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標題: | 以動物內耳功能檢測系統進行耳毒性評估之研究 Investigation of ototoxicity using inner ear monitoring system in the animal |
作者: | Ting-Hua Yang 楊庭華 |
指導教授: | 劉興華 |
關鍵字: | 耳毒性,天竺鼠,前庭功能,前庭誘發肌性電位,銀杏萃取物,器官培養, ototoxicity,guinea pigs,vestibular function,VEMP,extract of Ginkgo Biloba,organotypic culture, |
出版年 : | 2010 |
學位: | 博士 |
摘要: | 耳毒性是指因化學物質、藥物或某些外因性侵犯,造成內耳或相關神經系統的功能損害,主要症狀為聽力障礙、耳鳴、平衡障礙及眩暈。由於耳毒性傷害的初期臨床症狀不明顯,加上耳毒性物質種類繁多、對內耳功能影響的程度方式不一,而且過去研究耳毒性方法並不完全,導致準確性有偏差 。尤以今日又發展出耳內注射等新的給藥途徑,以往的耳毒性資料自需要重新再評估。舉例而言,以往的耳毒性動物實驗往往著重病理組織觀察,內耳功能監測未能涵蓋耳石器官 (橢圓囊及球囊),所以無法真實反映出耳毒性物質影響下的內耳狀態。即使至今,雖然聽力檢查的動物模型已完整建立,動物前庭功能的評估還是以行為觀察為主。有鑑於此,本論文主旨在建立天竺鼠的內耳測試系統,以檢測完整的內耳功能為目標。
首先,我們建立天竺鼠的眼性前庭誘發肌性電位 (oVEMP) 藉以評估橢圓囊功能。在安靜的環境下固定清醒天竺鼠,以手持振動器輕置於前額以刺激其前庭橢圓囊後,可於對側下眼外肌肉附近測得前庭眼反射造成的眼性前庭誘發肌性電位。實驗證明,耳內注射gentamicin破壞橢圓囊後,眼性前庭誘發肌性電位亦隨之消失。 其次,我們亦發展天竺鼠的頸性前庭誘發肌性電位 (cVEMP)以評估球囊功能;配合眼性前庭誘發肌性電位檢查,有效檢測所有耳石器官。我們分別選擇以氣倒音聲及骨導振動不同刺激產生前庭誘發肌性電位。實驗發現,120分貝以下的聲音刺激無法產生眼性前庭誘發肌性電位,而骨導振動刺激則能有效產生眼性前庭誘發肌性電位。當左耳灌注gentamicin引發耳毒性傷害後,所有天竺鼠均無法以振動刺激誘發源於左耳的眼性前庭誘發肌性電位,亦無法於左頸處產生受聲音刺激誘發的頸性前庭誘發肌性電位,但只有60%無法利用振動刺激誘發左頸處的頸性前庭誘發肌性電位。故推測聲音刺激誘發較合適於頸性前庭誘發肌性電位檢查,因為振動刺激可能會引起對側交叉反應引起偽陽性結果。眼性前庭誘發肌性電位檢查則適合以振動刺激誘發,聲音刺激的誘發率則不足,該結論亦與人類臨床報告結論相似。 接著,我們嘗試組合了聽性腦幹誘發反應 (auditory brainstem response)、溫差試驗(caloric test)及上述的眼性前庭誘發肌性電位、頸性前庭誘發肌性電位檢查,為天竺鼠進行完整的內耳功能檢測。相較於所有對照組 (未投藥的一邊正常耳)正常數值,所有受gentamicin耳毒性傷害的另一邊耳朵在聽性腦幹誘發反應均有聽力閾值上升現象,且其餘三者檢查全無反應。病理學檢查亦證實,受gentamicin耳毒性傷害的耳蝸及前庭組織耳毛細胞已大量死亡,與功能檢測結果相符。故利用我們建立的完整內耳功能檢測,的確能有效評估耳毒性物質對內耳器官的影響及傷害。 最後,我們以內耳器官體外培養進行耳毒性物質評估。器官體外培養系統不僅便於耳毒性物質大量篩選、有效節省時間經費外,更有利於耳毒性物質劑量反應分析及相關機制建立。我們利用體外培養耳蝸組織證明, 銀杏萃取物EGb761能透過抑制活性氧自由基及一氧化氮生成,降低gentamicin所引起的耳毛細胞自體凋亡比例及減少細胞死亡數目;另外,天竺鼠以口服方式投予EGb761也能減緩gentamicin所引起的耳毒性傷害。實驗同時篩選銀杏葉萃取物內含之活性成分,如槲黃素(quercetin)、白果內酯 (bilobalide)、銀杏內酯A (ginkgolide A)及銀杏內酯B (ginkgolide B)等,證實均有類似功效。 合併本研究所建立的動物實驗系統,將能有效篩檢並分析耳毒性物質,以提供詳細的毒性資料,同時也可評估具潛力可治療內耳疾病的藥物。此外,有鑑於耳石器官的檢查仍存在爭論,此內耳功能檢測系統也提供平台以進行相關的內耳生理及病理研究。 Ototoxicity implies the functional impairment of the inner ear or its neural pathway caused by certain substances or insults. As the range of ototoxic agents is diverse and extensive, it is hard to obtain their toxic information without some imprecision. Furthermore, as new drug delivery routes of medication, such as intratympanic delivery, were proposed, the existing toxic data should also be re-visited. The ways to evaluate ototoxicity in the past were mostly limited and inadequate. The main problem is the lack of thorough assessment of function status of the whole inner ear. The inner ear includes the auditory endorgan (the cochlea), and the vestibular endorgans (the utricle, saccule and semicircular canals). Up to date, only the auditory function tests are well established in the animals at most laboratories. On the contrary, vestibular function in the animal is frequently based on behavioral observation. Functional assessment of the inner ear that fails to include every endorgan results in inaccurate information of ototoxicity. In view of these deficits, we aimed to establish a system to completely evaluate inner ear functions in the guinea pigs. The work was divided into four parts and was described here separately. In the first part, we developed the ocular vestibular evoked myogenic potentials (oVEMP) test in the guinea pigs for the assessment of the utricular function. By gently fixing an alert guinea pig in a clamp, bone-conducted vibration (BCV) was given in the forehead. Normal oVEMP waveform was recorded near the inferior extraocular muscles bilaterally. The response comes from crossed vestibular-ocular reflex (VOR). When one utricle was destroyed by gentamicin, the oVEMP response vanished. We have also developed cervical VEMP (cVEMP) in the guinea pigs to evaluate saccular function. With cVEMP and oVEMP tests, the function of the otolith organs can be investigated. In order to efficiently record VEMPs in the guinea pigs, we applied both air-conducted sound (ACS) and bone-conducted vibration (BCV) to induce cVEMP and oVEMP. Guinea pigs were treated with intratympanic gentamicin in the left ear to induced ototoxicity. Using ACS mode, oVEMPs were absent in all animals despite the stimulus intensity increased up to 120 dB pe SPL. Conversely, using BCV mode, oVEMPs were present on the left (gentamicin-treated side) eye, and absent on the right (control) eye in all 10 (100%) animals. Via ACS mode, all (100%) right (control) neck showed clear cVEMPs, and all left (gentamicin-treated side) neck revealed absent cVEMPs. However, via BCV mode, except all (100%) right (control) neck showed clear cVEMPs, 6 (60%) animals also demonstrated clear cVEMP on the left neck. It appears that ACS mode is unreliable to elicit oVEMPs in guinea pigs, since the prevalence is low. BCV mode is not specific for inducing cVEMPs in guinea pigs, because a crossed response may occur. Thus, appropriate animal models for cVEMP and oVEMP in guinea pigs are via ACS and BCV modes, respectively, which are also consistent with those reported in humans. Next we combined auditory brainstem responses, cVEMP, oVEMP, and the caloric test to establish an inner ear monitoring system in the guinea pigs. Tested on the gentamicin-treated guinea pigs, we demonstrated normal results in all tests at all control ears, while elevated ABR threshold and absent responses in caloric, oVEMP and cVEMP tests were noted in all gentamicin-treated ears. The results of function tests were compatible with the observation of inner ear pathology. Apparently, the inner ear monitoring system including ABR, caloric, oVEMP and cVEMP tests helps to evaluate toxicity of agents on the inner ear of guinea pigs. Last, we use in vitro organotypic culture of inner ear endorgans to evaluate ototoxicity. For screening purpose, using organ culture has the advantages of time- and cost-saving and demonstration of dose response relationship. It is also a proper medium for the research of ototoxic mechanism. We showed that EGb761 (a standardized preparation of the extract of Ginkgo Biloba) reduced gentamicin-induced elevation of reactive oxygen species (ROS) and nitric oxide (NO) in cochlear cultures from postnatal rats. EGb761 also inhibited gentamicin-induced cellular apoptosis and reduced hair cell loss. In guinea pigs, EGb761feeding prevented gentamicin-induced cochleotoxicity. In the cochlear culture, EGb761 components quercetin, bilobalide, ginkgolide-A, and ginkgolide-B, but not kaempferol, significantly prevented the gentamicin-induced hair cell damage. The test battery in the animal model proves to be useful to screen and assess ototoxic drugs and to test potential therapeutic agents in the inner ear disorders. Moreover, the animal model provides a platform for the research to elucidate the controversy on the origin of VEMP tests and further studies on the inner ear. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/23429 |
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