparison of route of administrtion and use of facilitating agents[J]. Otolaryngol Head Neck Surg. 2000, 122(4): 521-528.
35. Parnes LS, Sun AH, Freeman DJ. Corticosteroid pharmacokinetics in the inner ear fluids: an animal study followed by clinical application[J]. Laryngoscope. 1999, 109(7 pt 2): 1-17.
36.杨军,吴皓.鼓室内给药治疗内耳疾病的基础与临床[J].中华耳鼻咽喉头颈外科杂志. 2004, 39(12): 770-772.
37.周宣岩,陶谦,吕凌燕,等.经鼓室插管注入地塞米松液治疗突发性耳聋[J].中国耳鼻咽喉颅底外科杂志. 2006, 12(1): 47-48, 51.
38. Becvarovski Z, Bojrab DI, Michaelides EM, et al. Round window gentamicin absorption:An in vivo human model[J]. Laryngoscope. 2002, 112(9): 1610-1613.
39. Sheppard WM, Wanamaker HH, Pack A, et al. Direct round window application of gentamicin with varying delivery vehicles:a comparison ototoxicity[J]. Otolaryngol Head Neck Surg. 2004, 131(6): 890-896.
40. Silverstein H. Use of a new device, the MicroWick, to deliver medication to the inner ear[J]. Ear Nose Throat J. 1999, 78(8): 595-598, 600.
41. Van Wijck F, Staecker H, Lefebvre PP. Topical steroid therapy using the Silverstein Microwick in sudden sensorineural hearing loss after failure of conventional treatment[J]. Acta Otolaryngol. 2007, 127(10): 1012-1017.
42. Charabi S, Thomsen J, Tos M. Round window gentamicin mu-catheter -- a new therapeutic tool in Meniere's disease [J]. Acta Otolaryngol. 2000, suppl(543): 108-110.
43. Shoji F, Yamasoba T, Magal E, et al. Glial cell line-derived neurotrophic factor has a dose dependent influence on noise-induced hearing loss in the guinea pig cochlea[J]. Hear Res. 2000, 142(1-2): 41-55.
44. Ylikoski J, Pirvola U, Virkkala J, et al. Guinea pig auditory neurons are protected by glial cell line-derived growth factor from degeneration after noise trauma[J]. Hear Res. 1998, 124(1-2): 17-26.
45. Yamasoba T, Schacht J, Shoji F, et al. Attenuation of cochlear damage from noise trauma by an iron chelator, a free radical scavenger and glial cell line-derived neurotrophic factor in vivo[J]. Brain Res. 1999, 815(2): 317-325.
46. Lalwani AK, Walsh BJ, Reilly PG, et al. Development of in vivo gene therapy for hearing disorders: introduction of adeno-associated virus into the cochlea of the guinea pig[J]. Gene Ther. 1996, 3(7): 588-592.
47. Brown JN, Miller JM, Altschuler RA, et al. Osmotic pump implant for chronic infusion of drugs into the inner ear[J]. Hear Res. 1993, 70(2): 167-172.
2. Juhn SK, Rybak LP. Labyrinthine barriers and cochlear homeostasis[J]. Acta Otolaryngol. 1981, 91(5-6): 529-534.
3. Juhn SK, Rybak LP, Prado S. Nature of blood labyrinth barrier in experimental conditions[J]. Ann Otol Rhinol Laryngol. 1981, 90(2): 135-141.
4. Suzuki M, Kaga K. Effect of cisplatin on the negative charge barrier in strial vessels of the guinea pig. A transmission electromicroscopic study using polyethyleneimine molecules[J]. Arch Otorhinolaryngol. 1996, 253(6): 351-355.
5. Auerbach R, Alby L, Morrissey LW, et al. Expression of organ-specific antigens on capillary endothelial cells[J]. Microvasc Res. 1985, 29(3): 401-411.
6. Sakagami M, Matsunaga T, Hashimito PH, et al. Fine structure and permeability of capillaries in the stria vascular and spiral ligament of the inner ear of the guinea pig[J]. Cell Tissue Res. 1982, 226(3): 5112-5221.
7. Kroll RA, Neuwelt EA. Outwitting the blood-brain barrier for therapeutic purposes: osmotic opening and othermeans[J]. Neurosurgery. 1998, 42(5): 10832-11001.
8. Sakagami M, Sano M, Matsunaga T. Ultrastructural study of the effect of acute hypertension on the stria vascularis and spiral ligament[J]. Acta Otolaryngol. 1984, 97(122): 532-611.
9. Zhang ZJ, Saito T, Kimura Y, et al. Disruption of mdr1ap-glycoprotein generesults in dysfunction of blood-inner ear barrier in mice[J]. Brain Res. 2000, 852(1): 1162-1261.
10. Lenhardt E, Plotzliche Horstomngen. Aufbeiden Seiten gleichzeitig oder nacheinander aufgetreten[J]. Z Laryngol Rhinol Otol. 1958, 37: 1-16.
11. McCabe BF. Autoimmune sensorineural hearing loss[J]. Ann Otol Rhinol Laryngol. 1979, 88(5 pt 1): 585 - 589.
12. Harris JP, Sharp PA. Inner ear autoantibodies in patients with rapidly progressive sensorineural hearing loss[J]. Laryngoscope. 1990, 100(5): 516- 524.
13. Harris JP, Ryan AF. Fundamental immune mechanisms of the brain and inner ear[J]. Otolaryngol Head Neck Surg. 1995, 112(6): 639 - 653.
14. Tomiyama S, Harris JP. The role of the endolymphatic sac in inner ear immunity[J ]. Acta Otolaryngol. 1987, 103(3-4): 182 - 188.
15. Revel JP. Hexagonal array of subunits in intercellular junctions of the mouse heart and liver[J]. J Cell Biol. 1967, 33(3): 7-12.
16. Banyard SH, Stammers DK, Harrison PM. Electron density map of apoferritin at 2.8-A resolution[J]. Nature. 1978, 271(5642): 282-284.
17.葛贤锡,沈云珍,刘建中.铁蛋白示踪观察甘油开放血-迷路屏障的作用[J].中华耳鼻咽喉科杂志. 1989, 21(2): 70-71.
18. Saria A, Lundberg JM. Evans blue fluorescence: quantitative and morphological evaluation of vascular permeability in animal tissues[J]. J Neu-rosci Mothods. 1983, 8(1): 41-49.
19.张学渊,汪吉宝.豚鼠内耳微循环障碍对血迷路屏障通透性的影响[J].中华耳鼻咽喉科杂志. 2000, 35(5): 339-341.
20. Gianoli GJ, Li JC. Transtympanic steroid for treatment of sudden hearing loss[J]. Otolaryngol Head Neck Surg. 2001, 125(3): 142-146.
21. Nordang L, Laurent C, Mollnes TE. Complement Activation in Sudden Deafness[J]. Arch Otolaryngol Head neck surgery. 1998, 124(6): 633-636.
22. Lopez IA, Ishiyama G, Lee M, et al. Immunohistochemical localization of aquaporins in the human inner ear[J]. Cell Tissue Res. 2007, 328(3): 453-460.
23. Fukushima M, Kitahara T, Fuse Y, et al. Changes in aquaporin expression in the inner ear of the rat after i.p. injection of steroids[J]. Acta Otolaryngol Suppl. 2004, 553: 13-18.
24. Beitz E, Zenner HP, Schultz JE. Aquaporin-mediated fluid regulation in the inner ear[J]. Cell Mol Neurobiol. 2003, 23(3): 315-329.
25. Trune DR, Kempton JB, et al. Mineralocorticoid receptor mediates glucocorticoid treatment effects in the autoimmune mouse ear[J]. Hear Res. 2006, 212((1-2): 22-32. .
26. Lee JH, Marcus DC. Nongenomic effects of corticosteroids on ion transport by stria vascularis[J]. Audiol Neurootol. 2002, 7(2): 100-106.
27. Trune DR, Kempton JB. Aldosterone and prednisolone control of cochlear function in MRL/MpJ-Fas(lpr) autoimmune mice[J]. Hear Res. 2001, 155(1-2): 9-20.
28. Nagashima R, Ogita K. Enhanced biosynthesis of glutathione in the spiral ganglion of the cochlea after in vivo treatment with dexamethasone in mice[J]. Brain Res. 2006, 1117(1): 101-108.
29. Stone JH, Francis HW. Immune-mediated inner ear disease[J]. Current Curr Opin Rheumatol. 2000, 12(1): 32-40.
30.Rahman MU, Poe DS, Choi HK. Autoimmune vestibulo-cochlear disorders[J]. Curr Opin Rheumatol. 2001, 13(3): 184-189.
31. Chandrasekhar, Sujans S. Intratympanic dexamethasone for sudden sensorineural hearing loss: Clinical and laboratory evaluation[J].Otolaryngology and Neurotology. 2001, 22(1): 18-23.
32. Scherer EQ, Herzog M, Wangemann P. Endothelin-1-induced vasospasms of spiral modiolar artery are mediated by rho-kinase-induced Ca(2+) sensitization of contractile apparatus and reversed by calcitonin gene-related Peptide[J]. Stroke. 2002, 33(12): 2965-2971.
33. Schuknecht HF. Ablation therapy for the relief of Meniere’s disease[J]. Laryngoscpe. 1956, 66(7): 859- 870.
34. Chandrasekhar SS, Rubinstein RY, Kwartler JA, et al. Dexamethasone pharmacokinetics in the inner ear : comparison of route of administrtion and use of facilitating agents[J]. Otolaryngol Head Neck Surg. 2000, 122(4): 521-528.
35. Parnes LS, Sun AH, Freeman DJ. Corticosteroid pharmacokinetics in the inner ear fluids: an animal study followed by clinical application[J]. Laryngoscope. 1999, 109(7 pt 2): 1-17.
36.杨军,吴皓.鼓室内给药治疗内耳疾病的基础与临床[J].中华耳鼻咽喉头颈外科杂志. 2004, 39(12): 770-772.
37.周宣岩,陶谦,吕凌燕,等.经鼓室插管注入地塞米松液治疗突发性耳聋[J].中国耳鼻咽喉颅底外科杂志. 2006, 12(1): 47-48, 51.
38. Becvarovski Z, Bojrab DI, Michaelides EM, et al. Round window gentamicin absorption:An in vivo human model[J]. Laryngoscope. 2002, 112(9): 1610-1613.
39. Sheppard WM, Wanamaker HH, Pack A, et al. Direct round window application of gentamicin with varying delivery vehicles:a comparison ototoxicity[J]. Otolaryngol Head Neck Surg. 2004, 131(6): 890-896.
40. Silverstein H. Use of a new device, the MicroWick, to deliver medication to the inner ear[J]. Ear Nose Throat J. 1999, 78(8): 595-598, 600.
41. Van Wijck F, Staecker H, Lefebvre PP. Topical steroid therapy using the Silverstein Microwick in sudden sensorineural hearing loss after failure of conventional treatment[J]. Acta Otolaryngol. 2007, 127(10): 1012-1017.
42. Charabi S, Thomsen J, Tos M. Round window gentamicin mu-catheter -- a new therapeutic tool in Meniere's disease [J]. Acta Otolaryngol. 2000, suppl(543): 108-110.
43. Shoji F, Yamasoba T, Magal E, et al. Glial cell line-derived neurotrophic factor has a dose dependent influence on noise-induced hearing loss in the guinea pig cochlea[J]. Hear Res. 2000, 142(1-2): 41-55.
44. Ylikoski J, Pirvola U, Virkkala J, et al. Guinea pig auditory neurons are protected by glial cell line-derived growth factor from degeneration after noise trauma[J]. Hear Res. 1998, 124(1-2): 17-26.
45. Yamasoba T, Schacht J, Shoji F, et al. Attenuation of cochlear damage from noise trauma by an iron chelator, a free radical scavenger and glial cell line-derived neurotrophic factor in vivo[J]. Brain Res. 1999, 815(2): 317-325.
46. Lalwani AK, Walsh BJ, Reilly PG, et al. Development of in vivo gene therapy for hearing disorders: introduction of adeno-associated virus into the cochlea of the guinea pig[J]. Gene Ther. 1996, 3(7): 588-592.
47. Brown JN, Miller JM, Altschuler RA, et al. Osmotic pump implant for chronic infusion of drugs into the inner ear[J]. Hear Res. 1993, 70(2): 167-172.