i

The changes of ERG channel

expression after administration of

antiepileptic drugs in the

Hippocampus of epilepsy gerbil model

Dong Hwa Heo

Department of Medicine

The Graduate School, Yonsei University

ii

The changes of ERG channel

expression after administration of

antiepileptic drugs in the

Hippocampus of epilepsy gerbil model

Directed by Professor Kum Whang

The Doctoral Dissertation

submitted to the Department of Medicine,

the Graduate School of Yonsei University

in partial fulfillment of the requirements for the degree

of Doctor of Philosophy

Dong Hwa Heo

June 2010

iii

This certifies that the Doctoral

Dissertation of Dong Hwa Heo

is approved.

------------------------------------ Thesis Supervisor : Kum Whang

------------------------------------

Seong-Woo Jeong : Thesis Committee Member#1

------------------------------------ Soo-Ki Kim: Thesis Committee Member#2

------------------------------------

Byung Ho Cha : Thesis Committee Member#3

------------------------------------ Tae-Cheon Kang : Thesis Committee Member#4;

The Graduate School

Yonsei University

June 2010

iv

ACKNOWLEDGEMENTS

박사 논문을 마치며

먼저 박사과정을 마칠 수 있도록 이끌어 주신 황금 교수님과

논문의 실험과 진행을 도와주신 강태천 교수님께 진심으로 감사

드립니다. 또한 연구와 논문작성에 있어 아낌없는 조언을 해주신

정성우 교수님, 김수기 교수님, 차병호 교수님께 감사 드립니다.

저를 신경외과 의사로 이끌어 주신 원주의과대학 신경외과학

교실, 척추외과의로 이끌어 주신 강남 세브란스 신경외과학 교실,

그리고 저의 꿈을 실현시킬 수 있도록 이끌어 주신 한림대학교

의과대학 신경외과학 교실, 이 모든 교수님들께 감사 드립니다.

그리고, 사랑하는 저의 아내 영주와 저에게 늘 힘이 되어 주시는

어머니, 저의 모든 가족들, 그리고 하늘에서 보고 있을

가족들에게 고맙다는 말을 전하고 싶습니다.

마지막으로 나를 가족처럼 도와주는 나의 친구들께 감사

드립니다.

2010년 7월에

저자 허동화 씀

v

<TABLE OF CONTENTS>

ABSTRACT ····························································································· 1

I. INTRODUCTION ·················································································· 4

II. MATERIALS AND METHODS ·························································· 8

1. Experimental animals ·········································································· 8

2. AED treatment ···················································································· 9

3. Electrophysiology ········································································· 10

4. Immunohistochemistry ···································································· 11

5. Western blot ····················································································· 12

6. Statistical analysis ········································································· 13

III. RESULTS ························································································ 15

1. ERG immunoreactivity in the gerbil hippocampus ····························· 15

2. The effect of AEDs on ERG immunoreactivity ·································· 15

3. Seizure activity and paired pulse responses ······································· 22

IV. DISCUSSION ················································································· 25

V. CONCLUSION ················································································ 28

REFERENCES ······················································································ 29

ABSTRACT (IN KOREAN) ································································ 39

vi

<LIST OF FIGURES>

Figure 1. ERG immunoreactivity in the SR gerbil

hippocampus ·······························································17

Figure 2. ERG immunoreactivity in the SS gerbil

hippocampus ·······························································18

Figure 3. Quantitative analysis of ERG immunoreactivity

in the SR and SS gerbil hippocampi ························ 19

Figure 4. Western blot of ERG in SR gerbils····························· 20

Figure 5. Western blot of ERG in SS gerbils ····························· 21

Figure 6. Effect of AEDs on paired-pulse inhibition of the

dentate gyrus of gerbils ·············································· 23

1

ABSTRACT

The changes of ERG channel expression after administration of

antiepileptic drugs in the Hippocampus of epilepsy gerbil model

Dong Hwa Heo

Department of Medicine

The Graduate School, Yonsei University

(Directed by Professor Kum Whang)

Ether-a-go-go (ERG) K+ channel is a channel of potassium inward

rectification. ERG channelopathy can induce sudden cardiac death, and

channelopathy related with ERG may be a cause of sudden unwanted

death. The purpose of our study is to assess the effect of AED on the

expression of ERG K+ channel in the hippocampus using seizure

2

resistant (SR) and seizure sensitive (SS) gerbils.Four kinds of drugs

were used in this study; Topiramate, Lamotrigine, Valproate and

Carbamazepine. Each AED was injected in seven SS and SR gerbils,

and AEDs were injected total 28 SS gerbils and 28 SR gerbils. For

comparing the effect of AEDs, normal saline was injected at 7 SS

gerbils and 7 SR gerbils. Immunoreactivity for ERG K+ channels was

checked and its quantitative analysis was performed using the western

blot. Hippocampal paired pulse response was checked in SS gerbils and

AED injected SS gerbils. In principal neuron of hippocampus,

immunoreactivity for ERG channel was significantly decreased after

administration of AEDs in SS and SR gerbils comparing to SS and SR

gerbils which were given saline (P<0.05). The expression of ERG

channel was significantly decreased in AEDs treated SS and SR gerbil

rather than the saline injected SS and SR gerbil in Western blot of

hippocampus (P<0.05). After administration of AEDs, seizure activities

were not observed in SS gerbils. Compared to saline treated SR and SS

gerbils, population spike in response to the second stimulus

disappeared, thus population spike amplitude ratio was significantly

reduced to zero in both SR and SS gerbils. According to our results,

3

AEDs reduced the expression of ERG potassium channel in the

hippocampus of the SR and SS gerbils. And, AEDS mainly influenced

on the principal neuron of hippocampus.

--------------------------------------------------------------------------------------

Key Words: ERG1 channel, Potassium, Antiepileptic drugs, Epilepsy,

Immunohistochemistry, Gerbil

4

The changes of ERG channel expression after administration of

antiepileptic drugs in the Hippocampus of epilepsy gerbil model

Dong Hwa Heo

Department of Medicine

The Graduate School, Yonsei University

(Directed by Professor Kum Whang)

1. INTRODUCTION

Spike-frequency adaptation is a property shown by neurons.1 Briefly, a

gradual increase in K+ currents is to produce a gradual increase in a K+

conductance that counteracts the Na+ currents leading to either a longer

interspike interval or a complete block of the burst. In this process, inwardly

rectifying K+ (Kir) channels exert a remarkable action in producing spike-

5

frequency adaptation.1 Inwardly rectifying K+ (Kir) channels are widely

expressed in excitable and nonexcitable cells. These channels play a major

role in maintaining the cellular resting membrane potential near the K+

equilibrium potential and permitting long-lasting depolarization in excitable

cells owing to inward rectification at depolarized potential.2-4 Interestingly,

ERG current (encoded by the human ether-a-go-go-related gene (hERG)

identified from a hippocampal cDNA)5 is believed to be involved in spike-

frequency adaptation, since ERG mutations cause the type 2 form of

congenital cardiac arrhythmia long QT syndromes (LQT2).6, 7

ERG is

identical to the cardiac inwardly K+ rectifier,8 is strongly modulated by the

extracellular K+ concentration ([K+]o) and is specifically blocked by class III

antiarrhythmic drugs, such as sotalol and its analogue E 4031,9 as well as by

WAY 123,398.10 Therefore, ERG channel is fundamental elements for cardiac

repolarization. However, ERG also regulates the noncardiac cell excitability;

ERG is abundantly expressed in brain.11 ERG also modulates the firing of

endocrine cells,12 and the firing frequency adaptation in cultured neurons.

1

Furthermore, some drugs including antipsychotics produce acquired long QT

syndrome by blocking ERG.13

In Drosophila, a homologue of the ERG K+ channel is encoded by the sei

(seizure) locus,14, 15

providing an alternative explanation for the severe

6

neuronal hyperactivity. Clinically, function of ERG K+ channel was related

with cardiac arrhythmia and seizure attack.16, 17

Furthermore, patient with

chronic epilepsy expired due to long QT syndrome, and mutation of ERG was

revealed.17 Therefore, dysfunction of ERG may be related to seizure activity

and possible cause of sudden unexpected death in epilepsy (SUDEP).16, 17

Indeed, antiepileptic drug (AED) can influence to ERG channels in vitro

study,16, 18, 19

although the in vivo electrical properties of ERG current in

neurons is for the most part poorly understood.

On the other hand, Mongolian gerbils exhibit spontaneous seizures in

response to a variety of stimuli without the neuronal degeneration that is

associated with the use of neurotoxins such as kainate. Moreover, this model

allows epileptic and non-epileptic animals to be directly compared, which

allows for the identification of differences in brain anatomy and

electrophysiology associated with seizure behavior.2, 20-23

We recently

reported that the differential expression of Kv1-4 channels in the seizure

sensitive (SS) gerbil hippocampus is relevant to seizure activity.2, 24

Thus, we

supposed that the alteration of ERG channel expression in the hippocampus

might be also related to seizure activity of SS gerbils, although it has not been

fully elucidated. Therefore, to confirm our hypothesis, we investigated (1)

whether the ERG immunoreactivity in the SS gerbil hippocampus differs from

7

that in SR gerbil hippcoampus and (2) whether AED treatment affects ERG

expression in the gerbil hippocampus.

8

II. MATERIALS AND METHODS

1. Experimental animals

This study utilized the progeny of Mongolian gerbil (Meriones

unguiculatus) obtained from the Experimental Animal Center, Hallym

University, Chunchon, South Korea. The animals were housed at constant

temperature (23 °C) and relative humidity (60%) with a fixed 12-h light/dark

cycle and free access to food and water. The procedures involving animals

and their care were conducted in conformity with the institutional guidelines

and in compliance with international laws and policies (NIH Guide for the

Care and Use of Laboratory Animals, NIH Publication No. 80-23, 1996).

Each adult animal (4–5 months old) was stimulated by vigorous stroking on

the back with a pencil as described by Paul et al.25 and tested a minimum of

three times.

According to the seizure severity rating scale,26, 27

seizures were

classified; grade 1, a brief pause in normal activity accompanied by vibrissae

twitches and retraction of the pinnae; grade 2, motor arrest with twitching of

the vibrissae and retraction of the pinnae; grade 3, motor arrest with

myoclonic jerks; grade 4, clonic–tonic seizures; grade 5, clonic–tonic seizures

9

with body rollover. Only animals with a consistent stage 4 or 5 seizure score

were included in the present study as SS gerbils. In addition, SS gerbils used

in the present study did not showed seizure activity at least 36 h prior to the

experimental procedure. SR gerbils never demonstrated the seizure activity,

thus they were assigned seizure severity score of 0.28-33

2. AED treatment

For drug treatment procedures, Both SR and SS adult gerbils were sub-

divided into 5 groups, and each group (n = 7) was given the following AEDs

one time a day during 7 days, respectively: (1) carbamazepine (25 mg/kg, I.P.),

(2) lamotrigine (20 mg/kg, I.P.), (3) topiramate (40 mg/kg, I.P.), (4) valproic

acid (30 mg/kg, I.P.) and (5) saline as controls. Three hours after the last drug

treatment, animals were used for electrophysiological study. The dosage of

each AEDs was determined as anti-epileptic dose that completely inhibits

seizure activity of SS adult gerbil in the preliminary and our previous

studies.34-38

The injection procedures were performed using aseptic devices

and manners under clean environment.

10

3. Electrophysiology

For recording hippocampal EEG and evoked potentials, both SR and SS

adult gerbils (n = 30, respectively) were anesthetized (urethane, 1.5 g/kg, IP)

and placed in stereotaxic frames. Holes were drilled through the skull for

introducing electrodes according to gerbil brain atlas.27 The coordinates (in

mm) referenced to bregma were as follows. For the recording electrode (to the

stratum radiatum of CA1 region, 50 µm Teflon insulated stainless steel wire,

AM system, Washington State, USA): – 1.6 anterior–posterior, 1.3 lateral, 1.2

depth (from dura mater). The reference electrode was placed in the cerebellum.

For the stimulating electrode (to the stratum radiatum of CA2–3 regions,

bipolar tungsten stimulating electrodes): – 1.6 anterior–posterior, 2.4 lateral,

1.8 depth (from dura mater) in the contralateral hippocampus. Electrode

depths were finally determined by optimizing the evoked response. Stimuli

were applied as DC square pulses at 0.1 Hz with pairs of 150-µs constant

current stimuli at 30-ms interstimulus intervals (stimulus intensity; 1.5×

threshold) depending on the threshold intensity (range = 15–500 µA)

consistently evoking the population spike in response to the first stimulus

during the baseline period. Signals were recorded with DAM 80 differential

amplifier (0.1–3000 Hz bandpass, World Precision Instruments, FL, USA)

and data were digitized (10 kHz) and analyzed on MacChart 5 (AD

11

Instruments, NSW, Australia). Baseline responses were recorded for 30 min.

Evoked responses and EEG were recorded for 2 h after drug treatment. To

analyze changes in evoked responses following drug treatment, the average of

the population spike amplitude of the first response during the baseline

measurement was used to normalize all of the population spike amplitude

measurement during the recording session from the same animal. After the

recording, animals were used for immunohistochemistry (n = 3, respectively)

and western blot (n = 4. respectively).

4. Immunohistochemistry

Animals were anesthetized with urethane, and perfused via the ascending

aorta with 200 mL of 4% paraformaldehyde in phosphate buffer (pH 7.4). The

brains were removed, postfixed in the same fixative for 4 h and rinsed in PB

containing 30% sucrose at 4 °C for 2 days. Thereafter the tissues were frozen

and coronally or horizontally sectioned with a cryostat at 30 µm and

consecutive sections were collected in 6-well plates containing phosphate

buffered saline (PBS, pH 7.4). These free-floating sections were first

incubated with 10% normal goat serum for 30 min at room temperature. They

were then incubated in rabbit anti-HERG (diluted 1:200, Alomone lab,, Israel)

12

in PBS containing 0.3% triton X-100 and 2% normal goat serum overnight at

room temperature. After washing three times for 10 min with PBS, sections

were incubated sequentially, in goat anti-rabbit IgG (Vector, USA) and ABC

complex (Vector, USA), diluted 1:200 in the same solution as the primary

antiserum. Between the incubations, the tissues were washed with PBS three

times for 10 min each. The sections were stained with 3,3′-diaminobenzidine

(DAB) in 0.1 M Tris buffer and mounted on the gelatin-coated slides. All

immunohistochemical staining procedures in the present study were

performed under the same circumstance and in one run.28 In order to establish

the specificity of the immunostaining, a negative control test was carried out

with pre-immune serum instead of primary antibody, or a pre-absorption test

for HERG antibody was performed using control antigen peptides (Alomone

lab, Israel). The negative control resulted in the absence of immunoreactivity

in any structures (Data not shown). The immunoreactions were observed

under an Axiophot microscope (Carl Zeiss, Germany), and images were

captured using an Axiocam HRc camera and Axio Vision 3.1 software.

5. Western blot

Five animals in each drug-treated group were used in the immunoblot

13

study. For tissue preparation, animals were decapitated, hippocampus was

removed, and then each tissue was homogenized in 10 mM PB containing

0.1 mM EDTA, 1 mM 2-mercaptoethanol and 1 mM PMSF. After

centrifugation, the protein concentrations in the supernatants were determined

by using a Micro BCA protein assay kit with bovine serum albumin as the

standard (Pierce Chemical, USA). Aliquots containing 30 µg of total protein

were boiled with an equal volume of 2× SDS sample buffer and boiled for

3 min, and then, each mixture was loaded onto a 10% polyacrylamide gel.

After electrophoresis, the gels were transferred to nitrocellulose transfer

membranes (Schleicher and Schuell, USA). To reduced background staining,

the filters were incubated with 5% non-fat dry milk in PBS containing 0.1%

Tween 20 for 45 min, sequentially incubated with primary antisera (1:10,000),

peroxidase conjugated goat anti-rabbit IgG (Sigma, USA) and then with ECL

kit (Amersham, USA). Optical density was measured using NIH Image 1.59

software.

6. Statistical analysis

For quantification of immunohistochemical data, image of each section

(15 sections per each animal) was captured in the stratum pyramidale of the

14

CA1 region and the granule cell layer and hilar neuron layer of the dentate

gyrus at magnification of 100×. All measurements were carried out under the

same optical and light conditions. Each image was normalized by adjusting

the black and white range of the image using Adobe PhotoShop v. 8.0.

Thereafter, HERG immunodensity of each section was represented as the

number of a 256 gray scale using NIH Image 1.59 software. Optical density

values were corrected by subtracting the average values of background noise

obtained from 5 image inputs. The optical density was then standardized by

setting the threshold levels. All data obtained from the quantitative

measurements were analyzed using one-way ANOVA to determine statistical

significance. Bonferroni's test was used for post hoc comparisons. P values

below either 0.01 or 0.05 were considered statistically significant.

15

III. RESULTS

1. ERG immunoreactivity in the gerbil hippocampus

In the SR gerbil hippocampus of SR adult gerbils (Figures 1A), ERG

immunoreactivity was detected in CA1-3 pyramidal cells, dentate granule

cells and hilar neurons. The localization of ERG immunoreactivity in the

hippocampus of SS gerbils was similar to that of SR gerbils (Figures 2A).

2. The effect of AEDs on ERG immunoreactivity

In the present study, AED treatments affected HERT immunoreactivity in

both SR and SS gerbil hippocampi, as compared to saline treatment. In SR

gerbil, lamotrigine, topiramate and valproic acid reduced the density of ERG

immunoreactivity in the hippocampus to about 40 % of that in saline-treated

SR gerbils. Carbamazepine decreased the intensity of ERG immunoreactivity

in the hippocampus to ~ 60 % of that in saline-treated SR adult gerbils

(Figures 1B-E and 3). Similar to the immunohistochemical study, immunoblot

data revealed that reduced ERG protein level in the SR gerbil hippocampus

after the AED treatments as compared with saline-treated SR gerbil

16

hippocampus (Figures 4). However, ERG immunoreactivity in hilar neurons

was unaffected by AED treatment (Figure 3).

Similar to SR gerbils, AED treatment also reduced ERG immunoreactivity in

the SS gerbil hippocampus, as compared to saline-treated SS gerbils. However,

the action of each AED treatment on reducing ERG expression in SS gerbils

was more effective than that in SR gerbils. Lamotrigine, topiramate and

valproic acid reduced the density of ERG immunoreactivity in the

hippocampus to about 20 % of that in saline-treated SR gerbils.

Carbamazepine decreased the intensity of ERG immunoreactivity in the

hippocampus to ~ 40 % of that in saline-treated SR adult gerbils (Figures 2B-

E and 3). Immunoblot data was consistent with the immunohistochemical

study (Figures 5). ERG immunoreactivity in hilar neurons was unaffected by

AED treatment (Figure 3).

17

Figure 1 ERG immunoreactivity in the SR gerbil hippocampus. As

compared to saline-treated animals, ERG immunoreactivity is markedly

reduced following AED treatment. Bar = 400 (panels A) and 50 µm

(panels B-D).

18

Figure 2 ERG immunoreactivity in the SS gerbil hippocampus. As compared

to saline-treated animals, ERG immunoreactivity is markedly reduced

following AED treatment. Bar = 400 (panels A) and 50 µm (panels B-D).

19

Figure 3 Quantitative analysis of ERG immunoreactivity in the SR and SS

gerbil hippocampi. After AED treatments, ERG is significantly decreased in

the hippocampus, as comparing to animals given saline. Significant

differences from saline-treated animals (*P < 0.05).

20

Figure 4 Western blot of ERG in SR gerbils. (A) The expression of ERG is

significantly decreased in hippocampus after administration of each AED. (B)

Quantitative analysis of western blot data in the SR gerbil hippocampus.

Significant differences from saline-treated animals (*P < 0.05).

21

Figure 5 Western blot of ERG in SS gerbils. (A) The expression of ERG

is significantly decreased in hippocampus after administration of each

AED. (B) Quantitative analysis of western blot data in the SS gerbil

hippocampus. Significant differences from saline-treated animals (*P <

0.05).

22

3. Seizure activity and paired pulse responses

Consistent with previous studies , evoked responses in the dentate gyrus

showed no significant differences between SR and SS gerbils.39, 40

Briefly, the

thresholds of stimulus intensities to evoke a population spike in response to

the first stimulus in SR and SS gerbils were 27.8 ± 4.17 and 29.2 ± 5.35 µA,

respectively. In addition, the population spike amplitude ratio was similarly

detected between SR and SS gerbils. After administration of AEDs, seizure

activities were not observed in SS gerbils. Compared to saline treated SR and

SS gerbils, population spike in response to the second stimulus disappeared,

thus population spike amplitude ratio was significantly reduced to zero in both

SR and SS gerbils (Figures 6).

23

Figure 6 Effect of AEDs on paired-pulse inhibition of the dentate gyrus of

24

gerbils. (A) Representative evoked responses of the dentate gyrus. Inset at top

shows measurement of population spike amplitude (PS). Filled circles indicate

stimulus artifacts. There is no difference of paired-pulse reponses between

saline treated SR and SS gerbils. Following AED treatments, paired-pulse

inhibition is increased as compared with saline-treated animals. (B)

Quantitative analysis of paired-pulse inhibition in the dentate gyrus of SS

gerbils. Significant differences from saline-treated animals (*P < 0.05).

25

IV. DISCUSSION

Major findings in the present study are reduction of ERG

immunoreactivity in SR and SS gerbil hippocampus following AED treatment,

although its immunoreactivity was no difference between SR and SS gerbils.

In addition, the efficiencies of AEDs on alteration in ERG immunoreactivity

in SS gerbils were higher than those of SR gerbils. Unlike previous

presumptions,41 these findings indicate that at least in gerbils seizure activity

may not be related to the ERG channel functionality in the SS adult gerbil

hippocampus.

In gerbils, it has been reported that Kv1-4 and TWIK-related acid-

sensitive K+ channel-1 (TASK-1) channel expressions in the SS gerbil

hippocampus are distinct from those in the SR gerbil hippocampus. Briefly,

Kv1 subunit expressions are down-regulated in principal neurons within the

SS gerbil hippocampus.42 Kv3.1b and Kv3.2 immunoreactivities are also

significantly lower in SS gerbils than those in SR gerbils.2 Thus, delayed

rectifier K(+) channels (Kv1.1, Kv1.2, Kv1.5, Kv1.6, Kv2.1 and Kv3.1-2), not

A-type K(+) channels (Kv1.4, Kv3.4 and Kv4.x), are down-regulated in the

SS gerbil hippocampus, as compared to SR gerbils. In contrast, TASK-1

immunoreactivity in the SS gerbil hippocampus is higher than that in the SR

gerbil hippocampus. Considering the roles of K+ channels in epilepsy, these

26

previous findings imply that dysfunction of K+ channel may be involved in

seizure activity in the gerbils. Indeed, K+ channel inhibitors are powerful

convulsants.43-45

ERG is one of the delayed rectifier currents (IKR) of the heart.6, 7 In

addition, K+ curents recorded from glia resemble properties with the cardiac

delayed rectifier current, which is responsible for cardiac action potential

repolarization.6, 46

Therefore, we had expected that the difference of ERG

expression would be detected between the SR- and the SS gerbil hippocampus.

In the present study, ERG immunoreactivity was observed in neurons and

astrocytes in the gerbil hippocampus. However, there was no distinct between

SR and SS gerbils. These findings indicate that ERG may not play an

ictogenic role in SS gerbils, unlike other delayed rectifier K+ channels.

Chiesa et al.1 reported that that neuroblastoma cells expressing ERG show

spike-frequency adaptation when current clamped with long depolarization.

This is because enhancement of ERG expression/function restores firing

properties needed for accommodation to repetitive stimuli. Thus, they

suggested that defective or null expression of ERG channels could result in

hyperexcitability. In the present study, unexpectedly, AED treatments

decreased ERG expression in both the SR and SS gerbil hippocampus.

Furthermore, paired-pulse inhibition in response to each AED treatment was

27

enhanced, as compared to saline treatment. Therefore, our findings indicate

that in the adult brain reduced ERG channel may not be accompanied by

neuronal hyperexcitability.

On the other hand, Danielsson et al.16, 18

reported that some AEDs inhibit

ERG currents. Similar to these previous reports, the present study shows that

AED treatments significantly reduced ERG expression in the gerbil

hippocampus. Furthermore, the efficiencies of AEDs on alteration in ERG

immunoreactivity in SS gerbils were higher than those of SR gerbils.

Although the exact mechanisms/biological meanings of AEDs acting on ERG

expression in the present study, it is considerable that reduced ERG

expression may be one of compensatory consequence from AED treatments. It

has been reported that blockade of the putative ERG current is a reduction

in

the duration of a pause in spontaneous firing in dopamine neurons.47

Considering this report, it is likely that reduced ERG expression may play a

role in maintenance of spontaneous neuronal activity that would be inhibited

by AEDs. In this condition, however, reduced ERG channel may trigger

neuronal hyperexcitability when AEDs are suddenly withdrawn. Alternatively,

reduced ERG expression induced by AEDs may be an experimental evidence

explaining sudden unexpected death (SUD). Epilepsy patient have an

increased risk of SUD, being 20 times more common than in the general

28

population.48 SUD is rare in patients with new onset epilepsy and among those

in remission,49 but considerably more common in patients with chronic

refractory epilepsy.50 Furthermore, polytherapy with more than one AED was

associated with a higher risk of SUD in epilepsy (SUDEP) than monotherapy.

51, 52 Based on these clinical evidences, decreased ERG expression induced by

AEDs may provide evidence demonstrating the relationship between AEDs

and SUDEP. Further studies are needed to elucidate the interactions of ERG

and AEDs.

V. CONCLUSION

According to our results, AEDs reduced the expression of HERG potassium

channel in the hippocampus of the SR and SS gerbils. And, AEDS mainly

influenced on the principal neuron of hippocampus. We recommend that

newly developing AEDS should be checked about the influence of HERG in

not only heart but also brain.

VI. REFERENCES

29

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39

ABSTRACT (IN KOREAN)

유전성 간질 동물 모델의 해마 부위에서 항전간제 투여 후에

ERG 채널의 발현의 변화

< 지도교수 황 금 >

연세대학교 대학원 의학과

허동화

Ether-a-go-go K

+ channel (ERG) 은 K

+ inward rectification에 관여하며,

주로 심장과 뇌에 존재하고 있다. ERG channel의 이상은 갑작스런 심

장사를 유발 할 수 있으며, 갑작스런 심장사의 원인으로 ERG

channel과 관계된 channelopathy가 원하지 않는 갑작스런 죽음의 하나

의 원인으로 추측되고 있다. 이에 연구자들은 유전성 간질 모델인

gerbil을 이용하여, 항전간제 투여가 해마 (hippocampus) 부위에서

ERG 발현에 어떠한 영향을 주는 알아보고자 한다. 간질 발작이 없

는 seizure resistant gerbil (SR-gerbil) 유전 간질 모델인 seizure sensitive

gerbil (SS-gerbil)을 이용하여 실험하였다. 네 가지의 항전간제가 실험

에 사용되었다 (Topiramate Lamotrigine, Valproate and Carbamazepine).

한가지의 약물은 7마리의 SR-gerbil과 7마리의 SS-gerbil에게 투여하

40

여, 총 28마리의 SR-gerbil과 28마리의 SS-gerbil에게 항전간제가 투여

되었다. 대조군에는 생리식염수가 투여되었으며, 7마리의 SR-gerbil과

7마리의 SS-geribl이 대조군으로 사용되었다. 유전성 간질 모델은 항

전간제 투여 후에 뇌정위적 장치를 이용하여 Paired pulse response 측

정하였다. 면역형광염색을 시행하여 ERG K+ channels 발현 정도를 측

정하였고, 이미지로 저장하여 ERG K+ channels 발현 정도를 정량적으

로 분석하였다. 또한 Western blot 을 이용하여 ERG K+ channels 발현

정도를 정량적으로 분석하였다. SR-gerbil의 해마 부위에서 ERG 발현

은, 항전간제 투여 후에 principal neuron에서 감소한 소견을 보였으며,

정량적 분석상에서 대조군에 비하여 의미 있게 감소하였다 (P>0.05).

SS-gerbil의 해마부위에서도 ERG 발현은, 항전간제 투여 후에

principal neuron에서 감소한 소견을 보였으며, 정량적 분석상에서 대

조군에 비하여 의미 있게 감소하였다 (P<0.05). 실험대상이 된 SS-

gerbil은 항전간제 투여 후에 간질발작이 없었으며, paired pulse

response 검사상에서 population spike amplitude ratio가 의미 있게 감소

하였다 (P<0.05).

항전간제는 SR-gerbil과 SS-gerbil 모두에서, 해마 부위 principal

neuron의 ERG K+ channel발현을 감소시켰다. 새로운 항전간제 개발에

41

있어서 ERG K+ channel에 대한 영향을 알아보는 것이 필요하다고 사

료된다.

--------------------------------------------------

핵심 되는 말: : ERG1 채널, 칼륨, 항전간제, 간질, 면역 조직 화학,

유전성 간질 모델