「癲癇」屬於神經疾病的一種，當中樞神經系統出現異常的神經元活性時，會異常的放電導致自發性的激起抽蓄。造成癲癇發作的病因相當的複雜，包括化學誘導、腦傷與遺傳因子等，這些多樣的病因也使得癲癇的分析變得相對困難。因為缺乏對於癲癇發作相關機制的了解，想預判癲癇的發生目前仍然不可能。先前我們實驗室的研究發現腦創傷的斑馬魚游動的途徑與戊四氮(PTZ)誘使斑馬魚癲癇發作時的游動模式相似。此外斑馬魚腦創傷後各癲癇相關基因表現的趨勢也有顯著的變化。因此我們假設腦創傷後的斑馬魚可能會導致癲癇，同時探討參與斑馬魚癲癇發作的機制可能有哪些。
為了驗證這個假說，我們首先針對小腦創傷後的斑馬魚進行行為上的分析並利用相對定量的方式檢測已知的癲癇指標c-fos和GABAA受體gabrg2的相對基因變化。結果發現腦創傷後的斑馬魚大約有35%會出現類似癲癇的游動途徑，而且這些異常的游動途徑與癲癇指標都可透過抗癲癇藥物丙戊酸(VPA)的治療而抑制。就分子層次，我們利用microarray尋找斑馬魚腦創傷後造成癲癇發作的相關基因，在腦創傷後斑馬魚與戊四氮誘使癲癇的斑馬魚共同比對下，發現atf3與c-jun會顯著的增加，此結果暗示atf3與c-jun在癲癇發作的過程中扮演了潛在的角色，同時我們利用相對定量的方式作驗證。再者我們分析腦創傷的的斑馬魚是否會透過atf3與c-jun的訊號傳遞而對戊四氮誘使癲癇發作的現象更加敏感，最後我們發現腦創傷後有癲癇較無癲癇的c-fos, atf3與c-jun確實有明顯增加的趨勢。
綜合上述結果，我們成功的建立帶有癲癇發作性狀的腦創傷斑馬魚模式。同時我們也是第一個報導斑馬魚在癲癇發作時有atf3與c-jun參與的現象，這些發現或許可以提供腦創傷後是否有可能誘發癲癇的早期診斷。

Epilepsy is a brain disorder that develops spontaneously. Recurring seizures are caused by abnormal neuronal activity in the central nervous system (CNS). The complex etiology of epilepsy is shown to contain multiple causes, including chemical induction, brain injury and genetic factors, which further complicate the analysis. Thus, it is still not possible to predict the occurrence of epileptic seizure due to lack of understanding of its disease mechanism. Previously, we have found the abnormal swimming patterns in traumatic brain injury (TBI) zebrafish resemble the pentylenetetrazol (PTZ)-induced seizure phenotypes. Moreover, expression profiles of several epilepsy-related genes are significantly changed in TBI zebrafish. Therefore, we hypothesize that TBI zebrafish may be used as model to resolve the disease mechanism of brain injury-induced epileptic seizures.
To investigate this hypothesis, we first monitored the behavioral phenotypes of TBI zebrafish and examined the expression profile of known epilepsy markers, c-fos and GABAA receptors gabrg2, in the damaged cerebellum using PCR method. The result indicated 35% of TBI zebrafish displayed seizure-like phenotypes. The seizures-like swimming patterns could be rescued by the administration of valproic acid (VPA), which is a common anti-epileptic drug. Next, we moved to identify genes associated with the TBI-induced seizures by microarray approach. A comparison between TBI and PTZ-treated zebrafishes revealed atf3 and c-jun were significantly up-regulated, implying their potential roles in epileptogenesis. This finding was validated by qPCR assay. We next verified whether TBI may sensitize PTZ-induced seizures via the Atf3 and C-jun signaling pathways. Our data indicated that both Atf3 and C-jun might regulate the development of epileptic seizures as their up-regulations sensitize zebrafish to subsequent PTZ treatment. A comparison between TBI with and without seizures groups further revealed c-fos, atf3 and c-jun were all significantly up-regulated.
In conclusion, we established a TBI model in zebrafish with reproducible epileptic seizures. We have also reported for the first time that Atf3 and C-jun may be involved in the TBI-dependent epileptogenesis. Our findings may be used to develop assays for early diagnosis of TBI-induced epilepsy.

Table of contents
中文摘要 I
Abstract II
致謝 IV
Abbreviations IX
1. Introduction 1
1.1 Epilepsy 1
1.2 Traumatic brain injury and post-traumatic epilepsy in human 1
1.2.1 Traumatic brain injury 1
1.2.2 Post-traumatic epilepsy 2
1.3 The interaction between hormones and epilepsy 3
1.4 Experimental models of post-traumatic epilepsy 4
1.5 The advantages of zebrafish model in PTS research 5
1.5.1 Diagnosis of epileptogenesis 5
1.5.2 Easy for molecular and cellular observation in neuroscience research 5
1.5.3 High regeneration capacity 6
1.6 The aim of this thesis 6
2. Materials and Methods 8
2.1 Animals and maintenance 8
2.2 Stab lesion/ Traumatic brain injury (TBI) 8
2.3 PTZ treatment/Chemical-induced seizure 8
2.4 Behavior analysis 9
2.5 Microarray 10
2.6 Real time-quantitative PCR analysis 10
3. Results 12
3.1 TBI induces seizures-like phenotypes in adult zebrafish 12
3.1.1 Establishment of zebrafish TBI model 12
3.1.2 Behavior and molecular analyses in the TBI zebrafish 13
3.2 GABA signaling is involved in PTS zebrafish 15
3.3 atf3 and c-jun as novel seizure biomarkers 17
3.3.1 The rationale of experimental plan 17
3.3.2 Microarray and Time-series gene expression profiling 17
3.3.3 Significant gene enriched pathway analysis 18
3.3.4 Target gene expression and association analysis 18
3.3.5 Validation of atf3 and c-jun gene expression by qPCR 19
3.3.6 VPA suppresses TBI-induced atf3 and c-jun increase 20
3.3.7 TBI sensitizes zebrafish in response to PTZ treatment 20
3.4 The role of atf3 and c-jun in nervous system and seizures generation 21
3.4.1 Protein-protein interaction among C-fos, Atf3 and C-jun in human, mouse and zebrafish 21
3.4.2 The neurohormone GnRH regulates atf3 and c-jun expression 21
3.4.3 Higher epileptic and PTS marker gene expression in TBI zebrafish model with seizure 22
4. Conclusion and Discussion 24
4.1 Establishment of novel PTS model in zebrafish 24
4.2 Hypothetical model for TBI-induced epileptic seizures 24
4.3 Clinical application 26
5. Reference 28
List of tables
Table 1. Experimental design for microarray analysis 33
Table 2. RNA QA/QC information for microarray 34
Table 3. Primer list of Real Time-quantitative PCR analysis 35
List of Figures
Fig. 1 Altered swimming pattern in TBI zebrafish model 36
Fig. 2 Behavior analysis of TBI induced and PTZ-treated zebrafish 37
Fig. 3 Expression of known epileptic marker c-fos in TBI zebrafish 38
Fig. 4 Expression of GABA synaptic relative gene in TBI zebrafish 39
Fig. 5 Anti-epileptic drug suppressed TBI induced epileptic seizures 40
Fig. 6 Analysis of microarray data by STEM and PANTHER 41
Fig. 7 Identification of novel epileptic markers from PTZ-induced and TBI zebrafish 42
Fig. 8 Validation of atf3 and c-jun as novel epileptic markers in TBI 43
Fig. 9 Protein-protein interaction among C-fos, Atf3 and C-jun 45
Fig. 10 Up-regulation of epileptic/PTS marker gene expression in post-traumatic seizure zebrafish 46
Fig. 11 Proposed model for etiological mechanism of TBI- induced epileptic seizures 47

Supporting Information
Fig. S1 Abnormal swimming pattern in zebrafish model of TBI to the cerebellum 48
Fig. S2 Histological illustration of traumatic brain injury 49
Fig. S3 The rationale of identifying genes associated with TBI-induced epileptic seizures 50
Fig. S4 Significant expression patterns among control, TBI, PTZ-treated and tandem TBI plus PTZ-treated groups 51
Fig. S5 Wnt signaling pathway/PCP signaling pathway (retrieved from KEGG) 52
Fig. S6 cyp11a1 and cyp17a1 are increased after TBI in zebrafish 53

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