臺灣博碩士論文加值系統

　　耳鳴是一種在無外在音源下所產生的聽覺，目前對於其生理病理成因尚未清楚。在人類及動物以證實在過量水楊酸下會造成可逆性耳鳴。在中耳局部投與水楊酸實驗中已發現由對聲音反應的聽腦幹誘發電位受到抑制亦支持水楊酸在耳蝸之作用。全身性投與水楊酸則造成中樞聽神經元的過度活化。對於此周邊及中樞作用之矛盾現象目前仍未有清楚的解釋。本研究欲在探討經由兩種不同途徑長期持續投與水楊酸對中樞聽覺核活性所造成的反應，藉由即時表現基因c-fos 之蛋白質產物Fos 為神經元活性標記，利用Fos免疫染色法呈現出聽腦幹核之活性改變。
　　本研究實驗大鼠(Sprague Dawley rat)分成兩組分別進行無菌手術。腹腔持續釋藥系統組以將注滿不同濃度(250及360 mg/kg/day)的水楊酸溶液之滲透幫浦(osmotic pump)置入腹腔內7 天;中耳持續釋藥系統組則以吸收不同濃度(5, 50,及250 mM)的水楊酸膠海綿放入中耳腔內6天，並分別以溶媒組實驗動物為對照組。實驗動物最後取腦做冷凍切片及Fos免疫染色並採用ABC呈色法。經顯微鏡觀察聽腦幹區耳蝸核(cochlear nucleus)、下丘(inferior colliculus)、及側上橄欖核(lateral superior olivary nucleus)區之Fos免疫染色分布及量化結果來觀察神經活性變化。另外為得知水楊酸局部(外耳淋巴液)及中樞(腦脊液)濃度對Fos表現之影響以及不同時間點之濃度，本研究另利用電灑游離串聯式質譜分析方法進一步來分析水楊酸濃度。
　　實驗結果顯示經由中耳及腹腔持續釋藥這兩種給藥方式均能觀察到Fos表現在下丘及側上橄欖核均有似連續5天單一劑量水楊酸注射的明顯增加現象，此亦證實耳鳴發生的可能性。中耳釋藥組中外耳淋巴液水楊酸含量將快速上升而隨著時間腦脊液中亦有緩升的水楊酸濃度，此證實在被動性通透的存在，例如耳蝸導水管。腹腔投與組中外耳淋巴液水楊酸含量亦快速上升且高於腦脊液，其可能因耳蝸有著主動分泌機制。因此周邊耳蝸水楊酸投與，即使是單側，可引起之耳鳴現象，其可支持周邊作用可能亦包括著但需中樞作用的存在。此活化現象主要是作傳導閥值的微調。對照組動物的Fos相對高數量可能來自於寂靜環境處理下之生理性耳鳴，其可能於多日後因適應而消失。在耳蝸核中，所有實驗動物Fos的表現並不會增加，此亦意味著耳鳴的來源主要來自於中樞而非周邊傳入。因此本研究亦證實了水楊酸引起之耳鳴成因是先作用於外周耳蝸進而影響中樞聽覺系統之過度活化所致。

　　Tinnitus is a phantom sensation of sound in the absence of external stimulations and its pathogenesis is unclear. Overdose of salicylate (SA) is known to cause tinnitus in both human and animals. Local application of SA in the bulla suppresses evoked potential to sound suggesting a peripheral action of SA at the cochlea. Systemic application of SA also induces over-activities of central auditory neurons suggesting a central action. In this study, the effects of SA applied at two different routes were compared in order to elucidate the action sites. Fos-immunohistochemistry was used to reveal the hyper-activity in the brainstem nuclei. Adult rats were divided into two groups for aseptic surgery. The first group was intraperitoneally implanted with osmotic pumps that would sustain release SA for 7 days. The second group had a SA-presoaked gelfoam placed over the round window in the bullae for 6 days. Controls received similar procedures with vehicle-treatments. Brains were processed for Fos-immunohistochemistry on thick frozen sections according to the conventional ABC method. For comparison, a limited number of animals also received a 5-daily SA injection. For some animals, SA levels in the perilymph and cerebrospinal fluid were measured at different time points with mass spectrometry (ESI-MS/MS).
　　Both the osmotic pump and gelfoam applications that released SA slowly were effective in raising Fos-activity labeling both in the inferior colliculus and lateral superior olivary complex in a manner rather similar to the multiple-daily injection protocol, suggesting a possible occurrence of tinnitus. Following intra-bullar applications there was a rapid increase of SA in perilymph followed by a delayed increase in CSF to about the level, suggesting an effective fluid passage linking the two compartments likely through the cochlear aqueduct. Intra-peritoneal applications also led to a rapid rise in SA level in the perilymph at a level higher than the CSF, suggesting the presence of an active secretion mechanism in the cochlea. The fact that SA when applied locally to the cochlea, even on one side, was effective suggested that a peripheral site was likely involved but may also require the action at the central nervous system. The relatively high level of Fos-expression at the IC of control rats was discussed with respect to a physiological tinnitus, which might subside in Fos-expression after animals were adapted to a silent environment.

Abstract
Abstract (in Chinese)

1. Introduction
1.1 Tinnitus 1
1.2 What is the relationship between salicylate
(SA) and tinnitus in human? 1
1.3 Animal studies on SA-induced tinnitus 2
1.4 The auditory brainstem and midbrain versus
SA-induced tinnitus 3
1.4.1 Inferior colliculus (IC) 3
1.4.2 Lateral olivary nucleus (LSO) and cochlea
nucleus (CN) 3
1.4.3 SA & auditory nuclei 3
1.5 Fos as a neural activity marker 4
1.6 Methods of SA measurement 5
1.7 Aims of this study 6

2. Materials
2.1 Instruments for online clean-up device with
ESI-MS/MS system 8
2.2 Chemicals and materials 9
2.3 Immunohistochemistry 10
2.4 Anesthetics 10
2.5 Solutions preparation 11

3. Methods
3.1 Establishing the SA analytical method 15
3.1.1 Online clean-up device and ESI-MS/MS system
15
3.1.2 Optimization of MS instrument 15
3.1.3 Online clean-up system 17
3.1.4 Validation of the analytic method 17
3.1.5 Analysis of the biological samples 20
3.1.6 Pre-treatment of the bio-sample 20
3.2 Animal preparation 21
3.2.1 Acute experiments 21
3.2.2 Chronic experiments 22
3.3 Perfusion and tissue preparation for Fos
Immunohistochemistry 23
3.4 Fos Immunohistochemistry 23
3.5 Data analysis 24
3.5.1 The counts and location of Fos-labeled
neurons 24
3.5.2 3D reconstruction of the Fos-labeled
neurons 24

4. Results
4.1 Establishing the SA analytical method 26
4.1.1 Validation of the analytic method 26
4.1.1.1 Selectivity 26
4.1.1.2 MDL (method detection limit) 26
4.1.1.3 Calibration and Linearity 26
4.1.1.4 Precision 27
4.1.1.5 Recovery 27
4.2 Time profiles of SA levels in perilymph and
cerebral spinal fluid (CSF) after acute
applications of SA 28
4.2.1 Intra-bulla application with SA-presoaked
gelfoam 28
4.2.2 Peritoneal application with SA single dose
injection 28
4.3 Residual SA levels in perilymph and CSF
after chronic application of SA via
intra-bulla or intra-peritoneal routes
28
4.4 Side effects of chronic SA application 29
4.5 Fos-activity changes in the inferior
colliculus (IC) 30
4.5.1 Chronic intra-bulla application of SA 30
4.5.2 Chronic intra-peritoneal application of SA
30
4.6 Fos-activity changes in the cochlear
nucleus (CN) 30
4.7 Fos-activity changes in the lateral
superior olive (LSO) 31

5. Discussion
5.1. The validation results of the analytic
method for SA 32
5.1.1. Recovery 32
5.1.2. Precision 32
5.2. The profile of SA in perilymph and CSF
after SA acute application 32
5.3. SA residual in perilymph and CSF at the end
of SA chronic applications 33
5.4. SA dose is enough to induce tinnitus-like
pattern 33
5.5. Side effects of chronic SA application 33
5.6. The possible mechanism of salicylate-
induce tinnitus-like pattern in the IC 34
5.7. Fos expression in the high-frequency region
at the CIC 34
5.8. Tinnitus-like Fos pattern in the IC of
control rats 35

6. Summary 37
7. References 38
8. Tables 44

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