The Pivotal Role of Nitric Oxide and Peroxynitrite Imbalance in ...

The Pivotal Role of Nitric Oxide and Peroxynitrite Imbalance in Epileptic Seizures

A dissertation presented to

the faculty of

the College of Arts and Sciences of Ohio University

In partial fulfillment

of the requirements for the degree

Doctor of Philosophy

Lu-Lin Jiang

August 2014

© 2014 Lu-Lin Jiang. All Rights Reserved.

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This dissertation titled


by

LU-LIN JIANG

has been approved for

the Department of Chemistry and Biochemistry

and the College of Arts and Sciences by

Tadeusz Malinski

Marvin & Ann Dilley White Distinguished Professor of Chemistry and Biochemistry

Robert Frank

Dean, College of Arts and Sciences

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ABSTRACT

JIANG, LU-LIN, Ph.D., August 2014, Chemistry


Director of Dissertation: Tadeusz Malinski

Epilepsy is one of the most severe neurological disorders. However, the detailed

molecular mechanism in triggering epileptic seizures is still unclear. Nitric oxide (NO) is

a versatile neurotransmitter in the brain; it acts as a messenger and antiplatelet

aggregation agent in the cerebral vasculature. Peroxynitrite (ONOO-), a cytotoxic

compound, can be easily produced by the diffusion-controlled reaction between NO and

superoxide anion (O2·-).

This study used a nanomedical approach to elucidate the role of NO and ONOO-

in epileptic seizures. The nanomedical approach involving a system of nanosensors

(diameter 200 300nm) has been used to measure directly in vivo release of NO and

ONOO- in the brains of Sprague-Dawley (SD) rats during the process of pilocarpine-

induced epileptic seizure events. Seizure events were simultaneously monitored by

electroencephalography (EEG). Pilocarpine stimulated both NO and ONOO- production

in the brain. The ratio of NO to ONOO- concentration ([NO]/ [ONOO-]) that reflected the

balance between NO and ONOO- shifted with time. Epileptic seizures were observed

only at the relatively low ratio of [NO]/ [ONOO-]. The latency, duration, and frequency

of seizure events have also been influenced by the balance between NO and ONOO-.

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DEDICATION

To my family,

To my beloved ones,

To my friends.

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ACKNOWLEDGMENTS

I would like to first thank my advisor, Dr. Tadeusz Malinski. He gave me this

great opportunity to work in his lab and a wonderful challenging project to work with.

Every time when I had problem, he was always there trying to help me, give me the ideas,

and lead me to think. His working attitude inspired me a lot. I really appreciate the great

support from him for all these years.

I also would like to thank my dissertation committee members, Dr. Marcia J.

Kieliszewski, Dr. Lauren E. H. McMills, and Dr. Tiao J. Chang. They helped me on my

research, and encouraged me when I have difficulties. Thank you very much for your

guidance.

I would like to thank Dr. Ruslan Kubant, Dr. Adam Jacoby, and Dr. Krystian

Jasinski for teaching me all the techniques in the lab and much discussion on my project

when I just started here. I would like to thank Dr. Jose Corbalan and Farina Mahmood for

all the help on animal protocols and animal surgeries. I also would like to thank Daniel

Nething and Michael Wagner for proof-reading my dissertation. And I would like to

thank all my labmates and many others in the Department of Chemistry and Biochemistry:

Dr. Han Wang, Yuanyuan Tang, Jiangzhou Hua, Elaine Saulinskas, Collin Arocho, Dr.

Salah Awad, Dr. Frazier Nyasulu, Dr. Barlag Rebecca, Paul Schmittauer, French Bascom,

Aaron Dillon, Rollie Merriman, Marlene Jenkins, Carolyn Khurshid, etc. I’m so proud to

work with you as a team.

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My special thanks to Paula Hale for all her support that helped me to conquer

many difficulties. Paula, you will be always in my heart. I will remember the kindness

and courage that you gave me.

Last but not least, I would like to thank all my family members for your great

support and encouragement. I would also like to thank my fiancé, Dening Ye. I’m so

thankful that I came here and met you. We’ve gone through a lot, and we will get through

more and more. Thank you for being with me. And I would like to thank all my friends.

I’ve learned a lot from you. Thank you for making me to be a better one.

I cannot finish this work without the help from all of you.

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TABLE OF CONTENTS

Page

Abstract ..................................................................................................................................... 3

Dedication ................................................................................................................................. 4

Acknowledgments .................................................................................................................... 5

List of Tables .......................................................................................................................... 11

List of Figures......................................................................................................................... 12

List of Abbreviations ............................................................................................................. 20

1. Introduction .................................................................................................................... 24

1.1 Epilepsy ........................................................................................................................ 24

1.1.1 The Classification of Epileptic Seizures ............................................................. 24

1.1.2 The Pathogenesis of Epileptic Seizures .............................................................. 27

1.2 Nitric Oxide.................................................................................................................. 30

1.2.1 The Physics and Chemistry of Nitric Oxide ....................................................... 30

1.2.2 The Biological Synthesis of NO .......................................................................... 31

1.2.3 The Biological Function of Nitric Oxide ............................................................ 34

1.3 Peroxynitrite (ONOO-) ................................................................................................ 37

1.4 Oxidative/ Nitroxidative Stress ................................................................................... 39

1.5 Epilepsy and Nitric Oxide ........................................................................................... 41

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1.6 Research Goals............................................................................................................. 45

1.6.1 Goal 1: To Understand the Role of NO and ONOO- in the Mechanism of

Epileptic Eeizures .......................................................................................................... 46

1.6.2 Goal 2: To Elucidate the Effect of [NO] / [ONOO-] Balance in Epileptic

Seizures ........................................................................................................................... 46

1.6.3 Goal 3: To Study an Environmental Effect of Different Molecules on NO/

ONOO- Balance in Epileptic Seizures .......................................................................... 46

1.6.4 Goal 4: To Propose the Potential Pharmacological Method of Intervention to

Minimize/ Eliminate Epileptic Seizures ....................................................................... 47

2. Materials and Methods .................................................................................................. 48

2.1 Nanosensor Fabrication ............................................................................................... 48

2.2 NO Nano-sensor Preparation ...................................................................................... 49

2.3 Preparation of NO Standard Solution ......................................................................... 50

2.4 NO Sensor Calibration ................................................................................................ 54

2.5 ONOO- Nano-sensors Preparation .............................................................................. 57

2.6 ONOO- Standard Solution Synthesis .......................................................................... 58

2.7 ONOO- Sensors Calibration ........................................................................................ 61

2.8 Animal Surgery ............................................................................................................ 64

2.9 Animal Models and Treatments .................................................................................. 66

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2.10 NO and ONOO- in vivo Measurement ..................................................................... 69

2.12 Protein Sample Preparation ....................................................................................... 72

2.13 Total Protein Concentration Measurement .............................................................. 72

2.14 Immunoblotting ......................................................................................................... 73

2.15 Statistical Analysis .................................................................................................... 77

3. Results............................................................................................................................. 79

3.1 NO and ONOO- Release in the Brain after PILO Induced Seizure. ......................... 79

3.2 Modulation of Seizure Events by Regulating the Release of NO and ONOO- ....... 89

3.2.1 L-Arg Treatment ................................................................................................... 90

3.2.2 L-NAME Treatment ............................................................................................. 95

3.2.3 MnTBAP Treatment ............................................................................................. 99

3.2.4 VAS2870 Treatment........................................................................................... 103

3.3 cNOS and NADPH Oxidase Protein Expression................................................. 106

3.3.1 eNOS Expression ................................................................................................ 107

3.3.2 nNOS Expression ............................................................................................... 108

3.3.3 NADPH Oxidase Expression ............................................................................. 109

3.4 Seizure Episodes Triggered by Artifact NO / ONOO- Ratio .................................. 111

3.4.1 SIN-1 Treatment ................................................................................................. 112

3.4.2 Synthetic NO and ONOO- Solution Treatment ................................................ 115

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3.4.3 O2·- Treatment ..................................................................................................... 119

4. Discussion..................................................................................................................... 124

4.1 The Establishment of Nanomedical System to Measure NO and ONOO - Release in

vivo during Epileptic Seizures ......................................................................................... 124

4.2 Abnormal NO and ONOO- Release in PILO Induced Epileptic Seizures. ............ 125

4.3 PILO-induced Epileptic Seizures Can be Regulated by the [NO]/ [ONOO-]

Modulators........................................................................................................................ 128

4.3.1 The Effect of NOS Modulators in PILO-induced Epileptic Seizures ............. 128

4.3.2 The Effect of ONOO- Scavenger in PILO-induced Epileptic Seizures .......... 130

4.3.3 The Effect of NADPH Oxidase Inhibitor in PILO-induced Epileptic Seizures

....................................................................................................................................... 131

4.4 Low [NO]/ [ONOO-] Can Trigger Seizures. ............................................................ 133

4.4.1 Low [NO]/ [ONOO-] Induced by SIN-1 Triggered Seizure-like Events ........ 133

4.4.2 Exogenous Low [NO]/ [ONOO-] Triggered Seizure-like Events ................... 134

4.4.3 Endogenous Low [NO]/ [ONOO-] Induced by O2·- Triggered Seizure-like

Events ........................................................................................................................... 135

4.5 Conclusion.................................................................................................................. 135

5. References .................................................................................................................... 137

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LIST OF TABLES

Page

Table 1.1 The effect of L-Arg and NOS inhibitors on epileptic seizures ........................... 43

Table 2.1 SDS-PAGE gel ..................................................................................................... 75

Table 2.2 Summerized primary antibody and secondary antibody information ................ 77

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LIST OF FIGURES

Page

Figure 1.1Classification of Epilepsy (adapted and modified from ILAE commission

report) 4 ................................................................................................................................... 26

Figure 1.2 Mutation of ion channels alters the ion channels function (adapted and

modified from Steinlein, O. K., Genetic mechanisms that underlie epilepsy. Nat Rev

Neurosci 2004, 5 (5), 400-8)7 ................................................................................................ 29

Figure 1.3 The Lewis structure of NO .................................................................................. 31

Figure 1.4 The biosynthesis of NO (adapted and modified from Freire, M. A.; Guimaraes,

J. S.; Leal, W. G.; Pereira, A., Pain modulation by nitric oxide in the spinal cord. Front

Neurosci 2009, 3 (2), 175-81) 26 ........................................................................................... 32

Figure 1.5 Structure of nitric oxide synthase (NOS) monomer .......................................... 33

Figure 1.6 The Biological Function of NO in Nerve System (adapted and modified from

Boehning, D.; Snyder, S. H., Novel neural modulators. Annu Rev Neurosci 2003, 26, 105-

31)38 ......................................................................................................................................... 37

Figure 1.7 The interplay of nitric oxide, superoxide anion, and peroxynitrite (adapted and

modified from Malinski, T., Nitric oxide and nitroxidative stress in Alzheimer's disease. J

Alzheimers Dis 2007, 11 (2), 207-18)21 ................................................................................ 39

Figure 2.1 Schematic diagram of NO nanosensor (adapted and modified from Malinski,

T.; Taha, Z., Nitric oxide release from a single cell measured in situ by a porphyrinic-

based microsensor. Nature 1992, 358 (6388), 676-8)19 ....................................................... 50

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Figure 2.2 Schematic diagram of set-up for NO synthesis (adapted and modified from

Malinski, T.; Huk, I., Measurement of nitric oxide in single cells and tissue using a

porphyrinic microsensor. Curr Protoc Neurosci 2001, Chapter 7, Unit7, 14) 111 ............. 52

Figure 2.3 An example of UV-Vis spectra of (a) oxyHb and (b) metHb solutions in PB

buffer ....................................................................................................................................... 54

Figure 2.4 Three-Electrode electrochemical system used for NO calibration ................... 55

Figure 2.5 (a) A typical NO response curve, (b) The calibration curve of NO standard

solution (R2=0.9586). ............................................................................................................. 56

Figure 2.6 Schematic diagram of ONOO- nanosensor (adapted and modified from

Malinski, T.; Taha, Z., Nitric oxide release from a single cell measured in situ by a

porphyrinic-based microsensor. Nature 1992, 358 (6388), 676-8 and Xue, J.; Ying, X.;

Chen, J.; Xian, Y.; Jin, L., Amperometric ultramicrosensors for peroxynitrite detection

and its application toward single myocardial cells. Anal Chem 2000, 72 (21), 5313-21)

19,113 ......................................................................................................................................... 58

Figure 2.7 Schematic diagram of set-up for ONOO- synthesis (adapted and modified from

Koppenol, W. H.; Kissner, R.; Beckman, J. S., Syntheses of peroxynitrite: to go with the

flow or on solid grounds? Methods Enzymol 1996, 269, 296-302 and Reed, J. W.; Ho, H.

H.; Jolly, W. L., Chemical Syntheses with a Quenched Flow Reactor -

Hydroxytrihydroborate and Peroxynitrite. Journal of the American Chemical Society

1974, 96 (4), 1248-1249) 114-115 ............................................................................................. 60

Figure 2.8 A UV-Vis spectrum of ONOO- (λmax= 302nm) solution in PB buffer ............. 61

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Figure 2.9 (a) A typical amperogram showing nanosensor response to ONOO- response

after ONOO- standard solution injection, (b) A calibration curve of ONOO- (R2=0.9911).

................................................................................................................................................. 63

Figure 2.10 The schematic diagram showing NO, ONOO- and EEG electrodes

localization in a brain. ............................................................................................................ 65

Figure 2.11 The flowchart for animal treatment .................................................................. 71

Figure 3.1 Typical amperograms showing real-time NO and ONOO- release during

normal brain activity in the SD rat brain .............................................................................. 80

Figure 3.2 Typical amperograms showing NO and ONOO- real-time release during

epileptic seizures induced by PILO (300 mg/kg, IP) in the SD rat brain. .......................... 81

Figure 3.3 (a) Typical amperograms showing NO and ONOO- real-time release during

epileptic seizures induced by PILO (1.6 mg/kg, IH) in the SD rat brain. (b) The high

resolution amperograms and EEG signals of NO and ONOO- release during one seizure

episode onset (the time frame between vertical red dot line in Figure 3.3 a)..................... 83

Figure 3.4 (a) Maximal NO and (b) ONOO- concentration produced in the brains of SD

rats in the absence or presence of epileptic seizures. Epileptic seizures were induced by

0.82 mg/kg; or 1.6 mg/kg PILO injected IH, or 300 mg/kg PILO injected IP (**p < 0.01,

***p< 0.0001 vs saline group). (c) The ratio of NO to ONOO- maximal concentration

measured in the brains of SD rats in the absence of or the presence of epileptic seizures.

Epileptic seizures were induced by 0.82 mg/kg, or 1.6 mg/kg PILO injected IH, or 300

mg/kg PILO injected IP (*p< 0.05, **p < 0.01, ***p< 0.0001 vs saline group). .............. 86

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Figure 3.5 Seizure events induced by PILO at the dosage of 0.82 mg/kg (solid bar), 1.6

mg/kg (gray bar) PILO through IH injection or PILO at the dosage of 300mg/kg (open

bar) through IP injection. (a) Latency from PILO injection to the first seizure event onset.

(b) Seizure duration (***p<0.0001 vs PILO IP group). (c) Number of seizure events per

hour (frequency of seizure events). ....................................................................................... 89

Figure 3.6 (a) Maximal NO and (b) ONOO- concentration produced in brains of epileptic

SD rats. Epileptic seizures were induced by IH injection of 1.6 mg/kg PILO in the

absence or presence of L-Arg (***p< 0.0001, vs saline group, ^^^p<0.0001 vs PILO

group). (c) The ratio of maximal NO to ONOO- concentration released in the brains of

epileptic SD rats. Epileptic seizures were induced by IH injection of 1.6 mg/kg PILO in

the absence or presence of L-Arg (***p< 0.0001 vs saline group). ................................... 92

Figure 3.7 Epileptic seizure events were observed in the brain of a SD rat induced by

PILO (1.6 mg/kg, IH) in the presence of L-Arg (5.7 μg/kg). .............................................. 93

Figure 3.8 Seizure events induced by PILO (1.6 mg/kg) through IH injection in the

absence (close bar) or presence (open bar) of L-Arg (5.7 μg/kg). (a)Latency from PILO

injection to the first seizure event onset. (b) Seizure duration (*p<0.05 vs PILO group).

(c) Number of seizure events per hour (frequency of seizure events). ............................... 94

Figure 3.9 (a) Maximal NO and (b) maximal ONOO- concentration produced in the

brains of epileptic SD rats. Epileptic seizures were induced by PILO (1.6 mg/kg, IH) in

the absence or presence of L-NAME (*p<0.05, ***p< 0.0001, vs saline group,

^^^p<0.0001 vs PILO group). (c) The ratio of maximal NO to ONOO- concentration

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released from the epileptic SD rats induced by PILO (1.6 mg/kg, IH) in the absence or

presence of L-NAME (***p< 0.0001 vs saline group, ^^^p<0.0001 vs PILO group). ..... 96

Figure 3.10 Epileptic seizure events were observed in the brain of SD rat induced by

PILO (1.6 mg/kg, IH) in the presence of L-NAME (7.3 μg/kg). ........................................ 97

Figure 3.11 Seizure events were induced by PILO (1.6 mg/kg, IH) in the absence (close

bar) or presence (open bar) of L-NAME (7.3 μg/kg). (a) Latency from PILO injection to

the first seizure event onset (*p<0.05 vs PILO group). (b) Seizure duration (*p<0.05 vs

PILO group). (c) Number of seizure events per hour. ......................................................... 98



the absence or presence of MnTBAP (***p< 0.0001 vs saline group, ^^^p<0.0001 vs

PILO group). (c) The ratio of maximal NO to ONOO- concentration released from

epileptic SD rats induced by PILO (1.6 mg/kg, IH) in the absence or presence of

MnTBAP (***p< 0.0001 vs saline group, ^^p< 0.01 vs PILO group). ............................ 100

Figure 3.13 Epileptic seizure in the brain of SD rat induced by PILO (1.6 mg/kg, IH) in

the presence of MnTBAP (64 μg/kg). ................................................................................. 101


bar) or presence (open bar) of MnTBAP. (a) Latency from PILO injection to the first

seizure event onset. (b) Seizure duration. (c) Number of seizure events per hour (*p< 0.05

vs PILO group). .................................................................................................................... 102



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the absence or presence of VAS2870 (***p< 0.0001 vs saline group, ^^^p<0.0001 vs

PILO group). (c) The ratio of maximal NO to ONOO- concentration released in the

epileptic brain of SD rats. Epileptic seizures were induced by PILO (1.6 mg/kg, IH) in the

absence or presence of VAS2870 (***p< 0.0001 vs saline group, ^^^p< 0.0001 vs PILO

group). ................................................................................................................................... 105

Figure 3.16 Epileptic seizure in the brain of a SD rat induced by 1.6 mg/kg PILO in the

presence of VAS2870 and 1.6 mg/kg PILO. ...................................................................... 106

Figure 3.17 eNOS expression in native form (dimer and monomer) and eNOS total

expression level. ................................................................................................................... 108

Figure 3.18 nNOS expression in native form (dimer and monomer) and nNOS total

expression level. ................................................................................................................... 109

Figure 3.19 NOX4 expression in the absence or presence of modulators after PILO-

induced epileptic sezures. .................................................................................................... 110

Figure 3.20 p67phox expression in the absence or presence of modulators after PILO-

induced epileptic sezures. .................................................................................................... 111

Figure 3.21 Example amprograms showing NO and ONOO - release measured with

nanosensors, and EEG showing brain activity change after SIN-1 (1.2 µg/kg, IH) injected

into the brain of a SD rat...................................................................................................... 113

Figure 3.22 Maximal NO (close bar) and ONOO- (open bar) concentration produced in

the brains of SD rats during the epileptic seizure-like events induced by 0.6 µg/kg and 1.2

µg/kg SIN-1 IH..................................................................................................................... 114

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Figure 3.23 The ratio of NO to ONOO- concentration released from SD rats during

seizure events induced by 0.6 µg/kg and 1.2 µg/kg SIN-1 (close bar) and induced by 1.6

mg/kg PILO (open bar) . ...................................................................................................... 114

Figure 3.24 The duration of seizure events induced by 0.6 µg/kg and 1.2 µg/kg SIN-1

(close bar) and induced by 1.6 mg/kg PILO (open bar). ................................................... 115

Figure 3.25 Seizure episodes triggered by the artifact change of the [NO] / [ONOO-].

Example of amperograms (a) NO and (b) ONOO- release after the exogenous injection of

NO and ONOO- solution (verticle dot lines represent the time when injection done). (c)

EEG recording shows seizure-like brain discharge (I, II, III) and non-seizure-like brain

discharge (IV) after each injection. ..................................................................................... 117

Figure 3.26 NO (open bars) and ONOO- (solid bars) concentration measured by

nanosensors during the time of seizure-like events. .......................................................... 118

Figure 3.27 The ratio of maximal NO to ONOO- concentration measured in the presence

or absence of seizure-like events. ........................................................................................ 118

Figure 3.28 Duration for seizure-like events after each injection. .................................... 119

Figure 3.29 Seizure-like episodes triggered by exogenous O2·-. Amperograms of (a) NO

and (b) ONOO- release after exogenous injection of O2·- solution (vertically dotted lines

indicate the time of injection, arrows indicate the time of seizure-like events). (c) EEG

recording shows two seizure-like brain discharges change (I and II) after injections of

O2·-. ........................................................................................................................................ 121

Figure 3.30 Maximal NO (open bars) and ONOO- (solid bars) concentration measured by

nanosensors at when seizure-like events were observed. .................................................. 122

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Figure 3.31 The ratio of maximal NO to ONOO- concentration measured at when seizure-

like events were observed. ................................................................................................... 122

Figure 3.32 Duration for seizure-like events after O2∙- (0.52 µg, IH) injection. .............. 123

Figure 4.1 Schematic diagram of the role of NO and ONOO- imbalance in PILO induced

epileptic seizures. ................................................................................................................. 127

Figure 4.2 Schematic diagram showing the mechanism of the prevention of epileptic

seizures by VAS 2870 (NADPH oxidase inhibitor) .......................................................... 133

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LIST OF ABBREVIATIONS

Ach acetylcholine

ALS amyotrophic lateral sclerosis

APS ammonium persulfate

BCA bicinchoninic acid

BH4 tetrahydrobiopterin

BSA bovine serum albumin

CaM calmodulin

cGMP cyclic guanosine-3’,5’-monophosphate

CLC voltage-gated chloride channel

cNOS constitutive nitric oxide synthase

C-terminal carboxyl terminal

CV cyclic voltammetry

DMSO dimethyl sulfoxide

EDRF endothelium-derived relaxing factor

EEG electroencephalogram

eNOS or NOS III endothelial nitric oxide synthase

FAD flavin adenine dinucleotide

FMN flavin mononucleotide

GABA γ-aminobutyric acid

GABAA subtype A of γ-aminobutyric acid receptor

GOF gain of function

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GSH glutathione

GSNO S-nitrosoglutathione

Hb hemoglobin

HPLC high-performance liquid chromatography

IBE International Bureau for Epilepsy

IH interhippocampus(ly)

ILAE International League Against Epilepsy

iNOS or NOS II inducible nitric oxide synthase

IP intraperitoneal(ly)

KCNQ voltage-gated potassium channel

L-Arg L-arginine

L-NAME L-NG- nitroarginine methyl ester

L-NMMA L-NG-monomethylarginine

L-NNA L-nitroarginine

LOF loss of function

LPS lipopolysaccharide

MES maximal electroshock

metHb methemoglobin

Mn porphrin manganese (III)- [2, 2] paracyclophenyl-porphyrin

MnTBAP manganese (III) tetrakis (4-benzoic acid) porphyrin chloride

MPT mitochondrial permeability transition

mtDNA mitochondrial DNA

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nAChR neuronal nicotinic acetylcholine receptor

NADPH nicotinamide adenine dinucleotide phosphate

Ni porphyrin nickel (II) tetrakis (3-methoxy-4-hydroxyphenyl) porphyrin

NMDA N-methyl-D-aspartate

nNOS or NOS I neuronal nitric oxide synthase

NOS nitric oxide synthase

N-terminal amino terminal

oxyHb oxyhemoglobin

PB phosphate buffer

PILO pilocarpine

pKa logarithmic acid dissociation constant

PSD95 postsynaptic density protein

PTZ pentylenetetrazol

PVDF polyvinyl difluoride

PVP poly (4-vinylpyridine)

RIPA radioimmunoprecipitation

RNS reactive nitrogen species

ROS reactive oxygen species

SCN voltage-gated sodium channel

SD Sprague-Dawley

SDS-PAGE sodium dodecyl sulfate polyacrylamide

SEM standard error of the mean

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sGC soluble guanylate cyclase

SIN-1 3-Morpholinosydnonimine

SOD superoxide dismutase

TBAP tetrabutylammonium perchlorate

TBI traumatic brain injury

TBS tris-buffered saline

TBST TBS solution with 0.1 % tween-20

TEMED tetramethylethylenediamine

TG tris/glycine

TGS tris/glycine/SDS solution

TLE temporal lobe epilepsy

VAS 2870 1,3-benzoxazol-2-yl-3-benzyl-3H-[1,2,3] triazolo [4,5-d]-

pyrimidin-7-yl sulfide

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1. INTRODUCTION

1.1 Epilepsy

Epilepsy is one of the most severe health problems around the world. There are

about 65 million people worldwide1 and about 3 million people in the United States2

suffering from epileptic seizures. About 200,000 new cases of epileptic seizures occur

every year2.

The word epilepsy comes from Ancient Greek, which means “to seize”. The

definition for “epilepsy” proposed by the International League Against Epilepsy (ILAE)

and the International Bureau for Epilepsy (IBE) is “a disorder of the brain characterized

by an enduring predisposition to generate epileptic seizures and by the neurobiologic,

cognitive, psychological, and social consequences of this condition”3. A diagnosis of

epilepsy requires a history of at least one seizure, and evidence of enduring alteration to

the brain that increases the possibility of future seizures, as well as other associated

symptoms. These symptoms can be neurobiological, cognitive, psychological, and

social3. Therefore, epilepsy describes a family of neurological disorders that

predisposition to seizures is increased abnormally, and is accompanied by cognitive

impairment and behavior problems3.

1.1.1 The Classification of Epileptic Seizures

Based on the ILAE and IBE report, the definition of an epileptic seizure is “a

transient occurrence of signs and/or symptoms due to abnormal excessive or synchronous

neuronal activity in the brain”3. Mode of onset and termination, clinical manifestations,

and abnormal enhanced synchrony are essential to define an epileptic seizure3. Thus, the

25

ILAE has proposed the classification (Figure 1.1) of epileptic seizures based on the

clinical symptoms and electroencephalogram (EEG).

The two main classes of seizures are generalized seizures and focal seizures.

Generalized seizures are seizures originating within bilaterally distributed networks and

rapidly spread to the whole hemispheres. The bilateral networks may include both

cortical and subcortical structures. Individual generalized seizures may differ from case

to case because the location and lateralization may not be consistent between seizures4.

Focal epileptic seizures may originate in subcortical structures. The onset of a focal

seizure is more likely to be consistent in all subtypes4.

Any seizures not falling under generalized or focal are in a third “unknown”

classification, due to lack of knowledge about the seizure type. This mainly covers

epileptic spasms. Epileptic spasms, like infantile spasms, may happen during infancy.

More work is needed to be done to decide the classification of this type of epileptic

seizure.

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Figure 1.1Classification of Epilepsy (adapted and modified from ILAE commission report) 4

Epileptic seizures

Generalized seizures

Tonic-clonic

Absense

Typical

Atypical

Absence with special features

Myoclonic absence

Eyelid myoclonia

Myoclonic

Myoclonic

Myoclonic atonic

Myoclonic tonic

Clonic

Tonic

Atonic

Focal seizures

Unknown Epileptic spasms

27

1.1.2 The Pathogenesis of Epileptic Seizures

Epilepsy is a dynamic disease, and progresses with time5. Recurrent epileptic

seizures can cause apoptosis and necrosis of neurons in the brain6. Neuronal networks can

be permanently altered7, especially after status epilepticus, a neurologic emergency

caused when the brain is in a state of prolonged seizures. This process may affect

emotional, cognitive and behavioral characteristics and even cause death8.

Many factors may cause epilepsy, such as genetic mutation, developmental

conditions, tumors, head trauma and central nervous system infection6. Based on the

etiology, underlying causes of epilepsy can be classified as genetic,

“structural/metabolic”, and “unknown cause”. The best understood form is genetic

epilepsy, in which seizures are “the core symptom of the disorder”3. More than 200 single

gene disorders have been identified in association with epilepsy7, which include genes

that encode ion channels or their subunits. Mutations in any of the following may be

associated with genetic epilepsy (Figure 1.2)7: neuronal nicotinic acetylcholine receptor

(nAChR); subtype A of γ-aminobutyric acid receptor, (GABAA,); voltage-gated sodium

channel (SCN); voltage-gated potassium channel (KCNQ); voltage-gated chloride

channel (CLC) . Since neuronal communication and signal transduction are based on the

action of various ion channels, mutant channels may alter ion permeability of the

membrane, change excitability of the membrane, and regulate neurotransmitter release.

When the balance between excitatory and inhibitory synaptic input of the brain is

disrupted, uncontrolled hyperexcitability accompanys, which part of the brain may

receive excessive excitatory synaptic input9. As a result, seizures may occur.

28

“Structural/metabolic” epilepsy may be caused by structural lesions such as those caused

by stroke, trauma and infection4; these are collectively known as acquired epilepsy. It has

been reported that approximately 60% of all cases of epilepsy are acquired epilepsy10.

Status epilepticus, stroke and traumatic brain injury (TBI) are the common brain lesions

which may lead to the development of acquired epilepsy11. Studies have shown a

common molecular mechanism that induces brain damage after these brain lesions. Under

the pathogenic conditions, the increase in extracellular glutamate concentration

associated with calcium ion (Ca2+) overflow into neurons resulted in neuronal death and

brain damage, which is also known as excitotoxicity12. Conditions like these can

gradually elevate the risk of epilepsy onset. Forms of epilepsy with “Unknown cause”

may have a genetic defect or the consequence of a disorder that has not been recognized

yet4.

Epilepsy is one of the oldest recognized disorders, which was first described by

Hippocrates in the 5th century BC13. Much effort has been contributed to investigate new

types of antiepileptic drugs or antiepileptic treatments to decrease the frequency and

severity of seizures in people with epilepsy since potassium bromide was introduced as

the first anti-seizure drug in 185714. Unfortunately, about one third of epileptic patients

are not responsive to the current medications14-15. It is urgent to find innovative direction

for more effective and better tolerated treatments that can prevent, stop, or reverse the

development of epilepsy13-15.

29

Normal

nAChR GABAA SCN KCNQ CLC

K+; Ca2+ ; Na+

Cl- Na+

K+ Cl-

Mutant

GOF LOF LOF LOF GOF

K+; Ca2+ ; Na+

Cl- Na+

K+ Cl-Type of Mutation:

Figure 1.2 Mutation of ion channels alters the ion channels function (adapted and

modified from Steinlein, O. K., Genetic mechanisms that underlie epilepsy. Nat Rev

Neurosci 2004, 5 (5), 400-8)7

GOF, Gain of function; LOF, Loss of Function; nAChR, neuronal nicotinic acetylcholine

receptor; GABAA, subtype A of γ-aminobutyric acid receptor; SCN, voltage-gated

sodium channel; KCNQ, voltage-gated potassium channel; CLC, voltage-gated chloride

channel.

30

1.2 Nitric Oxide

1.2.1 The Physics and Chemistry of Nitric Oxide

Nitric oxide (NO) is one of the simplest biologically active molecules. Despite its

simplicity, this molecule has very complicated biosynthesis and regulation systems inside

the biological systems16. The rate of NO production, distance of diffusion, rate of

consumption, and local microenvironment all contribute to the role that NO plays in

physiological and pathological responses17.

NO is a colorless gas at room temperature, but it can react rapidly and

spontaneously with oxygen (O2) to form nitrogen dioxide (NO2), a brown gas, the main

component of air pollution18. The solubility of NO in water at room temperature is low,

about 2mM17. NO is a lipophilic molecule, so it is more concentrated in hydrophobic

regions (such as membranes) rather than aqueous regions19. This allows NO to easily

diffuse through cell membranes and act as a signaling molecule over distances in the

body20.

The Lewis structure (Figure 1.3) of NO reveals that NO has one unpaired

electron. NO can react quickly with other radicals, such as ·NO2, O2, and superoxide

anion (O2·-)21, to terminate radical chain reactions20. Therefore, NO is considered as an

antioxidant. NO can also serve as a ligand in several metal complexes17. One of the

important metal complexes that NO binds strongly to is the ferrous ion (Fe2+) – heme

complex. The binding between NO and Fe2+–heme groups allows conformation changes

in the enzyme that initializes activation17. And the high-affinity of NO for Fe2+ – heme

groups allows NO targets to be in other cells or even different tissue20. The Fe2+ – heme

31

containing protein includes soluble guanylate cyclase (sGC), cytochrome P-450, nitric

oxide synthase (NOS), and hemoglobin17, 20. The reactions between NO and Fe2+ – heme

containing protein are of biological significance. For example, the NO/sGC/ cyclic

guanosine-3’,5’-monophosphate (cGMP) pathway has been found to be vitally important

in preventing platelet aggregation and inhibition of platelet adhesion to endothelium

cells22. Additionally, oxidation of NO can yield electrophilic nitrosating species, such as

dinitrogen trioxide (N2O3), which can nitrosate thiols to form S-nitrosothiol compounds

(RS-NO)17. This process has been thought to play an important role in regulating both the

oxidative/nitroxidative stress phenomenon and protein misfolding23.

Figure 1.3 The Lewis structure of NO

1.2.2 The Biological Synthesis of NO

In the biological system, NO is produced by a five-electron oxidation of L-

arginine (L-Arg) to L-citrulline. This oxidation reaction involves O2 and is catalyzed by

nitric oxide synthase (NOS) (Figure 1.4). There are three types of NOS found in

biological systems: neuronal NOS (nNOS or NOS I); inducible NOS (iNOS or NOS II);

and endothelial NOS (eNOS or NOS III). NOSs have been named according to the order

and primary localization that they have been isolated and purified. These three isozymes

32

share 5 60 % amino acid sequence identity, retaining almost identical domain structure

and catalytic mechanisms24.

In NOS, the amino terminal (N-terminal) is an oxygenase domain and the

carboxyl terminal (C-terminal) is a reductase domain. There is a binding site for heme on

the oxygenase domain. Flavin mononucleotide (FMN), flavin adenine dinucleotide

(FAD), and nicotinamide adenine dinucleotide phosphate (NADPH) can bind to the

reductase domain of NOS. In between the oxygenase and reductase domains, there is a

binding site for calmodulin (CaM) (Figure 1.5).

Gel filtration experiments reveal that the molecular mass for the nNOS monomer

is 161 kDa, while the iNOS monomer is 131 kDa, and the eNOS monomer is 133 kDa17.

The catalytically active form of each enzyme is a homodimer, so functional NOS units

should be twice as large. This dimerization involves a cofactor called tetrahydrobiopterin

(BH4), and its binding is essential for NOS dimerization25. There are also additional

differences among the three enzymes besides molecular weight.

Figure 1.4 The biosynthesis of NO (adapted and modified from Freire, M. A.; Guimaraes,

J. S.; Leal, W. G.; Pereira, A., Pain modulation by nitric oxide in the spinal cord. Front

Neurosci 2009, 3 (2), 175-81) 26

33

Figure 1.5 Structure of nitric oxide synthase (NOS) monomer

nNOS was the first NOS to be purified from rat cerebellum27. This work was done

by Drs. Bredt and Snyder. The majority of nNOS has been found in the central and

peripheral nerve system27. eNOS was the third form to be purified, and has been mainly

found in endothelial cells and pyramidal cells in the hippocampus of the brain. The

expression of nNOS and eNOS are constitutive and low-output, and both are Ca2+ -

dependent enzymes. The Ca2+ CaM complex is essentially required for nNOS and eNOS

activation. With elevated Ca2+ in neurons or endothelium, Ca2+ binds to CaM and

changes the binding conformation between CaM and nNOS or eNOS, which allows

NADPH derived electrons to pass onto the catalytic heme, and leads to NO production

over several minutes28.

However, iNOS is different. Because of the strong binding between iNOS and

CaM, Ca2+ is not required for iNOS activation and iNOS activity is insensitive to the

cellular Ca2+ concentration. iNOS has a much higher output than the other two NOS

enzymes. The expression of iNOS can be induced under inflammatory conditions to

release NO for days, and can be triggered by bacterial lipopolysaccharide (LPS) and

cytokines 16, 28.

34

In addition to the component mentioned above, NOSs are subject to many

modifications that affect their function. Certain posttranslational modifications, such as

phosphorylation, are required for NOS activity16, 29. eNOS is also myristoylated, which

anchors eNOS to the membrane. Acylation, palmitoylation, and Golgi localization are

required for eNOS function and subcellular distribution17. Moreover, the presence of a

zinc-sulfur (Zn-S) cluster can help to stabilize the binding between BH4 and NOS

dimer30.

1.2.3 The Biological Function of Nitric Oxide

Nitrocompounds have been used to clinically treat angina pectoris for over 100

years without knowing the mechanisms involved17. Later, researchers found that

regulation of cGMP concentration in mammalian tissues can cause vascular smooth

muscle relaxation and increase in blood flow31. The link between nitrocompounds and

cGMP was the next connection. Drs. Furchgott and Zawadzki did pioneering work in

noticing an endothelium-derived relaxing factor (EDRF) that could be produced by

acetylcholine-treated endothelial cells to relax blood vessels through activating guanylate

cyclase32. In 1986, Drs. Furchgott33, Ignarro34, and Moncada35 proposed that NO was the

EDRF. In 1992, Dr. Malinski used a porphyrin-based microsensor to measure NO release

in situ, which was the first time NO has been measured from biological milieu and

provided the final proof that NO was EDRF19.

NO has been found to regulate several biological systems. The circulatory system

aspects of NO were the first to be discovered. NO can inhibit platelet aggregation,

regulate blood pressure, prevent platelet adhesion to endothelial cells, and relax smooth

35

muscle cells in the cardiovascular systems22. NO is also a mediator in the immune system

that responds to cytokine stimulation16, 28.

In the nervous system, NO serves as a neurological messenger involved in

excitatory signaling transduction pathway36-37. This gas molecule is different from other

biogenic transmitters, such as glutamate and γ-aminobutyric acid (GABA). NO does not

require synaptic vehicles for storage38. NO is hydrophobic, and it can easily diffuse

through the cell lipid bilayers and affect adjacent cells. All three NOS isoforms exist in

the brain, but the predominant source of NO is provided by nNOS in neurons23. The PDZ

binding domain on the C-terminal of nNOS helps it to bind to the N-methyl-D-aspartate

(NMDA) receptor through postsynaptic density protein PSD95. NMDA receptor is a

predominant ion channel that is involved in synaptic plastics, learning, and memory.

NMDA receptors are highly permeable to Ca2+. Excitatory neurotransmitters, like

glutamate, bind to NMDA receptor and activate the receptor, which causes the receptor

open. Extracellular Ca2+ enters into the cytosol, and Ca2+ – CaM complex is formed to

activate nNOS to produce NO release27, 36 (Figure 1.6).

NO participates in signal transduction pathways by binding to sGC. With NO

binding to the heme-containing transition metal (Fe2+) center, the structural conformation

of sGC changes to stimulate cGMP production. cGMP is found to mediate NO-dependent

vasodilation and neuronal plasticity38-39. This signal transduction pathway is essential for

synaptic development, neuronal plasticity, learning, and memory.

NO can also react with the sulfhydryl groups on proteins to form nitrosothiol

compounds (SNOs). This post-translational modification is known as S-nitrosylation.

36

Research suggests this modification may have a similar function to phosphorylation in

signal transduction processes40-44. The Lipton group was the first to find that excessive

activity of the NMDA receptor can be downregulated by S-nitrosylation on cysteine

residue (Cys399) in the NR2A subunit of NMDA receptor45, in which glutamate induced

excitotoxity can be inhibited; NO yields a neuroprotective effect. Moreover, NO can also

S-nitrosylate caspases, which can inhibit the protease activity of caspases, and prevent

neuronal cell apoptotic death46. S-nitrosoglutathione (GSNO), which is the product from

reduced glutathione (GSH) S-nitrosylation, has been found to have 100 times more

effective antioxidant ability than GSH in suppressing iron-induced generation of

hydroxyl radical (·OH) 47, the major toxin produced in vivo20.

In addition, NO produced by eNOS from the brain serves as a modulator in

neurogenic vasodilation48.

37

CaM nNOS

PSD95

NMDA receptor

Ca2+

Glutamate

NO

L-Arginine L-Citrulline

sGC GTP

cGMP

Presynaptic Neuron

Postsynaptic Neuron

Figure 1.6 The Biological Function of NO in Nerve System (adapted and modified from

Boehning, D.; Snyder, S. H., Novel neural modulators. Annu Rev Neurosci 2003, 26, 105-

31)38

1.3 Peroxynitrite (ONOO-)

At the time when EDRF was discovered, it was also found that addition of

superoxide dismutase (SOD) could extend the half-life of EDRF49. Since SOD can

diminish endogenous superoxide anion (O2·-) production, O2

·- was involved in the

breakdown of EDRF. The protective effect from EDRF was considered to scavenge O2·-,

which was also one of the criterions of EDRF20. After NO was identified as the EDRF,

38

peroxynitrite (ONOO-), the product from the reaction between NO and O2·-, was also

noticed to yield many cytotoxic effects50-51.

Peroxynitrite (ONOO-) is produced by reaction between NO and O2·-, which is

controlled by diffusion (average rate at 6.7 ± 0.9×109 M-1s-1 )52 (Figure 1.7). This anion

has a short half-life, less than 1 second. The logarithmic acid dissociation constant (pKa)

of ONOO- is 6.8. Thus, there are two forms, peroxynitrite anion (ONOO-) and

peroxynitrous acid (ONOOH) under physiological conditions (pH 7.4) 50. ONOO-/

ONOOH are strong oxidants that can directly react with biomolecules, such as

metalloproteins and protein thiols. ONOO- can rapidly react with carbon dioxide (CO2) to

form ·NO2 and a carbonate radical (CO3·-). ONOOH, the protonated form of ONOO-, can

readily produce ·OH and ·NO2 through homolytic cleavage, hydroxide ion (OH-) and

nitronium ion (NO2+) through heterolytic cleavage, and hydrogen ion (H+) and nitrate ion

(NO3-) by isomerization (Figure 1.7). The radical products from ONOO-, such as ·NO2;

CO3·- and ·OH, are more potent oxidants than ONOO - itself, which contributes to the

oxidative/nitroxidative stress on the system21.

39

NO + O2·-

ONOO-

ONOOH HO· + · NO2

ONOOCO2- CO3

·- + · NO2CO2

H+

HO- + NO2+

H+ + NO3-

Homolytic cleavageHeterolytic cleavage

Isomerization

Figure 1.7 The interplay of nitric oxide, superoxide anion, and peroxynitrite (adapted and

modified from Malinski, T., Nitric oxide and nitroxidative stress in Alzheimer's disease. J

Alzheimers Dis 2007, 11 (2), 207-18)21

1.4 Oxidative/ Nitroxidative Stress

Reactive oxygen species (ROS), such as O2·-, ·OH, and hydrogen peroxide

(H2O2), are important molecules that are involved in both normal cell function and the

development of disease53. Oxidative stress describes the imbalance between ROS and

antioxidants in the biological system. Overproduction of ROS, especially the formation

of O2·- , has been found in many diseases, such as Parkinson’s54, Alzheimer’s disease55,

amyotrophic lateral sclerosis (ALS)56, and a majority of cardiovascular diseases57, like

atherosclerosis, heart failure and hypertension. O2·- itself can serve as an oxidant (by

accepting an electron) or as a reducing agent (by donating an electron). In fact, O2·- acts

as a mild reductant under physiological conditions, and becomes a strong oxidant when it

binds to proteins20. In biological systems, O2·- is the precursor to produce other ROS.

40

Since O2·- cannot freely diffuse across the cells like NO and has to permeate through

anion channels58, the function of O2·- is limited to the location where it was produced.

O2·- is primarily produced in mitochondria as a byproduct of the mitochondrial respiration

chain. Under physiological conditions, SOD inside the mitochondria can scavenge O2·- to

detoxify the cells. If mitochondria become dysfunctional, or other oxidases (such as

NADPH oxidase or xanthine oxidase) are activated under certain conditions, O2·- and

ROS are overexpressed59. This imbalance between active oxidants and endogenous

antioxidants induces oxidative stress.

The non-enzymatic interaction between NO and O2·- is so rapid that it can

outcompete O2·- scavenger action from SOD20. As a result, when cellular oxidative stress

increases, excessive O2·- can react with NO to synthesize ONOO-. ONOO- is a more

powerful oxidant species than O2·- and NO. It was first found to trigger cell death by

necrosis60. Later, more studies revealed ONOO- is also involved in cell apoptosis by

inducing mitochondrial permeability transition (MPT)20, which is the prominent feature

of ONOO- mediated cell apoptosis. The efflux of proapoptotic signaling molecules from

mitochondria to the cytosol can induce cell death. This occurs through permeability

transition pores during MPT61. ONOO- can also react directly with electron-rich groups,

such as sulfhydryls, iron-sulfur clusters, zinc-thiolates, and the active sulfhydryl groups

in tyrosine phosphatases.

ONOO- has a number of downstream impacts once present in the cell. First,

ONOO- can readily oxidize transition metals, such as iron, zinc, and copper. Those metals

are usually cofactors in certain enzymes, like copper, zinc, and manganese based SODs.

41

The oxidation reaction between ONOO- and SOD would deactivate the enzyme, which

can cause cells to lose the ability to diminish ROS damage62-63. Second, ONOO- can also

react with eNOS, which triggers eNOS uncoupling and switches eNOS from NO

producing enzyme to synthesize O2·-64. Third, ONOO- can react with lipid to cause lipid

peroxidation, which can alter membrane bilayers integrity to trigger cell apoptosis62.

Fourth, ONOO- can cause mitochondrial dysfunction by reacting with mitochondrial

cytochrome oxidase (Complex I and Complex III)65. Finally, ONOO- can react with

purine nucleotides in DNA, which causes DNA strands to break66.

The homolytic and heterolytic cleavage products from ONOO-/ ONOOH, such as

·NO2 and NO2+; are more potent oxidants than ONOO- itself21. Collectively, these

compounds contribute to the nitroxidative stress of the biological system. Oxidative/

nitroxidative stress together shift the redox state of the biological milieu. The resulting

overproduction of ROS and reactive nitrogen species (RNS) has been found to play a

vital role in vascular disease67, neurodegenerative disease21, and aging68.

1.5 Epilepsy and Nitric Oxide

Excitotoxicity is a term used to describe the neuronal damage caused by excessive

activation of glutamate receptors, including the NMDA receptor69-70. Neurological

damage caused by epileptic seizures has been strongly linked to excessive excitatory

amino acid neurotransmitters69, 71-73. The release of NO is also thought to be the result of

NMDA receptor activation27. When excitatory amino acids and neurotransmitters, such as

glutamate, bind to NMDA receptors, Ca2+ influx is stimulated. Then, the Ca2+ -sensitive

nNOS and eNOS are activated to release NO74-76.

42

V. Mollace et.al77 published the first paper about the role of NO in epilepsy in

1991. They injected L-Arg prior to NMDA injection into rat brains, and found the

convulsant effect from NMDA had been significantly potentiated. L-Arg can lower the

threshold of epileptic seizure events induced by NMDA. Thus, L-Arg serves as a

proconvulsant agent77.

Later, many scientists studied the effect of NO in seizures, NO precursors, NO

donors, and NOS inhibitors on living organisms. The results turned out to be

controversial. Table 1 summarized the studies on regulation of NO levels in different

epileptic models. Pentylenetetrazol (PTZ), a GABA antagonist, and maximal

electroshock (MES) therapies have been used to induce generalized seizures in rodents.

Pilocarpine (acetylcholine agonist), kainic acid (glutamate agonist), and NMDA

(glutamate agonist) have been used to induce temporal lobe epileptic seizure in rodents.

The elevation of NO levels showed proepileptic, antiepileptic or even no effect

depending on models.

Due to the short half-life (3 5 s), no direct methods were used to measure in situ

NO release in epileptic brain. NO production was estimated through NOS expression

measured by NADPH-diaphorase histology methods78-79. Other methods measured the

concentration of L-Arg, L-citrulline and cGMP by microdialysis through high-

performance liquid chromatography (HPLC) or by radiological tracking of the activity of

NOS80. NO metabolites, NO2- and NO3

- (NOx), were also measured as indirect

quantifications of NO production during the seizures81. Moreover, fewer interests have

43

been addressed on ONOO- production in epileptic conditions due to lack of direct

methods to measure ONOO-.

Table 1.1 The effect of L-Arg and NOS inhibitors on epileptic seizures Epileptic

animal models

PTZ Pilocarpine Kainic

acid

NMDA MES

L-Arg -82 -83 +77

L-NAME

(NOS

inhibitor)

-84-85

+86

+83, 87-88

-89

+90

-77

No effect91

L-NNA

(NOS

inhibitor)

No

effect 92

+86, 93

+74, 93 +74, 94 No effect92

L-NMMA

(NOS

inhibitor)

-85 +83

7-nitroindazole

(nNOS

inhibitor)

-95 +87 +90

“+” sign indicates the severity of seizure events has been increased.

“-” sign indicates the severity of seizure events has been decreased.

L-NAME: L-NG- nitroarginine methyl ester

L-NNA: L-nitroarginine

L-NMMA: L -NG-monomethylarginine

44

Since there was no direct evidence to show NO production during seizures, the

role of NO in epilepsy can be argued. The effect of NO may result from drug interactions

and pharmokinetics92, 96-97. NOS inhibitors themselves may react with other chemicals

that induce epilepsy in animals. This means that the anticonvulsant or proconvulsant

effects from NOS inhibitor can be NO independent. The route of drug delivery (systemic

versus central), and animal species were also considered to contribute to differing

results81, 96.

Several studies also suggested oxidative stress was involved in the development

of epileptic seizures. After prolonged epileptic seizures, ROS production increased 98, 99-

100. Oxidative damage was observed as a decrease of mitochondrial DNA (mtDNA) copy

number, deactivation of Complex I and Complex IV of respiration chain101, and lipid

peroxidation102.

With the presence of excessive O2·-, it is very likely that NO could react with O2

·-

to form ONOO-. However, fewer studies were conducted to investigate the effect of

ONOO- in epilepsy. The indirect measurement methods of NO mentioned above cannot

distinguish between the effects of NO and ONOO- in seizures. This may explain a

controversial effect of NO. ue to the short half-life (3 5 s) and rapid-diffusion of NO,

and also extremely short life (< 1.0 s) of ONOO-, no direct in situ measurements of NO

and ONOO- concentration in epileptic brains have been reported. This study is the first

one to apply nanomedical approach on direct measurement of NO and ONOO - in

epileptic brain.

45

1.6 Research Goals

Temporal lobe epilepsy (TLE) is a form of focal seizure which often happens in

adults, and is the most frequent form of acquired epilepsy in humans. An initial injury,

such as an episode of prolonged seizures or status epilepticus, hypoxia, or trauma may

precede the onset of the disease103. Head trauma and stroke are thought to be the cause of

a majority of TLE cases 104. The onset area of TLE is believed to be in the

hippocampus105, which plays an important role in learning and memory. A unique pattern

of hippocampal damage is found in most TLE patients 104. Continuous abnormal

hippocampal discharge causes significant cerebral damage, resulting in long-term

behavioral changes and cognitive decline 106-107. This type of epilepsy is poorly controlled

with current anticonvulsant drugs. Severe TLE patients may need surgical removal of

lesion foci.

Most epileptic studies are focused on the regulation of ion channels and receptors

to alter net excitability in the brain. Antiepileptic drugs were also designed to modulate

the function of ion channels and receptors to restore the ion balance. However, seizures

are not quite controlled in a third of all affected individuals14. A new insight in epilepsy

research has been proposed to emphasize the prevention of chronic epilepsy development

rather than controlling seizures per se with antiepileptic drugs14, 103. In order to achieve

the goal, the mechanism in the development of epileptic seizures should be addressed and

new biomarker is also needed for prediction of the disease108.

In the study presented here, pilocarpine (PILO), a muscarinic receptor agonist, has

been used to induce epileptic seizure events in Sprague-Dawey (SD) rats. The usage of

46

PILO in rodents has been found to develop similar epileptic seizure characteristics when

compared to temporal lobe epilepsy in humans. This includes neuronal loss and aberrant

mossy fibers sprouting in hippocampus. Electroencephalography (EEG) was used to

monitor rat brain activity for the whole study.

1.6.1 Goal 1: To Understand the Role of NO and ONOO- in the Mechanism of

Epileptic Eeizures

It is crucially important to know about minute level of NO and ONOO- during the

epileptic seizure events. Therefore, a nanomedical system has been prepared to measure

NO and ONOO- directly in the brain in this study. Two types of nanoscale porphyrin-

based electrochemical nanosensors (diameter 200 300 nm) were used to monitor NO and

ONOO- release in vivo, which helped us to better understand the role of NO and ONOO-

in epileptic seizures.

1.6.2 Goal 2: To Elucidate the Effect of [NO] / [ONOO-] Balance in Epileptic

Seizures

Since the function of NO can be altered by the rate of NO production, distance of

diffusion, rate of consumption, and redox cellular states, a ratio of NO concentration to

ONOO- concentration ([NO]/ [ONOO-] ) was used as a criterion to evaluate the

relationship between cytoprotective NO and cytotoxic ONOO- in epileptic seizures.

1.6.3 Goal 3: To Study an Environmental Effect of Different Molecules on NO/

ONOO- Balance in Epileptic Seizures

Nitric oxide synthase (NOS) substrate, L-Arg; nonselective NOS inhibitor, L-

NAME; NADPH oxidase inhibitor, 1,3-Benzoxazol-2-yl-3-benzyl-3H-

47

[1,2,3]triazolo[4,5-d]pyrimidin-7-yl sulfide (VAS 2870); cell-permeable superoxide

dismutase (SOD) mimetic and ONOO- scavenger, Mn(III)tetrakis(4-benzoic

acid)porphyrin Chloride (MnTBAP); ONOO- donor, 3-Morpholinosydnonimine (SIN-1);

synthetic solution of NO, ONOO- , and O2·- have been administered into the brain to

study the change of NO and ONOO- concentrations and their influence on epileptic

seizures. [NO]/ [ONOO-] release was directly correlated with the severity and intensity of

epileptic seizures.

1.6.4 Goal 4: To Propose the Potential Pharmacological Method of Intervention to

Minimize/ Eliminate Epileptic Seizures

Current anticonvulsant medications have saved the lives of many epileptic people,

but the medications target the suppression of seizure severity. In this study, the pivotal

role of NO, ONOO-, and their imbalance in epileptic seizures was addressed. A new

pharmacological intervention by increasing/restoring a balance between NO and ONOO-

and reducing an effectiveness of the oxidative stress was proposed to provide a pathway

for mollification and treatment of epileptic seizures.

48

2. MATERIALS AND METHODS

2.1 Nanosensor Fabrication

Real-time NO and ONOO- release was measured with nanosensors (diameter 200

300 nm). The design of the nanosensors was based on previously developed chemically

modified carbon fiber technology 19, 109.

1. The glass capillary (KIMAX-51, Kimble Glass, INC.) with 1.5 ~ 1.8mm

diameter open-end was heated in a Bunsen burner flame and pulled. A

300-µm-diameter tip was formed.

2. Single carbon fiber (original diameter ~ 7 µm) were put through the end of the

capillary with 1~2 cm of the fiber protruding.

3. The carbon fiber was sealed and electrically connected to copper wires with

conductive sliver epoxy.

4. Assembled electrode was cured in the vacuum oven at ~ 80 ºC for 3 hrs.

5. The interstitial space between glass tip and the carbon fibers was sealed by

bee wax and rosin.

6. The diameter of carbon fiber tip was reduced to ~ 300 nm and the length of

carbon fiber tip was reduced to ~ 0.5 cm by gradual burning of the fiber using

propane microburner.

7. The assembled electrodes were kept in 0.10 M sodium hydroxide (NaOH)

before the poly-metalloporphyrin coating to remove extra beeswax and other

organic residues.

49

2.2 NO Nano-sensor Preparation

After the electrodes have been assembled, a three-electrode cell set-up as

following was used to coat the sensors: a platinum wire served as a counter electrode; a

silver/ silver chloride (Ag/AgCl) was the reference electrode; the assembled carbon fiber

as the working electrode. The exposed suface of the conical shape of the carbon fiber tip

was electrochemically cleaned in 0.10 M NaOH while potential was alternatively kept at

1.5 V or - 1.5 V vs Ag/AgCl to remove the impurities. Then, the carbon fiber tip was

electrochemically covered with conductive polymeric porphyrin: polymeric nickel (II)

tetrakis (3-methoxy-4-hydroxyphenyl) porphyrin (Ni porphyrin, Frontier Scientific) for

NO sensors in the repeated cyclic voltammetry (CV) mode. The potential from - 0.20 V

to + 1.0 V vs Ag/AgCl and the scan rate of 100 mV/sec were applied. Two glowing

voltammetric peaks were observed at 0.54 V and 0.40 V vs Ag/AgCl, which indicated the

amount of polyporphyrin film that had been deposited. After the Ni porphyrin coating,

the electrode was covered with another thin layer of Nafion (Sigma-Aldrich) by

immersing the electrodes in 1% Nafion solution (5% Nafion solution mixed with absolute

ehanol and stir for ≥ 1 hr) for ~ 10 sec. and repeated 3 times. The integrity of Nafion

coating can be also tested by CV in 0.10M NaOH from – 0.2 V to 0.8 V vs Ag/AgCl at

100 mV/sec scan rate. The absence of peaks at 0.54 V and 0.4 V vs Ag/AgCl can confirm

the good coating of Nafion110.

As Figure 2.1 shows, Nafion is negative charged, which can repel NO2-, NO3

-

species but highly permeable to NO. High conductive Ni polyporphyrin serves as a

50

catalyst in NO oxidation reaction, which allows the rapid movement of the electrons.

Both of the factors ensure the sensors are highly selective and rapidly responsive to NO.

Figure 2.1 Schematic diagram of NO nanosensor (adapted and modified from Malinski,

T.; Taha, Z., Nitric oxide release from a single cell measured in situ by a porphyrinic-

based microsensor. Nature 1992, 358 (6388), 676-8)19

2.3 Preparation of NO Standard Solution

The sensors were calibrated by using NO standard solution. NO gas was

generated by dripping 6M sulfuric acid (H2SO4, Fisher) into the solid sodium nitrite

51

(NaNO2, Sigma-Aldrich) in the presence of iron sulfate (FeSO4, Sigma-Aldrich) as a

catalyst (Equation 2.1).

The setup for NO synthesis is shown in Figure 2.2. The whole setup was

deo ygenated for .5 1 hr by flowing through nitrogen (N2) gas before starting and

during the reaction to prevent NO from rapidly oxidation to form NO2, a brown gas.

NaNO2 (4 50g) and FeSO4·7H2O (10 15g) was added into the dry two-arm flask.

Vacuum grease and parafilm were applied on all the connections to prevent gas leakage.

The speed of H2SO4 dripping into NaNO2 was adjusted to 1 drop every 10 seconds. Later,

the mixture gas produced by the reaction between NaNO2 and H2SO4 accompanied with

N2 went through two reaction vessels which contain 4M and 2M NaOH respectively to

remove the impurities produced by the reaction, such as NO2, NO3, N2O3 and N2O5. NO

was collected in a 5-mL conical Pyrex reaction vial with a rubber septum in the ice bath.

Each of the collecting vials was filled with 3 mL 0.1 M sodium phosphate buffer (PB, pH

7.4) and deoxygenated for ~ 20 min before the collection. NO was collected for ~ 15 min

in each vial with stirring all the time.

52

Figure 2.2 Schematic diagram of set-up for NO synthesis (adapted and modified from Malinski, T.; Huk, I., Measurement of

nitric oxide in single cells and tissue using a porphyrinic microsensor. Curr Protoc Neurosci 2001, Chapter 7, Unit7, 14) 111

53

After the collection, the vials of NO solution were kept in the refrigerator. The

concentration of the standard solution was measured by the oxyhemoglobin (oxyHb)

assay112. The assay is based on the reaction shown below:

Hemoglobin (Hb, Sigma-Aldrich, 20mg) was dissolved in 1 mL nanopure water.

A small amount (about 10 µg) of sodium hydrosulfite (Na2S2O4, Sigma-Aldrich) was

added to Hb solution to reduce iron (III) to iron (II). The pink solution of reduced Hb was

oxygenated by O2 for ~ 20min until the solution turned bright red. UV-vis

spectrophotometer (DU 640, Beckman Coulter) has been used to record the absorbance

from 380 nm to 450 nm wavelength (Figure 2.3). Absorbance change at wavelength

401nm before and after NO addition (ΔA401nm (metHb-oxyHb)) reflects the concentration

change of methemoglobin (metHb). The isobestic point of oxyHb and metHb is at

wavelength 410.5nm.

The concentration of metHb ([metHb]) that formed was based on the amount of

NO solution. Knowing the molar absorptivitity coefficient (ε) for oxyHb is 131.0 mM -

1cm-1 and for metHb is 49 mM-1 cm-1, NO concentration ([NO]) can be determined by the

following equations:

(2.4)

(2.3)

(2.2)

54

Figure 2.3 An example of UV-Vis spectra of (a) oxyHb and (b) metHb solutions in PB

buffer

In the presence of NO, oxyHb is converted to metHb; the spectrum of oxyHb

shifts to left and becomes the spectrum of metHb. The difference in absorbance of metHb

and oxyHb at wavelength 401 nm (ΔA401nm (metHb-oxyHb)) was taken to calculate the

concentration of metHb and the concentration of NO.

2.4 NO Sensor Calibration

The calibration of the sensor was performed by using the aliquots of NO standard

solution to the electrolytic cell. A three-electrode system was utilized for

amperometerically record of NO release (Figure 2.4). Platinum wire was used as a

counter electrode, Ag/AgCl was used as a reference electrode, and NO sensor was the

working electrode. Gamry VFP600 multichannel potentiastat was used to record the NO

response curve at a constant potential of 700mV vs Ag/AgCl. A linear calibration curve

55

was constructed for each sensor in the range of 20 nM to 1 µM using NO standard

solution (Figure 2.5 a and b).

Figure 2.4 Three-Electrode electrochemical system used for NO calibration

56

Figure 2.5 (a) A typical NO response curve, (b) The calibration curve of NO standard

solution (R2=0.9586).

y = 577.55x + 0.5307 R² = 0.9586

0

50

100

150

200

250

300

0 0.1 0.2 0.3 0.4 0.5

Cu

rre

nt (

nA

)

[NO] µM

a)

b)

57

2.5 ONOO- Nano-sensors Preparation

The design of ONOO- electrodes was based on Xue et.al.113 with some

modification (Figure 2.6). The assembled carbon fiber electrode was electrochemically

cleaned in 0.50 M H2SO4. The potential range from - 1.0 V to + 1.0 V vs Ag/AgCl and

the scan rate of 100 mV/sec have been used to remove impurities from the surface of

carbon fibers. Then, the carbon fiber tip was electrochemically covered with a film of

conductive polymeric porphyrin: manganese(III)-[2,2] paracyclophenyl-porphyrin (Mn

porphrin, Frontier Scientific). A solution of monomer Mn porphrin (5 mM) dissolved in

dimethyl sulfoxide (DMSO, Sigma-Aldrich) and 0.1 M tetrabutylammonium perchlorate

(TBAP) as a supporting electrolyte was used in this process. The potential from - 0.50 V

to + 1.0 V vs Ag/AgCl and the rate of 100 mV/sec were applied for electrochemical

coating. After cleaning with acetone and distilled water, the electrode was immersed in

poly (4-vinylpyridine) (PVP) solution (1% w/v, PVP/methanol) for ~10 sec. This process

was repeated 3 times and allowed to dry in between. The last step for ONOO- nanosensor

preparation involved a repeating CV scan - 0.2 V to 1.0V vs Ag/AgCl (scan rate of 100

mV/sec) in buffer solution (PB solution, pH 7.4) for ~ 5 min to activate the surface

coating of the electrode.

Figure 2.6 shows the diagram of the construction and mechanism for ONOO-

measurement. Mn polyporphyrin is selective to ONOO-. PVP is positively charged

barrier and prevents diffusion of positively charged molecules like dopamine interference

to the sensor.

58

Figure 2.6 Schematic diagram of ONOO- nanosensor (adapted and modified from

Malinski, T.; Taha, Z., Nitric oxide release from a single cell measured in situ by a

porphyrinic-based microsensor. Nature 1992, 358 (6388), 676-8 and Xue, J.; Ying, X.;

Chen, J.; Xian, Y.; Jin, L., Amperometric ultramicrosensors for peroxynitrite detection

and its application toward single myocardial cells. Anal Chem 2000, 72 (21), 5313-21)

19,113

2.6 ONOO- Standard Solution Synthesis

ONOO- can be synthesized by following fast reaction (equation 2.5, 2.6 and 2.7)

between hydrogen peroxide (H2O2) and NaNO2, and then quenched by NaOH solution.

59

Figure 2.7 shows the setup for ONOO- synthesis. 0.6 M H2O2 was made with 0.7

M hydrochloride (HCl) solution. 0.6 M NaNO2 and 0.9 M NaOH solution were made

with distilled water. All the solution was kept in ice or fridge after preparation to lower

the temperature to 0 ºC. A vacuum pump was used in the end of the system to drive H2O2

and NaNO2 to the first T-junction at where ONOOH solution was formed. The flow

speed of H2O2 and NaNO2 solution was adjusted to ensure the two reagents arrival in that

T-junction at the same time. The formed ONOOH solution kept flowing to the second T-

junction and was quenched by excess NaOH to stabilize ONOO- anion. A strong yellow

product solution indicated the formation of ONOO - was collected in the end of the

system. Other gas phase waste was collected in the waste container. After the synthesis,

ONOO- standard solution was stored in the - 80 ºC freezer to prevent ONOO-

decomposing to NO2- and O2.

(2.7)

60

Figure 2.7 Schematic diagram of set-up for ONOO- synthesis (adapted and modified from Koppenol, W. H.; Kissner, R.;

Beckman, J. S., Syntheses of peroxynitrite: to go with the flow or on solid grounds? Methods Enzymol 1996, 269, 296-302 and

Reed, J. W.; Ho, H. H.; Jolly, W. L., Chemical Syntheses with a Quenched Flow Reactor - Hydroxytrihydroborate and

Peroxynitrite. Journal of the American Chemical Society 1974, 96 (4), 1248-1249) 114-115

61

The concentration of the ONOO- standard solution was determined by a UV-vis

spectrophotometer (DU640, Beckman Coulter). Maximal absorbance reading of ONOO-

at wavelength 302nm (λmax= 302nm) was observed. A molecular absorptivity coeffient

(ε) for ONOO- is 1670 M-1cm-1. ONOO- (10 µL) standard solution was added to 990 µL

0.1 M PB. The absorbance was recorded between 5 400 nm (Figure 2.8). The

concentration of ONOO- ([ONOO-]) was calculated based on the absorbance at

wavelength 302 nm (equation 2.8).

(2.8)

Figure 2.8 A UV-Vis spectrum of ONOO- (λmax= 302nm) solution in PB buffer

2.7 ONOO- Sensors Calibration

A calibration of the sensor was performed by adding aliquots of ONOO- standard

solution to the electrolytic cell. The similar three-electrode system for NO measurement

λmax= 302nm

62

was utilized to amperometerically record ONOO- release. Gamry VFP600 multichannel

potentiastat has been used to record the ONOO- response curve at a consant potential at -

450mV vs Ag/AgCl. A linear calibration curve was constructed for each sensor in the

range from 1 µM to 10 µM of ONOO- standard solution (Figure 2.9 a and b).

63

Figure 2.9 (a) A typical amperogram showing nanosensor response to ONOO- response

after ONOO- standard solution injection, (b) A calibration curve of ONOO- (R2=0.9911).

y = 48.427x + 0.885 R² = 0.9911

0

50

100

150

200

250

300

350

0 1 2 3 4 5 6

Cu

rren

t (n

A)

[ONOO-] µM

a)

b)

64

2.8 Animal Surgery

Male Spragure- Dawey (SD) rats (Harlan, Indianapolis, IN) 5 350g were used

in our study and were provided with standard chow and water ad libitum. All procedures

were approved by the Institutional Animal Care and Use Committee and complied with

the National Institutes of Health standard. All the animals were anesthetized by Ketamine and Xylazine (100mg/kg

+10mg/kg, intramuscular). The animals were placed on the stereotaxic frame (Nashique,

Japan) when lack of corneal reflex and toe pinch were found. Five holes were drilled in

the skull by a sterile dental drill with a drill stopper to avoid brain piercing: (I) the hole

for NO/ONOO- working electrode, AP - 4.3mm posterior to bregma, ML + 2.0mm lateral

to the midline of the skull, V 2.5mm ventral to the dura surface, according to the

stereotactic atlas of rat brain. (II) The hole for drug delivering IH, AP - 4.3mm, ML

0mm, V 2.5mm. (III) The hole for reference and counter electrode, AP - 10mm, ML +

2.7mm, V 2mm. (IV) the hole for electroencephalography (EEG) measurement electrode,

AP - 4.3mm, ML - 3.0mm, and V 2.5mm. (V) The hole for EEG measurement reference

electrode, AP -10mm, ML 0mm, and V 2.5mm. (Figure 2.10)

65

Figure 2.10 The schematic diagram showing NO, ONOO- and EEG electrodes localization in a brain.

66

2.9 Animal Models and Treatments

Pilocarpine (PILO, Sigma-Aldrich) was used to induce epileptic seizures events in

SD rats. PILO is a muscarinic acetylcholine (Ach) receptor agonist. Dr. Turski et. al. first

found that PILO injections can generate acute epileptic seizures in naïve, healthy (non-

epileptic) animals116. Later, many studies were done on the usage of PILO in rodents.

They revealed that the epileptic seizures induced by PILO developed the similar epileptic

seizure aspects as temporal lobe epilepsy in humans, such as neuronal loss and aberrant

mossy fiber sprouting. Therefore, the PILO model has been widely used to study the

cellular and molecular mechanism of temporal lobe epilepsy, one of the most common

and severe type of seizures that occurs in adults.

Two drug delivery routes for PILO have been used in this study. 300mg/kg PILO

dissolved in 0.9% sterile physiological saline has been administered into the animal

intraperitoneally (IP). The IP injection has been considered as a systemic delivering

method. The drug was delivered into the peritoneum and reached the brain by blood

circulation. Another route is 0.16, 0.82, or 1.6mg/kg PILO dissolved in 2 µL 0.9% sterile

physiological saline injected interhippocampusly (IH) into animals. The IH injection was

a central drug delivery method in which the drug was directly applied into the

hippocampus; the area that has been thought to be one of the initiation places of epileptic

seizures. The results using both delivery methods have been compared in this study.

Several treatments have been utilized to modulate the production of NO and

ONOO- in the epileptic brain.

67

L-arginine (L-Arg, Sigma-Aldrich) is the substrate of NOS. 5.7 µg/kg of L-Arg

was dissolved in 2 µL 0.9% sterile physiological saline and injected into the animal brain

by IH.

L-NG-nitroarginine methyl ester (L-NAME, Santa Cruz) is a non-selective

inhibitor of nitric oxide synthase (NOS). L-NAME (7.3 µg/kg) was dissolved in 2µL 0.9%

sterile physiological saline and injected into the animal brain by IH.

1,3-Benzoxazol-2-yl-3-benzyl-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-yl sulfide

(VAS2870) is an inhibitor for NADPH oxidase. VAS2870 (35 µg/kg) was dissolved in

2µL DMSO and injected into the animal brain by IH.

Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP) is a cell-permeable

SOD mimetic and ONOO- scavenger. MnTBAP (64 µg/kg) was dissolved in 2µL DMSO

and injected into the animal brain by IH.

Since the brain is in a sealed environment, adding too much liquid would increase

intracranial pressure and cause brain damage. The volume for each injection in this study

was limited to 2 µL. An injection of modulator was performed 10min ahead of the PILO

injection.

3-Morpholinosydnonimine (SIN-1) is a ONOO- donor. Two different dosages of

SIN-1, 1.2 µg/kg and 0.6 µg/kg, dissolved in 2 µL 0.9% sterile physiological saline were

administrated to the animal brain by IH respectively.

Synthetic NO and ONOO- standard solution in physiological solution at different

concentrations were injected simultaneously into the animal brain by IH to increase

exogenous NO and ONOO- concentration in the brain.

68

O2·- standard solution of 3.6 mM was made by adding 5 g potassium superoxide

(KO2) to 5 mL dry DMSO with strong vortex mixing for 1 min under room temperature,

and then centrifuged at 1000 × g for 5 min to remove the excess solid of KO2117. In our

study, different dosages of O2·- standard solution have been injected into the animal brain

by IH.

ONOO- donor and standard solutions of NO, ONOO-, and O2·- were also used in

the absence of a PILO injection to investigate the relation between NO, ONOO- and O2·-

in the initiation of epileptic seizure events.

The summarized information below includes the different treatments. SD rats

(250-350g) were randomly assigned to 13 groups (n=8~10 in each group): Group , vehicle control ( μL, IH 0.9% sterile physiological saline);

Group 2, PILO (300 mg/kg, IP)-treated animals;

Group 3, PILO (1.6 mg/kg, 2 µL, IH)-treated animals;



Group 6, L-Arg (5.7 µg/kg, 2 µL, IH) + PILO (1.6 mg/kg, 2 µL, IH)-treated

animals;

Group 7, L-NAME (7.3 µg/kg, 2 µL, IH) + PILO (1.6 mg/kg, 2 µL, IH)-treated

animals;

Group 8, MnTBAP (64 µg/kg, 2 µL, IH) + PILO (1.6 mg/kg, 2 µL, IH)-treated

animals;

69

Group 9, VAS2870 (35 µg/kg, 2 µl, IH) + PILO (1.6 mg/kg, 2 µl, IH)-treated

animals;

Group 10, SIN-1 (1.2 µg/kg, 2 µL, IH)-treated animals;

Group 11, SIN-1 (0.6 µg/kg, 2 µL, IH) -treated animals;

Group 12, NO + ONOO- solution-treated animals.

Group 13, O2·- solution-treated animals.

Animals received DMSO (2 µL) in the absence or presence of 1.6 mg/kg PILO.

There was no obvious difference in EEG compared to those animals received saline or

1.6 mg/kg PILO. Those animals were grouped into group 1 (saline) and group 3 (1.6

mg/kg PILO). Therefore, DMSO (2 µL) did not interfere with the experiments. DMSO (2

µL) was used as the solvent for VAS2870 and MnTBAP.

2.10 NO and ONOO- in vivo Measurement

A Gamry 600 multichannel potentiostat was used to monitor in vivo the

simultaneous NO/ ONOO- release. The NO/ ONOO- concentrations were measured

continuously for a total of 90 min. After 10 min, the baseline stabilized, and the animals

from group 9 received saline, PILO, L-Arg, L-NAME, MnTBAP and VAS 2870,

respectively. Ten minutes after the first injection, PILO was administered to the animals

from group 6 9 and the NO/ ONOO- release was monitored for an additional 70 minutes.

Animals from group 10 and 11 received SIN-1 injection after baseline stabilized (~ 10

min), and the NO/ONOO- release was monitored for 80min without PILO injection.

Group 12 animals received the NO and ONOO- standard solution simultaneously every

15 min without PILO injection. Group 13 animals received the O2·- standard solution

70

every 15 min without PILO injection. NO/ONOO- release from Group 12 and 13 was

monitored for 90min. The flowchart for each group has been summarized in Figure 2.11.

2.11 EEG monitoring

The brain electrical activity was monitored by using a time-locked EEG

monitoring system (Pinnacle Technologies) at the same time when NO and ONOO- were

recorded (Figure 2.11). Two sterile, stainless steel bone screws were used (Figure 2.10).

Electrodes were locked in place by using dental acrylic. High-frequency, high voltage

synchronized polyspike or paroxysmal sharp waves with amplitude ≥ 2-fold of

background discharge that lasted for more than 6 sec were considered as seizure

episodes118.

71

Figure 2.11 The flowchart for animal treatment

72

2.12 Protein Sample Preparation

After the NO, ONOO- and EEG measurements, the rats were euthanized by

exsanguinations under anesthesia (Ketamine + Xylazine 100mg/kg + 10mg/kg, IM).

Brain hippocampus samples were quickly dissected on ice and washed with PB solution.

Ice-cold radioimmunoprecipitation (RIPA) buffer (1mL) (Cat# 24948, Santa

Cruz) with a HaltTM protease inhibitor cocktail solution (Cat# 1860932, Thermo) was

added to every hipposcampus sample. Samples were homogenized by ultra-turrax t8

homogenizer (IKA LABORTECHNIK) at maximal speed (3 × 5sec), with resting on ice

in between to prevent protein degradation caused by heat. After homogenizing, the

sample mixture was kept for mild shaking in the cold room for 2 hrs. Samples were later

centrifuged at 40,000 × g under 4 ºC for 20 min. Supernatant was collected and kept in

the - 80 º C freezer.

2.13 Total Protein Concentration Measurement

The total protein concentration from each sample was tested based on the

bicinchoninic acid (BCA) method. A PierceTM BCA protein assay kit (Cat# 23225,

Thermo) was utilized. The procedure has followed the manufacturer’s suggestion.

A set of calibration standard solutions were prepared from bovine serum albumin

(BSA) protein stock standard solutions (2000 µg/mL) diluted in nanopure water to yield

eight different concentrations of BSA standard solutions (2000, 1500, 1000, 750, 500,

250, 125, 25 µg/mL). The protein samples for testing were defrosted on ice. Each

standard solution was added (25 µL) in duplicate to a 96-cell microplate.

73

A protein sample (2 µL) was added in triplicate to the microplate with additional

23 µl of nanopure water (total volume 25 µL). 200 µL Working solution (200 µL) (made

by mixing BCA reagent A and regent B at ratio of 50:1) was added into each vial

(working solution: sample = 8:1). The plate was covered and shook on a plate shaker for

30 sec to mix the solution inside the vials. Then the plate was incubated at 37ºC for 30

min, and cooled down for 5 min. Absorbance at 562 nm was measured by a plate reader

(BioTek, Synergy HT). A calibration curve was plotted as absorbance versus BSA

protein concentration. The total protein concentration was calculated based on this

calibration curve.

2.14 Immunoblotting

Chemicals and Solution: 4×laemmli sample buffer (Cat# 161-0747, Bio-Rad); β-

mercaptoethanol (sigma-aldrich); sodium dodecyl sulfate (SDS, Cat# 161-0416, Bio-

Rad); ammonium persulfate (APS, Cat# 161-0700, Bio-Rad);

tetramethylethylenediamine (TEMED, Cat# BP 150-20, Fisher); 1.5M Tris-HCl (pH 8.8,

resolving gel buffer, Cat# 161-0798, Bio-Rad); 0.5M Tris-HCl (pH 6.8, stacking gel

buffer, Cat# 161-0799, Bio-Rad); 30% acrylamide/bis solution (29:1, Cat# 161-0156,

Bio-Rad); precision plus proteinTM dual color standard (protein ladder, Cat# 161-0374,

Bio-Rad); tris/glycine/SDS solution (TGS, Cat# 161-0772, Bio-Rad); tris/glycine (TG,

Cat# 161-0771, Bio-Rad); 10% Tween-20 (Cat# 161-0781, Bio-Rad); blotting-grade

blocker (non-fat milk, Cat# 170-6404, Bio-Rad); tris-buffered saline (TBS, Cat# 97062-

370, VWR); polyvinyl difluoride (PVDF) membrane (0.45 µm, Cat# 88518, Thermo);

SuperSignal West Pico chemiluminescent substrate (Cat# 34080, Thermo); HyBlot CL®

74

autoradiography film (E3012, Denvillie Scientific); Carestream® Kodak®

autoradiography GBX developer/replenisher (Cat# P4042, Sigma-Aldrich); Carestream®

Kodak® autoradiography GBX fixer/replenisher (Cat# P7167, Sigma-Aldrich).

After testing the total protein concentration, protein samples for electrophoresis

were prepared by mixing with 4 × volumes of laemmli solution plus 10% β-

mercaptoethanol. Denatured samples were boiled at 95 ºC for 5min. A discontinuous

electrophoresis system was utilized. Table 2.2 shows the composition of sodium dodecyl

sulfate polyacrylamide (SDS-PAGE) gel. Protein samples (60 µg ) were loaded in each

well and separated on a 7.5% SDS-PAGE gel for non-denatured samples (without

boiling) and 8% gels for denatured samples in TGS solution (25mM Tris, 192mM

Glycine, 0.1% (w/v) SDS, pH 7.4) for 120 min with a constant voltage at 120V and low

temperature at 4 ºC.

75

Table 2.1SDS-PAGE gel Separating Gel Stacking Gel

Solutions 7.5% 8% 4%

1.5M Tris-HCl (pH 8.8)

(mL)

2 2 -

0.5M Tris-HCl (pH 6.8)

(mL)

- - 1.25

30% Acrylamide (mL) 2 2.13 0.67

Water (mL) 3.8 3.7 3

10% (w/v) APS (µL) 80 80 50

10% (w/v) SDS (µL) 80 80 50

TEMED (µL) 8 8 5

After electrophoresis, the gel was washed in transferring buffer (TG/methanol

solution, 25mM Tris, 192mM Glycine, 10% (v/v) methanol, pH 7.4). A transferring

sandwich was assembled according to the order (spongefilter papergelPVDF

membranefilter paper sponge). The protein inside the gel was transferred into the

PVDF membrane with a constant voltage at 30V and low temperature condition at 4 ºC

for 16 hrs.

After transfer, the transferring sandwich was dissembled. Gel was stained with

Coomassie Blue to check the completion of transferring procedure. The PVDF membrane

(the blot) was dried at room temperature to ensure tight binding between the protein and

membrane matrix and then reactivated by methanol. The activated blot was washed with

76

nanopure water (2 × 5min) and washing solution (TBS solution with 0.1 % tween-20

(TBST), 1 × 5min) on the plate shaker to remove excess methanol from the membrane.

Then the blot was incubated in 5% non-fat milk made with TBST solution at room

temperature for 1 hr to block the non-specific protein binding sites.

After blocking, the blot was rinsed with TBST and then incubated with the

primary antibody (table 2.3) overnight at 4 °C. The next day, blots were washed with

TBST solution (4 × 5min) and incubated with secondary antibody (table) at room

temperature for 1 hr. Later, the blots were washed with TBST solution (4 × 5min) and

TBS solution (2 × 5min).

Last, the blots were incubated with West Pico SuperSignal chemiluminescence

reagent for 5 min in room temperature. The blot was put in the HypercassetteTM cassette,

and the autoradiography film was put on top of the blot. The film was exposed,

developed in the developing solution, and fixed in the fixing solution.

77

Table 2.2 Summerized primary antibody and secondary antibody information Primary

antibody Company Dilution

Secondary

antibody Dilution

Anti-eNOS

Developed in

rabbit

Santa Cruze

(Cat# sc-654) 1:250 (TBST)

Anti-rabbit

(Santa Cruz,

Cat# sc-2004)

1:5000

(TBST)

Anti-nNOS

Developed in

rabbit

Santa Cruze

(Cat# sc-648)

1:1000

(Blocking

solution)

Anti-NOX 4

Developed in

rabbit

Santa Cruze

(Cat# sc-30141)

1:1000

(Blocking

solution)

Anti-p67phox

Developed in

mouse

Becton

Dickinson (BD)

(Cat# 610912)

1:500 (TBST) Anti-mouse

(Santa Cruz,

Cat# sc-2005)

1:5000

(TBST)

Anti-β actin

Developed in

mouse

Sigma-Aldrich

(Cat# A5316)

1:25,000

(Blocking

solution)

1:10,000

(TBST)

2.15 Statistical Analysis

All data are presented as mean ± standard error of the mean (SEM) of n=8 10.

Statistical analysis of the mean difference between multiple groups was performed by

using one-way analysis of variance (ANOVA) with Student-Newman-Keuls multiple

78

comparisons post hoc analysis; and between two groups, using two-tailed Student’s t-test.

The alpha level for all the tests was 0.05. A P value < 0.05 was considered to be

statistically significant. All statistical analyses were performed with Origin (v 6.1 for

Windows; OriginLab, Northampton, MA).

79

3. RESULTS

3.1 NO and ONOO- Release in the Brain after PILO Induced Seizure.

In vivo production of NO and ONOO- has never been measured in the epileptic

brain. Therefore, the data presented here are the first ever reported. Our first question was

whether NO and ONOO- were produced during PILO-induced epileptic seizure events. In

order to monitor the release of NO and ONOO- during PILO-induced seizure, both NO

and ONOO- nanosensors were placed above the ipsilateral side of the hippocampus CA1

region. An EEG recording electrode was implanted above the contralateral side of

hippocampus CA1 region to record the brain activity to identify seizure episodes.

Two delivery routes for PILO—interhippocampal (IH) administration of PILO

and intraperitoneal (IP) injection of PILO— were utilized for different drug delivery. The

release of NO and ONOO- were monitored continuously together with EEG recording.

Figure 3.1 shows a typical current/ concentration vs time amperometric curve that

reflects near real time changes of NO and ONOO- in the brain after saline (2 µL) IH

injection. The simultaneously recorded EEG signal is also shown. About 2 min after the

saline injection, a NO peak was observed and the maximal NO concentration reached 814

± 16 nM. A small ONOO- peak with maximal concentration of 636 ± 117 nM was

recorded about 20 min after the saline injection. Normal brain discharge was showed in

the EEG signals. There was no difference in the amplitude and frequency of the EEG

signals before and after the saline injection.

80

Figure 3.1 Typical amperograms showing real-time NO and ONOO- release during

normal brain activity in the SD rat brain

Figure 3.2 shows a typical amperometric curves that reflect near real time changes

of NO and ONOO- in the brain after a PILO (300mg/kg) IP injection. The simultaneously

recorded EEG signal is also shown. A NO peak was observed about 4min after the PILO

injection, and the maximal NO concentration reached 753 ± 113 nM. The NO production

decayed to 234 ± 30 nM in 10 min. At the same time, ONOO- production increased

significantly; the maximal ONOO- production was recorded at 3392 ± 318 nM. Abnormal

discharges characterized as high-frequency and high-amplitude polyspikes were observed

81

simultaneously. The first seizure event occurred at 552 ± 42 s after the PILO IP injection;

the time when NO production decayed and ONOO- production increased.

Figure 3.2 Typical amperograms showing NO and ONOO- real-time release during

epileptic seizures induced by PILO (300 mg/kg, IP) in the SD rat brain.

Figure 3.3a shows the amperometric curves of NO and ONOO- in the brain after

PILO (1.6mg/kg) IH injection. The simultaneously recorded EEG signal is also shown. A

NO peak was observed about 4 min after the PILO injection into the hippocampus, and

the maximal NO concentration reached 826 ± 88 nM. The first seizure event was

observed at 18 ± 4 min after the PILO injection, when NO decreased to 283 ± 26 nM. NO

production was accompanied by a high production of ONOO- at 2181 ± 161 nM. A

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ONOO- peak was observed coincident with the seizure onset (Fig 3.3 b). Maximal NO or

ONOO- concentration were measured at the onset of the seizures.

83

Figure 3.3 (a) Typical amperograms showing NO and ONOO- real-time release during

epileptic seizures induced by PILO (1.6 mg/kg, IH) in the SD rat brain. (b) The high

resolution amperograms and EEG signals of NO and ONOO- release during one seizure

episode onset (the time frame between vertical red dot line in Figure 3.3 a).

b)

a)

84

Three dosages of PILO, 0.16 mg/kg; 0.82 mg/kg; and 1.6 mg/kg, were

administered into the animals through IH injection. The forth dosage of PILO, 300 mg/kg,

was injected into the animals through IP. The effect of these two different drug delivery

routes, IH and IP, were compared. No epileptic seizures were observed in the 0.16 mg/kg

PILO IH group. Maximal NO release was recorded at 722 ± 41 nM from this group.

However, all other PILO dosages we used induced epileptic seizures in SD rats. In 0.82

mg/kg PILO IH group, maximal NO release during the seizure was 566 ± 18 nM. In the

1.6 mg/kg PILO IH group, maximal NO release during the seizure was 283 ± 26 nM; and

in the 300mg/kg PILO IP injection group, maximal NO release was 234 ± 30 nM (Fig

3.4a). The NO production significantly reduced with the increase in PILO dosages

compared to the saline group accompanied by a dramatic elevation in ONOO- production

during the seizures (Fig 3.4b). Maximal ONOO- release increased with the increase in

PILO dosages. In 0.16 mg/kg PILO IH group, the ONOO- release was 1075 ± 37 nM. In

the 0.82 mg/kg PILO IH group, the maximal ONOO- release during the seizure events

was recorded as 1307 ± 72 nM. In the 1.6 mg/kg PILO IH group, the amount of ONOO-

release reached 2181 ± 161 nM, and the ONOO- concentration was measured at 3392 ±

318 nM in the PILO IP group. Additionally, the ratio between NO and ONOO- from

different groups was calculated: 1.37 ± 0.26 in the saline group, 0.67 ± 0.05 in the 0.16

mg/kg PILO IH group, 0.45 ± 0.01 in the 0.82 mg/kg PILO IH group, 0.15 ± 0.02 in the

1.6 mg/kg PILO IH group, and 0.07 ± 0.008 in the 300mg/kg PILO IP group (Fig 3.4c).

The ratio of the [NO]/ [ONOO-] decreased with the increase in dosage of PILO.

85

a)

b)

86

Figure 3.4 (a) Maximal NO and (b) ONOO- concentration produced in the brains

of SD rats in the absence or presence of epileptic seizures. Epileptic seizures were

induced by 0.82 mg/kg; or 1.6 mg/kg PILO injected IH, or 300 mg/kg PILO injected IP

(**p < 0.01, ***p< 0.0001 vs saline group). (c) The ratio of NO to ONOO- maximal

concentration measured in the brains of SD rats in the absence of or the presence of

epileptic seizures. Epileptic seizures were induced by 0.82 mg/kg, or 1.6 mg/kg PILO

injected IH, or 300 mg/kg PILO injected IP (*p< 0.05, **p < 0.01, ***p< 0.0001 vs

saline group).

EEG results from PILO IH groups and IP group were also compared. Seizure

episodes were defined as high-frequency, high-voltage synchronized polyspikes or

paroxysmal sharp waves with amplitude 2-fold greater than background discharge which

lasted more than 6 sec118. In the saline and 0.16 mg/kg PILO IH group, no seizure events

c)

87

were observed, while all other PILO dosages administrations successfully induced

epileptic seizures in the animals. To analyze the severity and intensity of the seizure

events, three characteristics were measured: latency from the PILO injection to first

seizure onset; duration for seizure events; and number of seizure events occurring per

hour (frequency of seizure events occurring). The latency in the IP group was reduced

compared to the two PILO IH injection groups but with no significance (p> 0.05, Fig 3.5

a). However, the duration in the IP group was significantly longer compared to the two

IH groups (Fig 3.5 b), which also resulted in a lower number of seizure events occurring

per hour (frequency of seizure events) in PILO IP group compared to the IH groups (Fig

3.5 c). Between the two IH injection groups, a shorter latency; a longer duration; and a

higher frequency of seizure events were observed in the 1.6 mg/kg PILO IH group

compared to the 0.82 mg/kg PILO IH groups (Fig 3.5 a, b and c). Thus, the IP injection

caused more severe seizure events than the IH injection. Within the PILO IH groups,

epileptic seizure events induced by PILO can be enhanced with the increase in the dosage

of PILO.

In order to limit drug usage to prevent the drug interaction that may affect the

results, PILO was used solely to induce epileptic seizures in animals, and no other drugs

were used to reduce mortality. Due to the high mortality (3/5 SD rats) in the PILO IP

injection group, we used a PILO IH injection at the dosage of 1.6m/kg as our study

model, which can also successfully induce seizure in SD rats, but with low mortality (0 /

10 SD rats) compared to the IP injection group.

88

a)

b)

89

Figure 3.5 Seizure events induced by PILO at the dosage of 0.82 mg/kg (solid bar), 1.6

mg/kg (gray bar) PILO through IH injection or PILO at the dosage of 300mg/kg (open

bar) through IP injection. (a) Latency from PILO injection to the first seizure event onset.

(b) Seizure duration (***p<0.0001 vs PILO IP group). (c) Number of seizure events per

hour (frequency of seizure events).

3.2 Modulation of Seizure Events by Regulating the Release of NO and ONOO-

In previous studies, low NO and high ONOO- production were observed

accompanying with the PILO-induced epileptic seizure events. Epileptic seizures

occurred at the time when NO production decreased and ONOO- production increased.

Our next question was whether the regulation of NO and ONOO- release can modulate

the intensity and severity of PILO-induced epileptic seizures. In order to investigate the

effect of the different amount of NO and ONOO- production endogenously in modulating

c)

90

seizure events, NOS substrate, NOS inhibitor, ONOO- scavenger, and NADPH oxidase

inhibitor were utilized to regulate the release of NO and ONOO- during the seizures.

3.2.1 L-Arg Treatment

L-Arg is the substrate for nitric oxide synthase (NOS). In the presence of elevated

L-Arg, endogenous NO production should increase. L-Arg (5.7 μg/kg) dissolved in

physical saline solution (2 µL) was injected IH after 10 min of baseline recording. Later,

PILO (1.6 mg/kg) was injected 10min after the L-Arg injection. Maximal NO release

increased from 283 ± 26 nM in the absence of L-Arg to 615 ± 52 nM in the presence of

L-Arg during the seizure events (Fig 3.6 a). NO production during the epileptic seizures

was significantly enhanced in the presence of L-Arg. This increase in NO was

accompanied with an increase in ONOO- production simultaneously from 2181 ± 161 nM

in the absence of L-Arg to 5005 ± 420 nM in the presence of L-Arg (Fig 3.6 b). The ratio

between the NO and ONOO- concentration was 0.14 ± 0.02 in the presence of L-Arg,

which was very close to the ratio of the [NO]/[ONOO-] (0.15 ± 0.02) in the absence of L-

Arg. Both of the ratios were significantly lower than the ratio that we found in saline

group (Fig 3.6 c).

91

a)

b)

92

Figure 3.6 (a) Maximal NO and (b) ONOO- concentration produced in brains of epileptic

SD rats. Epileptic seizures were induced by IH injection of 1.6 mg/kg PILO in the

absence or presence of L-Arg (***p< 0.0001, vs saline group, ^^^p<0.0001 vs PILO

group). (c) The ratio of maximal NO to ONOO- concentration released in the brains of

epileptic SD rats. Epileptic seizures were induced by IH injection of 1.6 mg/kg PILO in

the absence or presence of L-Arg (***p< 0.0001 vs saline group).

Example EEG signal in the presence of L-Arg is shown in Figure 3.7. The latency

from PILO injection to the first seizure onset was reduced from 1103 ± 246 sec in the

c)

93

absence of L-Arg to 355 ± 164 sec in the presence of L-Arg (Fig 3.8 a). The duration of

the seizure episode was significantly extended from 112 ± 12 sec in the absence of L-Arg

to 186 ± 38 sec in the presence of L-Arg (Fig 3.8b). However, the number of seizure

events occurring per hour was reduced in the presence of L-Arg compared to the number

in the absence of L-Arg, which may be caused by the longer duration for each seizure

episode (Fig 3.8 c). Therefore, elevated L-arg did not suppress the PILO-induced seizure

events, but lowered the threshold of seizure onset.

Figure 3.7 Epileptic seizure events were observed in the brain of a SD rat induced by

PILO (1.6 mg/kg, IH) in the presence of L-Arg (5.7 μg/kg).

94

Figure 3.8 Seizure events induced by PILO (1.6 mg/kg) through IH injection in the

absence (close bar) or presence (open bar) of L-Arg (5.7 μg/kg). (a)Latency from PILO

injection to the first seizure event onset. (b) Seizure duration (*p<0.05 vs PILO group).

(c) Number of seizure events per hour (frequency of seizure events).

a)

b)

c)

95

3.2.2 L-NAME Treatment

L-NAME is a non-selective NOS inhibitor. In the presence of L-NAME, NOS

activity was inhibited, which could cause endogenous NO production to reduce. This may

result in a decrease in ONOO- production. L-NAME (7.3 μg/kg) dissolved in physical

saline solution (2 µL) was injected IH into the animal after 10 min of baseline recording.

PILO (1.6 mg/kg) was injected IH into the animal 10 min after the L-NAME injection.

The maximal NO release during the seizure was significantly reduced from 283 ± 26 nM

in the absence of L-NAME to 65 ± 5 nM in the presence of L-NAME (Fig 3.9 a). A

significant decrease in maximal ONOO- release was also recorded to be 1099 ± 77 nM

during the seizures (Fig 3.9 b). The ratio between the NO and ONOO- concentrations in

the presence of L-NAME treatment was 0.06 ± 0.005, which was significantly lower than

the value that we found in the PILO (1.6 mg/kg, IH) group in the absence of L-NAME

(Fig 3.9 c).

a)

96



the absence or presence of L-NAME (*p<0.05, ***p< 0.0001, vs saline group,

^^^p<0.0001 vs PILO group). (c) The ratio of maximal NO to ONOO- concentration

released from the epileptic SD rats induced by PILO (1.6 mg/kg, IH) in the absence or

presence of L-NAME (***p< 0.0001 vs saline group, ^^^p<0.0001 vs PILO group).

b)

c)

97

An example EEG signal in the presence of L-NAME is shown in Figure 3.10. The

latency to seizure onset was significantly reduced from 1103 ± 246 in the absence of L-

NAME to 91 ± 26 sec in the presence of L-NAME (Fig 3.11 a). The seizure duration also

increased from 186 ± 38 in the absence of L-NAME to 211 ± 61 sec in the presence of L-

NAME (Fig 3.11 b). The number of seizure episodes per hour was reduced in the

presence of L-NAME, which may be caused by the longer duration of epileptic seizure

events in the presence of L-NAME (Fig 3.11 c). Thus, the presence of L-NAME assisted

PILO to induce epileptic seizures in the animals.

Figure 3.10 Epileptic seizure events were observed in the brain of SD rat induced by

PILO (1.6 mg/kg, IH) in the presence of L-NAME (7.3 μg/kg).

98


bar) or presence (open bar) of L-NAME (7.3 μg/kg). (a) Latency from PILO injection to

the first seizure event onset (*p<0.05 vs PILO group). (b) Seizure duration (*p<0.05 vs

PILO group). (c) Number of seizure events per hour.

a)

b)

c)

99

3.2.3 MnTBAP Treatment

In this study, the production of ONOO- was observed at a high concentration of

2181 ± 161 nM when seizures onset. Thus, a cell-permeable superoxide dismutase (SOD)

mimetic, MnTBAP, was used as an antioxidant and ONOO- scavenger to reduce the

amount of ONOO-. MnTBAP (64 μg/kg) dissolved in DMSO (2 µL) and was injected into

the animal after 10 min of baseline recording. PILO (1.6 mg/kg) was injected into the

animal 10 min after the MnTBAP injection. NO production was recorded as 118 ± 20 nM

in the presence of MnTBAP (Fig 3.12 a), and ONOO- production was 426 ± 45 nM (Fig

3.12 b). Both NO and ONOO- production were reduced significantly in the presence of

MnTBAP compared to those produced from the PILO (1.6 mg/kg, IH) group in the

absence of MnTBAP. The [NO]/ [ONOO-] ratio increased from 0.15 ± 0.02 in the

absence of MnTBAP to 0.26 ± 0.03 in the presence of MnTBAP, which was still

significantly lower than the value we found in the saline group (Fig 3.12 c).

a)

100



the absence or presence of MnTBAP (***p< 0.0001 vs saline group, ^^^p<0.0001 vs

PILO group). (c) The ratio of maximal NO to ONOO- concentration released from

epileptic SD rats induced by PILO (1.6 mg/kg, IH) in the absence or presence of

MnTBAP (***p< 0.0001 vs saline group, ^^p< 0.01 vs PILO group).

b)

c)

101

Electrographic seizures were still observed but with reduced intensity (Fig 3.13).

The latency to seizure onset was 628 ± 152 sec (Fig 3.14 a), which was reduced but

without significant difference from the latency in the absence of MnTBAP. However, the

duration of the seizure episodes was recorded as 81 ± 23 sec, which was reduced

compared to the one from the PILO (1.6 mg/kg, IH) group (Fig 3.14 b). The frequency of

the seizure events was significantly reduced as well (Fig 3.14 c). Thus, the severity and

intensity of epileptic seizure events were suppressed in the presence of MnTBAP, but the

onset of seizure cannot be prevented by MnTBAP.

Figure 3.13 Epileptic seizure in the brain of SD rat induced by PILO (1.6 mg/kg, IH) in

the presence of MnTBAP (64 μg/kg).

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bar) or presence (open bar) of MnTBAP. (a) Latency from PILO injection to the first

seizure event onset. (b) Seizure duration. (c) Number of seizure events per hour (*p< 0.05

vs PILO group).

a)

b)

c)

103

3.2.4 VAS2870 Treatment

NADPH oxidase is a family of oxidase which has been found in the cell

membrane, and can exclusively produce O2·-119. Since ONOO- is formed by the diffusion-

controlled reaction between NO and O2·-, a NADPH oxidase inhibitor---VAS2870---was

used to inhibit O2·- formation. Therefore, ONOO- production was expected to be reduced

in the presence of VAS2870. VAS2870 (35 µg/kg) was dissolved in DMSO (2 µL) and

injected IH into the animal after 10 min of baseline recording. PILO (1.6 mg/kg) was

injected IH into the animal 10 min after the VAS2870 injection. The amount of NO

release in the presence of VAS2870 was 679 ± 78 nM (Fig 3.15 a) accompanied by the

production of ONOO- at 719 ± 71 nM (Fig 3.15 b). The NO production in the presence of

VAS2870 was significantly recovered compared to the abnormal NO production in the

absence of VAS2870 during the epileptic seizures. The production of ONOO- in the

presence of VAS2870 was also significantly reduced compared to the ONOO- production

measured during the epileptic seizures in the 1.6 mg/kg PILO group. The [NO]/ [ONOO-]

ratio significantly increased from 0.15 ± 0.02 in the absence of VAS2870 to 1.14 ± 0.25

in the presence of VAS2870 (Fig 3.15 c), which indicated that the ONOO- production

was successfully suppressed by inhibiting the production of O2·-, and the amount of NO

can be restored by preventing ONOO- production.

104

a)

b)

105



the absence or presence of VAS2870 (***p< 0.0001 vs saline group, ^^^p<0.0001 vs

PILO group). (c) The ratio of maximal NO to ONOO- concentration released in the

epileptic brain of SD rats. Epileptic seizures were induced by PILO (1.6 mg/kg, IH) in the

absence or presence of VAS2870 (***p< 0.0001 vs saline group, ^^^p< 0.0001 vs PILO

group).

Moreover, no severe seizure episodes were observed in the EEG results (Fig

3.16). Only some oscillations at high frequency (~12 Hz) but with low amplitude (~ -10

c)

106

to 10 μV) were noticed. In the presence of VAS2870, the [NO]/ [ONOO-] ratio was

restored, and the seizure onset was prevented also.

Figure 3.16 Epileptic seizure in the brain of a SD rat induced by 1.6 mg/kg PILO in the

presence of VAS2870 and 1.6 mg/kg PILO.

3.3 cNOS and NADPH Oxidase Protein Expression

In previous sections, we showed that the NO production was significantly

suppressed during the process of PILO-induced epileptic seizures, while the ONOO-

production was significantly enhanced. The severity, intensity, and the onset of epileptic

seizures can be regulated by different agents which modulated NO and ONOO -

production. The activity and expression of proteins involved in the production of NO and

ONOO- were analyzed by western blotting. The optical density of each band was read by

ImageJ (NIH, MD). All the bands were normalized to the optical density of the internal

control, β-actin.

107

3.3.1 eNOS Expression

Two constitutive NOSs can be found to produce NO in the brain. NO produced by

endothelial NOS (eNOS) is involved in the regulation of cerebral blood flow. The

hippocampus samples were dissected quickly from the SD rats after the NO and ONOO-

measurement, and homogenized in RIPA buffer. Prepared protein samples were separated

by SDS-PAGE based on the molecular weight. Two different types of protein samples---

denatured (boiled) and native (non-boiled) ---were used to test the expression and activity

of eNOS respectively in the study.

Denatured protein samples were boiled at 95 °C for 5 min, which were used to

analyze the total expression level of eNOS. The expression level of eNOS in the absence

or presence of different treatments is showed in Figure 3.17. PILO didn’t induce the

change in eNOS expression compared to the control group. L-Arg, L-NAME, and

VAS2870 cannot alter the expression level of eNOS after exposure to PILO. However,

the expression of eNOS was inhibited by MnTBAP, which could explain the low

production of NO in the presence of MnTBAP during PILO-induced epileptic seizures.

Native protein samples without boiling treatment were used to analyze the

expression of functional and dysfunctional forms of eNOS. The functional form of eNOS

in the biological system is the homodimer, which is a SDS-resistant but temperature-

sensitive structure. The dysfunctional form of eNOS is the monomer under the natural

condition. The optical density ratio between the dimer and monomer bands was used to

analyze the functional state of eNOS. Native protein samples were separated in the low

temperature SDS-PAGE. PILO induced an increase in the dysfunctional state of eNOS

108

(dimer/monomer = 1.28) compared to the saline (dimer/monomer = 2.78). The

dysfunctional state of eNOS after PILO exposure can be reduced in the presence of L-

Arg, MnTBAP, or VAS2870. VAS2870 was the most efficient agent in improving the

dysfunctional eNOS to functional one. However, L-NAME induced an increase in

dysfunctional eNOS expression level even compared to PILO, which suggested the

inhibition of NO production through L-NAME was due to the alternation of functional

form of eNOS.

Figure 3.17 eNOS expression in native form (dimer and monomer) and eNOS total

expression level.

3.3.2 nNOS Expression

NO produced by neuronal NOS (nNOS) acts as a neurotransmitter in the

biological system. The nNOS total expression was up-regulated after PILO induced

epileptic seizures compared to that of saline samples (Fig 3.18). The up-regulation

109

expression level was enhanced in the presence of L-Arg, L-NAME, MnTBAP, or

VAS2870 after PILO exposure.

The functional nNOS structure in the biological system is a homodimer, which is

a SDS-resistant but temperature-sensitive structure, the same as eNOS dimer. PILO-

induced epileptic seizures increased the production of the dysfunctional nNOS (nNOS

monomer) compared to the control group. This increase can be reduced in the presence of

L-Arg, L-NAME, MnTBAP, or VAS2870 with different degrees.

Figure 3.18 nNOS expression in native form (dimer and monomer) and nNOS total

expression level.

3.3.3 NADPH Oxidase Expression

NADPH oxidase is a family of proteins which was found to be in the cell

membrane, and exclusively produce O2·- with the downstream reactive oxygen species120.

110

Two proteins, NOX4 and p67phox, were used in our study to investigate the expression of

NADPH oxidase.

NOX4 is a member of the NADPH oxidase family. Induction of NOX4 mRNA

has been found in the mouse experimental ischemia model121 and also in the response to

shear stress122 and endoplasmic reticulum stress123. In our study, the expression level of

NOX4 was up-regulated after PILO induced epileptic seizures compared to the control

group. This increase was enhanced in the presence of L-Arg after PILO exposure.

However, the up-regulation of NOX4 expression after PILO-induced epileptic seizures

was reduced in the presence of L-NAME, MnTBAP, or VAS2870 (Fig 3.19). VAS2870

induced the most inhibition of the up-regulation of NOX4 after PILO exposure.

Figure 3.19 NOX4 expression in the absence or presence of modulators after PILO-

induced epileptic sezures.

p67phox was identified as an activator subunit of NADPH oxidase120. The

expression of p67phox responded to different stimuli, such as zinc application in neurons

111

and astrocytes124. In our study, an increase in the expression of p67phox after PILO

induced epileptic seizures compared to the control group. But this up-regulation after

PILO exposure was reduced in the presence of L-Arg, L-NAME, MnTBAP, or VAS2870

(Fig 3.20). In the presence of L-NAME. MnTBAP or VAS2870, the expression level of

p67phox was even lower than the one in the control group.

Figure 3.20 p67phox expression in the absence or presence of modulators after PILO-

induced epileptic sezures.

3.4 Seizure Episodes Triggered by Artifact NO / ONOO- Ratio

In previous sections, the fact that the severity and intensity of PILO-induced

epileptic seizures can be modulated by the regulation of endogenous NO and ONOO-

production through different modulators was described. The negative correlation between

the ratio of NO and ONOO- production and seizure events was noticed. With a lower

[NO]/ [ONOO-] established, more server and intense epileptic seizures were recorded.

However, whether a low [NO]/ [ONOO-] can trigger seizure episodes was still unknown.

112

In order to answer this question, another three sets of experiments were performed:

ONOO- donor, SIN-1; synthetic NO and ONOO- standard solution mixture; and an O2·-

standard solution; were injected in the absence of PILO into animals IH, respectively.

The exogenous and artificial [NO]/ [ONOO-] was built to mimic the condition of the

abnormal endogenous NO and ONOO- production after PILO exposure.

3.4.1 SIN-1 Treatment

SIN-1 can readily produce both NO and O2·- under physiological conditions,

which leads to the production of ONOO-. In our study, two dosages of SIN-1 solution,

0.6 µg/kg and 1.2 µg/kg, were used to increase exogenous ONOO- concentration in the

brain of SD rats. During the measurement, SIN-1 (2 µL ) was injected after the 10 min of

baseline recording in the absence of a PILO injection.

Figure 3.21 shows the example amperometric curves for NO and ONOO-

production after SIN-1 (1.2 µg/kg) injection IH. The brain activity was also continuously

monitored by EEG (Fig 3.21). A seizure-like EEG signal was observed at ~ 100 sec after

the SIN-1 injection. This abnormal EEG signal disappeared at ~ 600 sec after the SIN-1

injection. Both of NO and ONOO- production during the abnormal EEG signal period are

shown in Figure 3.22. NO production was not affected by different dosages of SIN-1; the

release of NO was about the same in the presence of 0.6 µg/kg SIN-1 or 1.2 µg/kg SIN-1.

However, ONOO- production increased with higher dosage of SIN-1. The ratio of the NO

to ONOO- concentration found in the two SIN-1 groups was compared with the ratio we

found in the PILO (1.6 mg/kg) group (Fig 3.23). The [NO]/ [ONOO-] decreased with an

113

increase dosage of SIN-1, but the ratio was still higher than the one found in the PILO

group.

The abnormal seizure-like EEG signals were noticed after the injection of SIN-1

simultaneously. The duration of the seizure events was recorded to analyze the severity of

the seizure events (Fig 3.24). The duration of the seizure events increased with the

increase in dosage of SIN-1, but lower than the duration of seizure events which were

induced by PILO. Thus, SIN-1 can induce similar seizure brain activities like PILO.

More severe seizure events were found in higher dosage of SIN-1.

Figure 3.21 Example amprograms showing NO and ONOO- release measured with

nanosensors, and EEG showing brain activity change after SIN-1 (1.2 µg/kg, IH) injected

into the brain of a SD rat.

114

Figure 3.22 Maximal NO (close bar) and ONOO- (open bar) concentration produced in

the brains of SD rats during the epileptic seizure-like events induced by 0.6 µg/kg and 1.2

µg/kg SIN-1 IH.

Figure 3.23 The ratio of NO to ONOO- concentration released from SD rats during

seizure events induced by 0.6 µg/kg and 1.2 µg/kg SIN-1 (close bar) and induced by 1.6

mg/kg PILO (open bar) .

115

Figure 3.24 The duration of seizure events induced by 0.6 µg/kg and 1.2 µg/kg SIN-1

(close bar) and induced by 1.6 mg/kg PILO (open bar).

3.4.2 Synthetic NO and ONOO- Solution Treatment

Next, synthetic NO and ONOO- standard solutions were used to increase the

exogenous amount of NO and ONOO- in the hippocampus region of SD rats. After

surgery, the synthetic NO and ONOO- standard solution mixture was injected into animal

IH after 15 min of baseline recording. Due to the short half-lives for both compounds,

different compositions of NO and ONOO- mixture solutions were injected repeatly into

the animal every 15 min with a total volume of 2 µl each time. The example

amperometric curves for NO and ONOO- concentration after the injection and brain

activity is shown in Figure 3.25. Vertically-dotted lines in amperometric curves indicate

the time when the injections were done. The high resolution EEG signals were shown in

Figure 3.25c (I, II, III, and IV) during the time of the seizure-like events after the mixture

of NO and ONOO- solutions were injected. The concentration of NO and ONOO- were

measured simultaneously (Fig 3.26). The [NO]/ [ONOO-] ratio at the injection time was

116

calculated (Fig 3.27). At I, NO was measured at 194 nM and ONOO - was 466 nM. The

[NO]/ [ONOO-] ratio was at 0.42. The seizure episode lasted for 18 s after this injection

(Fig 3.28). At II, NO was measured at 76 nM and ONOO- was 1639 nM. The [NO]/

[ONOO-] ratio was at 0.05. The duration of this seizure event increased to 95 s compared

to the injection I (Fig 3.28). At III, NO was recorded as 87 nM, while ONOO- was 1215

nM. The [NO]/ [ONOO-] ratio was at 0.07. The duration of the seizure event after the

injection was 97 s (Fig 3.28). All seizure-like events were observed right after the

injection of the NO and ONOO- mixture solution. The amplitude of the EEG signals

stayed high during the period from III to IV, which indicated the activation of abnormal

brain waves under the low [NO]/ [ONOO-] condition. However, at IV, a high amount of

NO was injected with a low amount of ONOO-, which increased the [NO]/ [ONOO-]

ratio to 0.74. The abnormal high brain activity was suppressed (Fig 3.25c IV).

Therefore, a low [NO]/ [ONOO-] can trigger the onset of abnormal brain activities.

The lower [NO]/ [ONOO-] was applied, the longer duration of the abnormal seizure-like

events were recorded. With a restoration of a high [NO]/ [ONOO-] (>0.7), the abnormal

seizure-like events can be suppressed.

117

5 sec

50 µV

20 sec

50 µV

20 sec

50 µV

20 sec

50 µV

III

II

I

IV

Figure 3.25 Seizure episodes triggered by the artifact change of the [NO] / [ONOO-].

Example of amperograms (a) NO and (b) ONOO- release after the exogenous injection of

NO and ONOO- solution (verticle dot lines represent the time when injection done). (c)

EEG recording shows seizure-like brain discharge (I, II, III) and non-seizure-like brain

discharge (IV) after each injection.

118

Figure 3.26 NO (open bars) and ONOO- (solid bars) concentration measured by

nanosensors during the time of seizure-like events.

Figure 3.27 The ratio of maximal NO to ONOO- concentration measured in the presence

or absence of seizure-like events.

119

Figure 3.28 Duration for seizure-like events after each injection.

3.4.3 O2·- Treatment

Superoxide anion (O2·-) solution was also injected into animals IH in the absence

of PILO to investigate the relationship among NO, ONOO - and O2·-. After surgery, O2

·-

solution was injected into the animal IH after 15 min of baseline recording and the

injection repeatedly performed every 15 min with different O2·- dosage. Example

amperometric curves for NO and ONOO- production and EEG signal after the injection

are shown in Figure 3.29. Vertically dotted lines in the amperometric curves indicate the

time when the injections were performed. Red arrows indicate the time when abnormal

EEG signals were observed. The high resolution of EEG graphs were shown in Figure

3.29c (I and II) during the seizure-like events were observed after the O2·- solution was

injected. The abnormal brain discharges were shown ~ 270 s after a O2·- solution IH

injection at the dosage of 0.52 μg (Fig 3.29c). NO and ONOO- production were measured

120

at when epileptic seizure-like events were observed (Fig 3.30). The ratio of [NO]/

[ONOO-] dropped below 0.30 at when the epileptic seizure-like events were observed

(Fig 3.31). The duration of the seizure events extended with a lower ratio of [NO]/

[ONOO-] was observed (Fig 3.32). Thus, O2·- induced epileptic seizure-like events with

~270 sec delay after injection and low [NO]/ [ONOO -] was recorded when the seizure-

like events were observed.

121

I

50 µV

20 sec

50 µV

50 sec

II

Figure 3.29 Seizure-like episodes triggered by exogenous O2·-. Amperograms of (a) NO

and (b) ONOO- release after exogenous injection of O2·- solution (vertically dotted lines

indicate the time of injection, arrows indicate the time of seizure-like events). (c) EEG

recording shows two seizure-like brain discharges change (I and II) after injections of

O2·-.

122

Figure 3.30 Maximal NO (open bars) and ONOO- (solid bars) concentration measured by

nanosensors at when seizure-like events were observed.

Figure 3.31 The ratio of maximal NO to ONOO- concentration measured at when seizure-

like events were observed.

123

Figure 3.32 Duration for seizure-like events after O2∙- (0.52 µg, IH) injection.

124

4. DISCUSSION

The study presented here shows for the first time the pivotal role of the

concentration of NO and the concentration of ONOO- imbalance in epileptic seizures;

that seizures can be triggered at a concentration ratio of [NO]/ [ONOO-] lower than 0.7;

and seizure events become more intense and frequent with the decrease of [NO]/ [ONOO-

]. This study also explains a controversial role of NO in epilepsy published previously by

others. Both NO and ONOO- are the crucial molecules that maintain epileptic or

antiepileptic redox environment in the brain and link the effect of both nitroxidative and

oxidative stress under pathogenic conditions.

4.1 The Establishment of Nanomedical System to Measure NO and ONOO- Release

in vivo during Epileptic Seizures

The nanomedical system we developed including the electrochemical sensors for

both NO and ONOO- with diameters less than 300 nm was used as an efficient system to

study the real time NO and ONOO- in vivo release during the process of epileptic seizures.

There was no previous data in NO and ONOO- release during the seizures being reported.

Indirect methods, such as NOS expression analysis, production of L-citrulline, and

fluorescent dyes for free radicals staining, were used to estimate the production of NO.

The effect of ONOO- cannot be separated from the effect of NO through the indirect

measurements. Furthermore, the effect of ONOO- may be wrongly attributed to the effect

of NO in epilepsy and the controversial effects of NO in epileptic seizures reported

previously. Our nanomedical system is able to measure NO and ONOO- real time release

125

in vivo during the seizure events and to analyze the contribution of ONOO- and NO

separately.

Moreover, the nanomedical system with EEG measuring system applied together

in the animal study revealed the real time release of NO and ONOO- accompanied with

the abnormal brain activity during seizures. The direct evidence for free radicals release

in the seizure events was provided.

Thus, the same or similar nanomedical system can be applied to other epileptic

studies, or other in vivo neurological disorder studies to measure the real time free

radicals release. More detailed molecular mechanism can be studied with the utilization

of this nanomedical system compared to the indirect methods.

4.2 Abnormal NO and ONOO- Release in PILO Induced Epileptic Seizures.

Our in vivo measurements showed that NO production was abnormally

suppressed but ONOO- production was significantly enhanced during the onset of PILO

induced epileptic seizures. The protein expression results also showed that nNOS, NOX4

and p67phox expression level were upregulated responding to the seizures, which indicated

the activation of NO and O2∙- production system during the seizures. Due to the diffusion-

controlled reaction between NO and O2∙-, the likelihood for ONOO- production under the

condition of elevated NO and O2∙- was high. The ONOO- real-time-measurement results

proved our thought (Fig 3.4 b). The production of ONOO- was elevated as the increase in

PILO dosage, with which more severe and intense epileptic seizures were observed (Fig

3.5). Also, more dysfunctional monomers of eNOS and nNOS were found in the native

eNOS and nNOS structures after PILO induced epileptic seizures, which also indicated

126

the damage in protein function by the high production of ONOO- in the brains (Fig 3.17

and 3.18). The elevated production of ONOO- would cause the structures of eNOS and

nNOS to be dysfunctional, which leaded to change enzyme from NO production to

produce more ONOO-. With less NO and more ONOO- being formed, more damage

would be caused in the brain, not only to eNOS and nNOS, but also to other antioxidant

enzymes, such as SOD. Mitochondrial respiration chain, DNA structure, membrane lipids

could all be involved in the damage process induced by ONOO-, which eventually cell

apoptosis and necrosis would occur62.

Thus, we hypothesized that both of nitroxidative and oxidative systems were

working cooperatively in the PILO-induced epileptic model we used (Fig 4.1). The

NMDA receptor can be activated during the brain insult by PILO initially and

intracellular Ca2+ increased, which resulted in the activation of eNOS and nNOS to

produce NO. At the same time, NADPH oxidase was also activated to exclusively

produce O2·- to increase the oxidative stress in the brain as well. Elevated production of

NO which reacted rapidly with O2·- to form ONOO-, the more reactive neurotoxic agent

in this situation. The high production of ONOO- can significantly reduce the

bioavailability of NO production and shift the balance between oxidants and antioxidants

systems. Finally, the nitroxidative stress was greatly enhanced during the seizures.

The ratio of [NO]/ [ONOO-] was used in this study to evaluate the balance

between the production of NO and ONOO-. This ratio was found to negatively correlate

with the intensity and severity of the seizure events. That is more intense and severe

epileptic seizures were observed at a lower [NO]/ [ONOO-] ratio (Fig 3.4 and 3.5). The

127

lowest ratio of [NO]/ [ONOO-] below 0.3 found in our study indicated the most severe

redox imbalance state which was accompanying with the most severe epileptic seizure

events.

Figure 4.1 Schematic diagram of the role of NO and ONOO- imbalance in PILO induced

epileptic seizures.

128

4.3 PILO-induced Epileptic Seizures Can be Regulated by the [NO]/ [ONOO-]

Modulators

4.3.1 The Effect of NOS Modulators in PILO-induced Epileptic Seizures

Several treatments were utilized in our study to modulate [NO]/ [ONOO-] in order

to alter the seizure events onset and severity. Both L-Arg and L-NAME treatments were

utilized to modulate the production of NO released from the epileptic animals.

L-Arg is the substrate of NOS. The productions of both NO and ONOO- were

significantly elevated in the presence of L-Arg (Fig 3.6 a and b). The enhanced

production of NO by L-Arg may due to both substrate level and enzyme function level.

The increase in substrate of NOS could increase the production of NO. And the elevated

L-Arg reduced the dysfunctional NOS monomer form and increased the functional NOS

dimer form (Fig 3.17 and 3.18), which also could increase the production of NO.

However, the elevated L-Arg also enhanced the expression level of NADPH oxidase (Fig

3.19), which indicated an elevated O2∙- production. With the enhancement in both of the

NO and O2∙- production systems in the presence of L-Arg, an even higher production of

ONOO- compared to PILO only group was observed (Fig 3.6 b). And more severe PILO-

induced seizure events were recorded in the presence of L-Arg than those ones in the

absence of L-Arg (Fig 3.7 and 3.8). According to the EEG results, the duration of seizure

events was significantly increased in the presence of L-Arg. This result is consistent with

the study V.Mollace et.al77 published in 1991,which was the first paper about the effect

of NO in epilepsy. They reported that the convulsant effect induced by NMDA was

significantly potentiated with L-arg treatment. Therefore, L-Arg failed to improve the

129

severity of the epileptic seizures but enhanced it may due to the higher production of

ONOO- in the presence of L-Arg, which the low [NO]/ [ONOO-] was still existing.

L-NAME is a non-selective inhibitor of NOS. In our study, the production of NO

and ONOO- was significantly reduced in the presence of L-NAME during the PILO-

induced epileptic seizures (Fig 3.9). The inhibition in NO production was more

efficiently than the inhibition in ONOO- production. L-NAME is an arginine-based

inhibitor, which blocks the binding side of L-Arg in the NOS and thus inhibits the

production of NO. With the reduced amount of NO, less ONOO - was formed accordingly.

But the expression level of NADPH oxidase in the presence of L-NAME was still

upregulated compared to saline group. The O2∙- production system was still activated.

Huge amount of O2∙- reacted with NO, which still produced a large amount of ONOO-

and reduced bioavailable NO even more. The activity of eNOS and nNOS was not only

inhibited by L-NAME, but also ONOO- produced in the system. More dysfunctional

monomers of NOS (eNOS and nNOS) and less functional dimmers of them were found in

the brain hippocampus samples after PILO-induced epileptic seizures in the presence of

L-NAME. The ratio of [NO]/ [ONOO-] decreased significantly to 0.06 ± 0.005. More

severe and intense seizure events were observed in this treatment (Fig 3.10). The latency

of the seizure events was significantly reduced in the presence of L-NAME compared to

the one in the absence of L-NAME. The duration of the seizure events also significantly

increased (Fig 3.11). The failure of L-NAME in reducing PILO-induced epileptic

seizures may result from both of the two conditions: cytotoxic ONOO- remained high and

the neuroprotective NO depleted.

130

The presence of L-Arg or L-NAME decreased the frequency of seizure events

occurring because of the longer duration of each seizure event compared to the PILO

group. The modulation of NO level by L-arg or L-NAME cannot prevent the epileptic

seizure events’ onset nor reduce the severity of the seizure. The regulation in NO

production system was not efficient enough to improve the low [NO]/ [ONOO-] and to

reduce the intensity and severity of PILO-induced epileptic seizures.

4.3.2 The Effect of ONOO- Scavenger in PILO-induced Epileptic Seizures

In our study, the elevation of ONOO- production was found to accompany

suppressed NO production during the PILO-induced epileptic seizures. Therefore,

MnTBAP as an antioxidant and a ONOO- scavenger was used to reduce ONOO-

concentration to restore the low [NO]/ [ONOO-]. The release of ONOO- was significantly

lower in the presence of MnTBAP with suppressed NO release and seizure episodes (Fig

3.12). The reduction of ONOO- by MnTBAP was as expected. The dysfunctional

structures of eNOS and nNOS after the PILO exposure were restored to the functional

dimer forms in the presence of MnTBAP. No obvious change in NADPH oxidase

expression was observed in the samples of MnTBAP group compared to those of PILO

group, which indicated the effect of MnTBAP in reducing ONOO- was not via the

inhibition of O2∙- production system but through the scavenging of ONOO- itself. The

ratio of [NO]/ [ONOO-] was significantly improved in the presence of MnTBAP during

the PILO-induced epileptic seizures compared the one found in PILO group. Both

duration and frequency of seizures events were reduced significantly in the presence of

MnTBAP (Fig 3.14). However, the initiation of the seizure episode was not prevented,

131

which is consistent with previous reports that oxidative stress and hippocampus damage

were reduced but behavior seizures were still observed with MnTBAP treatment98. Since

NO reacts with O2·- to form ONOO- under a diffusion-controlled rate (6.7 ± 0.9×109 M-1

s-1 )52, MnTBAP scavenged a huge amount of ONOO- produced from the reaction of NO

and O2·-. But the bioavailability of NO was still not recovered, which could result in the

failure of preventing epileptic seizure initiation. The ratio of [NO]/ [ONOO-] at 0.26 ±

0.03 still indicated a significant dysfunctional imbalance of nitroxidative and oxidative

stress.

Moreover, the treatment of MnTBAP indicated the reduction of ONOO- amount

was able to improve the severity and intensity of the seizure events but not efficient

enough to inhibit the seizures. In order to prevent the onset of epileptic seizures, both of

the inhibition in ONOO- production and the restoration in NO release should be achieved

at the same time.

4.3.3 The Effect of NADPH Oxidase Inhibitor in PILO-induced Epileptic Seizures

In order to achieve the inhibition of ONOO- production and the restoration of NO

bioavailability at the same time, the usage of VAS2870 was applied to inhibit the activity

of NADPH oxidase in our study. It has been reported after NMDA receptor activation,

O2·- was primarily produced by NADPH oxidase119. VAS2870 is an inhibitor for NADPH

oxidase to inhibit the production of O2·- and to reduce the oxidative stress in many

diseases125-126. In our study, ONOO- production was significantly inhibited with a

significantly restored NO release in the presence of VAS 2870 (Fig 3.15). A significantly

improved ratio of [NO]/ [ONOO-] was at 1.14 ± 0.25, at which condition no obvious

132

seizure episodes were observed (Fig 3.16). This proved the hypothesis above: with

elevation of NO production and suppression of ONOO- release occurring simultaneously,

epileptic seizures can be prevented.

Furthermore, the inhibition of the O2·- producing enzyme is more effective than

the usage of antioxidants in reducing the production of O2·-. In the presence of VAS2870,

less O2·- being formed can limit the production of ONOO -. As a consequence, less NO

would react with less O2·-. The NO can be returned to perform physiological functions,

such as S-nitrosylating NMDA receptor NR1 and NR2 subunits44 to inhibit the

excitotoxicity due to the excessive activation of NMDA receptor, which epileptic seizures

can be prevented. Therefore, NADPH oxidase can be a potential therapeutic target for

antiepileptic drug development.

133

Figure 4.2 Schematic diagram showing the mechanism of the prevention of epileptic

seizures by VAS 2870 (NADPH oxidase inhibitor)

4.4 Low [NO]/ [ONOO-] Can Trigger Seizures.

Artifact [NO] / [ONOO-] in the absence of a proepiletic agent mimicked the

process of the seizure onset which indicated the pivotal role of the imbalance between

NO and ONOO- in epilepstic seizure. Three sets of solutions were used: ONOO- donors;

synthetic NO and ONOO- standard solutions; and O2·- standard solutions.

4.4.1 Low [NO]/ [ONOO-] Induced by SIN-1 Triggered Seizure-like Events

Two dosages of SIN-1 solution were administrated into SD rats IH. Seizure-llike

events were observed in ~ 200 sec after the SIN-1 injection due to the kinetics of ONOO-

formation from SIN-1. In the physiological condition, SIN-1 first decomposes into NO

134

and O2·-, which then reacts to form ONOO-. With a higher SIN-1 dosage used, higher

ONOO- production was observed, and a lower [NO]/ [ONOO -] ratio was noticed (Fig

3.23). Seizure events with longer duration induced by SIN-1 at the dosage of 1.2 µg/kg

were recorded. The abnormal brain signals, which were seizure-like events, lasted for

~500 sec. Later, the brain activity returned to normal ~700 sec after the SIN-1 injection

resulted from the depletion of SIN-1. Therefore, seizure-like events triggered by SIN-1

may due to the low [NO]/ [ONOO-] induced by SIN-1. The duration of the events was

correlated with the ratio of [NO]/ [ONOO-].

4.4.2 Exogenous Low [NO]/ [ONOO-] Triggered Seizure-like Events

The synthetic NO and ONOO- standard solutions at different compositions were

injected into animals. Due to the short half-life for these two molecules, repeat injections

were done every 15 min. It was clearly revealed that when a ratio of [NO]/ [ONOO-]

reaches below 0.7, seizure-like events were observed immediately (Fig 3.25 I, II, and III).

The duration for each seizure event correlated with the ratio of [NO]/ [ONOO-]. The

smaller the ratio of [NO]/ [ONOO-] recorded, the longer the duration of seizure event

observed (Fig 3.27 and 3.28). However, when high doses of NO solution were combined

with low doses of ONOO- solution and injected ([NO]/ [ONOO-] = 0.74), normal brain

discharge was restored (Fig 3.25 IV). Therefore, the low ratio of [NO]/ [ONOO-] which

indicated the imbalance between NO and ONOO- did induce epileptic seizure-like events.

When the [NO]/ [ONOO-] got extreme low, the duration of the seizure events was

prolonged. This was the similar situation which was observed in the regulation of PILO-

induced epileptic seizures via different modulators.

135

4.4.3 Endogenous Low [NO]/ [ONOO-] Induced by O2·- Triggered Seizure-like

Events

Superoxide anion (O2·-) standard solutions were also injected into animals IH in

the absence of PILO to investigate the relationship among NO, ONOO- and O2·-. The

abnormal brain discharges were shown ~ 270 s after 0.52 μg of O2·- standard solution IH

injection (Fig 3.30c). When the epileptic seizure-like events were observed, the ratio of

[NO]/ [ONOO-] dropped below 0.30 (Fig 3.31). The same tendency was also observed

with respect to seizure duration: when a smaller NO/ONOO- ratio was recorded, a longer

seizure was observed (Fig 3.32). The similar pattern of [NO]/ [ONOO-] and seizure

events was found in both NO and ONOO- mixture solution treatment and O2·- treatment.

However, the delayed abnormal brain signals induced by O2·- could suggest that O2

·- can

also induce epileptic seizure events but through an indirect route by reacting with NO to

form ONOO-. The role of O2·- in epileptic seizures involved the enhanced production of

ONOO- and reduction of NO release. And overall, the ratio of [NO]/ [ONOO-] reduced

and the frequency and severity of epileptic seizures increased.

4.5 Conclusion

Our study here revealed the cross-linking between oxidative and nitroxidative

stress in the pathogenesis of epileptic seizures. The ratio of [NO]/ [ONOO-] determine the

redox environment in the brain and at the ratio of [NO]/ [ONOO -] below 0.7, epileptic

seizures are triggered. The further decrease of the ratio of [NO]/ [ONOO-] below 0.7, the

more increase in the frequency and severity of epileptic seizures. Therefore, in order to

prevent the onset of epileptic seizures, the reduction in ONOO- production and elevation

136

of NO release would be required. Successful pharmacological treatments should involve

interventions leading to increase [NO]/ [ONOO-] in the brain. The inhibition of O2∙-

production is more efficient than increase of NO production or scavenging ONOO -

concentration in the restoration of [NO]/ [ONOO-] balance. NADPH oxidase inhibitors

may be potential prime therapeutic agents to prevent seizure onset.

5. REFERENCES

1. "About Epilepsy". http://www.epilepsyfoundation.org/aboutepilepsy/index.cfm

(accessed 12/16/2013).

2. "Incidence and Prevalence". http://www.epilepsyfoundation.org/aboutepilepsy/

whatisepilepsy/statistics.cfm (accessed 01/05/2014).

3. Fisher, R. S.; van Emde Boas, W.; Blume, W.; Elger, C.; Genton, P.; Lee, P.;

Engel, J., Jr., Epileptic seizures and epilepsy: definitions proposed by the International

League Against Epilepsy (ILAE) and the International Bureau for Epilepsy (IBE).

Epilepsia 2005, 46 (4), 470-2.

4. Berg, A. T.; Berkovic, S. F.; Brodie, M. J.; Buchhalter, J.; Cross, J. H.; van Emde

Boas, W.; Engel, J.; French, J.; Glauser, T. A.; Mathern, G. W.; Moshe, S. L.; Nordli, D.;

Plouin, P.; Scheffer, I. E., Revised terminology and concepts for organization of seizures

and epilepsies: report of the ILAE Commission on Classification and Terminology, 2005-

2009. Epilepsia 2010, 51 (4), 676-85.

5. Milton, J., Epilepsy as a Dynamic Disease. Springer: 2003.

6. Reynolds, E. H.; Rodin, E., The clinical concept of epilepsy. Epilepsia 2009, 50

Suppl 3, 2-7.

7. Steinlein, O. K., Genetic mechanisms that underlie epilepsy. Nat Rev Neurosci

2004, 5 (5), 400-8.

8. Wasterlain, C. G.; Fujikawa, D. G.; Penix, L.; Sankar, R., Pathophysiological

mechanisms of brain damage from status epilepticus. Epilepsia 1993, 34 Suppl 1, S37-53.

http://www.epilepsyfoundation.org/aboutepilepsy/index.cfm

138

9. Kumar, S. S.; Buckmaster, P. S., Hyperexcitability, interneurons, and loss of

GABAergic synapses in entorhinal cortex in a model of temporal lobe epilepsy. J

Neurosci 2006, 26 (17), 4613-23.

10. Delgado-Escueta, A. V.; Wilson, W. A.; Olsen, R. W.; Porter, R. J., New waves

of research in the epilepsies: crossing into the third millennium. Adv Neurol 1999, 79, 3-

58.

11. Delorenzo, R. J.; Sun, D. A.; Deshpande, L. S., Cellular mechanisms underlying

acquired epilepsy: the calcium hypothesis of the induction and maintainance of epilepsy.

Pharmacol Ther 2005, 105 (3), 229-66.

12. Choi, D. W., Glutamate receptors and the induction of excitotoxic neuronal death.

Prog Brain Res 1994, 100, 47-51.

13. Pitkanen, A.; Lukasiuk, K., Mechanisms of epileptogenesis and potential

treatment targets. Lancet Neurol 2011, 10 (2), 173-86.

14. Galanopoulou, A. S.; Buckmaster, P. S.; Staley, K. J.; Moshe, S. L.; Perucca, E.;

Engel, J., Jr.; Loscher, W.; Noebels, J. L.; Pitkanen, A.; Stables, J.; White, H. S.; O'Brien,

T. J.; Simonato, M., Identification of new epilepsy treatments: issues in preclinical

methodology. Epilepsia 2012, 53 (3), 571-82.

15. Loscher, W.; Schmidt, D., Modern antiepileptic drug development has failed to

deliver: ways out of the current dilemma. Epilepsia 2011, 52 (4), 657-78.

16. Nathan, C.; Xie, Q. W., Nitric oxide synthases: roles, tolls, and controls. Cell

1994, 78 (6), 915-8.

139

17. Ignarro, L. J., Nitric oxide : biology and pathobiology. 1st ed.; Academic Press:

San Diego, 2000; p xvii, 1003 p., [10] p. of col. plates.

18. "Nitrogen Dioxide" http://www.epa.gov/air/nitrogenoxides/. (accessed

01/03/2014).

19. Malinski, T.; Taha, Z., Nitric oxide release from a single cell measured in situ by

a porphyrinic-based microsensor. Nature 1992, 358 (6388), 676-8.

20. Pacher, P.; Beckman, J. S.; Liaudet, L., Nitric oxide and peroxynitrite in health

and disease. Physiol Rev 2007, 87 (1), 315-424.

21. Malinski, T., Nitric oxide and nitroxidative stress in Alzheimer's disease. J

Alzheimers Dis 2007, 11 (2), 207-18.

22. Ignarro, L. J., Endothelium-derived nitric oxide: actions and properties. FASEB J

1989, 3 (1), 31-6.

23. Nakamura, T.; Cho, D. H.; Lipton, S. A., Redox regulation of protein misfolding,

mitochondrial dysfunction, synaptic damage, and cell death in neurodegenerative

diseases. Exp Neurol 2012, 238 (1), 12-21.

24. Venema, R. C.; Ju, H.; Zou, R.; Ryan, J. W.; Venema, V. J., Subunit interactions

of endothelial nitric-oxide synthase. Comparisons to the neuronal and inducible nitric-

oxide synthase isoforms. J Biol Chem 1997, 272 (2), 1276-82.

25. Groves, J. T.; Wang, C. C., Nitric oxide synthase: models and mechanisms. Curr

Opin Chem Biol 2000, 4 (6), 687-95.

26. Freire, M. A.; Guimaraes, J. S.; Leal, W. G.; Pereira, A., Pain modulation by

nitric oxide in the spinal cord. Front Neurosci 2009, 3 (2), 175-81.

140

27. Bredt, D. S.; Hwang, P. M.; Glatt, C. E.; Lowenstein, C.; Reed, R. R.; Snyder, S.

H., Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450

reductase. Nature 1991, 351 (6329), 714-8.

28. Nathan, C.; Xie, Q. W., Regulation of biosynthesis of nitric oxide. J Biol Chem

1994, 269 (19), 13725-8.

29. Bredt, D. S.; Ferris, C. D.; Snyder, S. H., Nitric oxide synthase regulatory sites.

Phosphorylation by cyclic AMP-dependent protein kinase, protein kinase C, and

calcium/calmodulin protein kinase; identification of flavin and calmodulin binding sites.

J Biol Chem 1992, 267 (16), 10976-81.

30. Raman, C. S.; Li, H.; Martasek, P.; Kral, V.; Masters, B. S.; Poulos, T. L., Crystal

structure of constitutive endothelial nitric oxide synthase: a paradigm for pterin function

involving a novel metal center. Cell 1998, 95 (7), 939-50.

31. Ignarro, L. J.; Harbison, R. G.; Wood, K. S.; Kadowitz, P. J., Activation of

purified soluble guanylate cyclase by endothelium-derived relaxing factor from

intrapulmonary artery and vein: stimulation by acetylcholine, bradykinin and arachidonic

acid. J Pharmacol Exp Ther 1986, 237 (3), 893-900.

32. Furchgott, R. F.; Zawadzki, J. V., The obligatory role of endothelial cells in the

relaxation of arterial smooth muscle by acetylcholine. Nature 1980, 288 (5789), 373-6.

33. Furchgott, R. F.; Carvalho, M. H.; Khan, M. T.; Matsunaga, K., Evidence for

endothelium-dependent vasodilation of resistance vessels by acetylcholine. Blood Vessels

1987, 24 (3), 145-9.

141

34. Ignarro, L. J.; Adams, J. B.; Horwitz, P. M.; Wood, K. S., Activation of soluble

guanylate cyclase by NO-hemoproteins involves NO-heme exchange. Comparison of

heme-containing and heme-deficient enzyme forms. J Biol Chem 1986, 261 (11), 4997-

5002.

35. Palmer, R. M.; Ferrige, A. G.; Moncada, S., Nitric oxide release accounts for the

biological activity of endothelium-derived relaxing factor. Nature 1987, 327 (6122), 524-

6.

36. Garthwaite, J., Glutamate, nitric oxide and cell-cell signalling in the nervous

system. Trends Neurosci 1991, 14 (2), 60-7.

37. Bredt, D. S.; Hwang, P. M.; Snyder, S. H., Localization of nitric oxide synthase

indicating a neural role for nitric oxide. Nature 1990, 347 (6295), 768-70.

38. Boehning, D.; Snyder, S. H., Novel neural modulators. Annu Rev Neurosci 2003,

26, 105-31.

39. Chiueh, C. C., Neurobiology of NO. and .OH: basic research and clinical

relevance. Ann N Y Acad Sci 1994, 738, 279-81.

40. Chung, K. K.; Thomas, B.; Li, X.; Pletnikova, O.; Troncoso, J. C.; Marsh, L.;

Dawson, V. L.; Dawson, T. M., S-nitrosylation of parkin regulates ubiquitination and

compromises parkin's protective function. Science 2004, 304 (5675), 1328-31.

41. Gu, Z.; Kaul, M.; Yan, B.; Kridel, S. J.; Cui, J.; Strongin, A.; Smith, J. W.;

Liddington, R. C.; Lipton, S. A., S-nitrosylation of matrix metalloproteinases: signaling

pathway to neuronal cell death. Science 2002, 297 (5584), 1186-90.

142

42. Lipton, S. A.; Choi, Y. B.; Pan, Z. H.; Lei, S. Z.; Chen, H. S.; Sucher, N. J.;

Loscalzo, J.; Singel, D. J.; Stamler, J. S., A redox-based mechanism for the

neuroprotective and neurodestructive effects of nitric oxide and related nitroso-

compounds. Nature 1993, 364 (6438), 626-32.

43. Stamler, J. S.; Toone, E. J.; Lipton, S. A.; Sucher, N. J., (S)NO signals:

translocation, regulation, and a consensus motif. Neuron 1997, 18 (5), 691-6.

44. Jaffrey, S. R.; Erdjument-Bromage, H.; Ferris, C. D.; Tempst, P.; Snyder, S. H.,

Protein S-nitrosylation: a physiological signal for neuronal nitric oxide. Nat Cell Biol

2001, 3 (2), 193-7.

45. Choi, Y. B.; Tenneti, L.; Le, D. A.; Ortiz, J.; Bai, G.; Chen, H. S.; Lipton, S. A.,

Molecular basis of NMDA receptor-coupled ion channel modulation by S-nitrosylation.

Nat Neurosci 2000, 3 (1), 15-21.

46. Mannick, J. B.; Schonhoff, C.; Papeta, N.; Ghafourifar, P.; Szibor, M.; Fang, K.;

Gaston, B., S-Nitrosylation of mitochondrial caspases. J Cell Biol 2001, 154 (6), 1111-6.

47. Rauhala, P.; Lin, A. M.; Chiueh, C. C., Neuroprotection by S-nitrosoglutathione

of brain dopamine neurons from oxidative stress. FASEB J 1998, 12 (2), 165-73.

48. Chiueh, C. C., Neuroprotective properties of nitric oxide. Ann N Y Acad Sci 1999,

890, 301-11.

49. Gryglewski, R. J.; Palmer, R. M.; Moncada, S., Superoxide anion is involved in

the breakdown of endothelium-derived vascular relaxing factor. Nature 1986, 320 (6061),

454-6.

50. Beckman, J. S., Ischaemic injury mediator. Nature 1990, 345 (6270), 27-8.

143

51. Beckman, J. S.; Beckman, T. W.; Chen, J.; Marshall, P. A.; Freeman, B. A.,

Apparent hydroxyl radical production by peroxynitrite: implications for endothelial

injury from nitric oxide and superoxide. Proc Natl Acad Sci U S A 1990, 87 (4), 1620-4.

52. Beckman, J. S.; Koppenol, W. H., Nitric oxide, superoxide, and peroxynitrite: the

good, the bad, and ugly. Am J Physiol 1996, 271 (5 Pt 1), C1424-37.

53. Schramm, A.; Matusik, P.; Osmenda, G.; Guzik, T. J., Targeting NADPH

oxidases in vascular pharmacology. Vascul Pharmacol 2012, 56 (5-6), 216-31.

54. Mythri, R. B.; Venkateshappa, C.; Harish, G.; Mahadevan, A.; Muthane, U. B.;

Yasha, T. C.; Srinivas Bharath, M. M.; Shankar, S. K., Evaluation of markers of

oxidative stress, antioxidant function and astrocytic proliferation in the striatum and

frontal cortex of Parkinson's disease brains. Neurochem Res 2011, 36 (8), 1452-63.

55. Ansari, M. A.; Scheff, S. W., Oxidative stress in the progression of Alzheimer

disease in the frontal cortex. J Neuropathol Exp Neurol 2010, 69 (2), 155-67.

56. Barber, S. C.; Shaw, P. J., Oxidative stress in ALS: key role in motor neuron

injury and therapeutic target. Free Radic Biol Med 2010, 48 (5), 629-41.

57. Schnabel, R.; Blankenberg, S., Oxidative stress in cardiovascular disease:

successful translation from bench to bedside? Circulation 2007, 116 (12), 1338-40.

58. Fridovich, I., Superoxide radical and superoxide dismutases. Annu Rev Biochem

1995, 64, 97-112.

59. Xu, J.; Zou, M. H., Molecular insights and therapeutic targets for diabetic

endothelial dysfunction. Circulation 2009, 120 (13), 1266-86.

144

60. Bonfoco, E.; Krainc, D.; Ankarcrona, M.; Nicotera, P.; Lipton, S. A., Apoptosis

and necrosis: two distinct events induced, respectively, by mild and intense insults with

N-methyl-D-aspartate or nitric oxide/superoxide in cortical cell cultures. Proc Natl Acad

Sci U S A 1995, 92 (16), 7162-6.

61. Bouchier-Hayes, L.; Lartigue, L.; Newmeyer, D. D., Mitochondria:

pharmacological manipulation of cell death. J Clin Invest 2005, 115 (10), 2640-7.

62. Szabo, C.; Ischiropoulos, H.; Radi, R., Peroxynitrite: biochemistry,

pathophysiology and development of therapeutics. Nat Rev Drug Discov 2007, 6 (8),

662-80.

63. Alvarez, B.; Demicheli, V.; Duran, R.; Trujillo, M.; Cervenansky, C.; Freeman,

B. A.; Radi, R., Inactivation of human Cu,Zn superoxide dismutase by peroxynitrite and

formation of histidinyl radical. Free Radic Biol Med 2004, 37 (6), 813-22.

64. Forstermann, U.; Munzel, T., Endothelial nitric oxide synthase in vascular

disease: from marvel to menace. Circulation 2006, 113 (13), 1708-14.

65. Radi, R.; Cassina, A.; Hodara, R.; Quijano, C.; Castro, L., Peroxynitrite reactions

and formation in mitochondria. Free Radic Biol Med 2002, 33 (11), 1451-64.

66. Niles, J. C.; Wishnok, J. S.; Tannenbaum, S. R., Peroxynitrite-induced oxidation

and nitration products of guanine and 8-oxoguanine: structures and mechanisms of

product formation. Nitric Oxide 2006, 14 (2), 109-21.

67. Drummond, G. R.; Selemidis, S.; Griendling, K. K.; Sobey, C. G., Combating

oxidative stress in vascular disease: NADPH oxidases as therapeutic targets. Nat Rev

Drug Discov 2011, 10 (6), 453-71.

145

68. Finkel, T.; Holbrook, N. J., Oxidants, oxidative stress and the biology of ageing.

Nature 2000, 408 (6809), 239-47.

69. Meldrum, B.; Garthwaite, J., Excitatory Amino-Acid Neurotoxicity and

Neurodegenerative Disease. Trends in Pharmacological Sciences 1990, 11 (9), 379-387.

70. Olney, J. W., Brain lesions, obesity, and other disturbances in mice treated with

monosodium glutamate. Science 1969, 164 (3880), 719-21.

71. Kabuto, H.; Yokoi, I.; Habu, H.; Willmore, L. J.; Mori, A.; Ogawa, N., Reduction

in nitric oxide synthase activity with development of an epileptogenic focus induced by

ferric chloride in the rat brain. Epilepsy Res 1996, 25 (2), 65-8.

72. Hicks, T. P.; Conti, F., Amino acids as the source of considerable excitation in

cerebral cortex. Can J Physiol Pharmacol 1996, 74 (4), 341-61.

73. Aamodt, S. M.; Constantine-Paton, M., The role of neural activity in synaptic

development and its implications for adult brain function. Adv Neurol 1999, 79, 133-44.

74. Maggio, R.; Fumagalli, F.; Donati, E.; Barbier, P.; Racagni, G.; Corsini, G. U.;

Riva, M., Inhibition of nitric oxide synthase dramatically potentiates seizures induced by

kainic acid and pilocarpine in rats. Brain Res 1995, 679 (1), 184-7.

75. Lapouble, E.; Montecot, C.; Sevestre, A.; Pichon, J., Phosphinothricin induces

epileptic activity via nitric oxide production through NMDA receptor activation in adult

mice. Brain Res 2002, 957 (1), 46-52.

76. Noyan, B.; Jensen, M. S.; Danscher, G., The lack of effects of zinc and nitric

oxide in initial state of pilocarpine-induced seizures. Seizure 2007, 16 (5), 410-6.

146

77. Mollace, V.; Bagetta, G.; Nistico, G., Evidence That L-Arginine Possesses

Proconvulsant Effects Mediated through Nitric-Oxide. Neuroreport 1991, 2 (5), 269-272.

78. Hamani, C.; Tenorio, F.; Mendez-Otero, R.; Mello, L. E., Loss of NADPH

diaphorase-positive neurons in the hippocampal formation of chronic pilocarpine-

epileptic rats. Hippocampus 1999, 9 (3), 303-13.

79. Nagatomo, I.; Akasaki, Y.; Uchida, M.; Tominaga, M.; Kuchiiwa, S.; Nakagawa,

S.; Takigawa, M., Kainic and domoic acids differentially affect NADPH-diaphorase

neurons in the mouse hippocampal formation. Brain Res Bull 1999, 48 (3), 277-82.

80. Fedele, E.; Raiteri, M., In vivo studies of the cerebral glutamate

receptor/NO/cGMP pathway. Prog Neurobiol 1999, 58 (1), 89-120.

81. Uchida, M.; Nagatomo, I.; Akasaki, Y.; Tominaga, M.; Hashiguchi, W.;

Kuchiiwa, S.; Nakagawa, S.; Takigawa, M., Nitric oxide production is decreased in the

brain of the seizure susceptible EL mouse. Brain Res Bull 1999, 50 (4), 223-7.

82. Tsuda, M.; Shimizu, N.; Yajima, Y.; Suzuki, T.; Misawa, M., Role of nitric oxide

in the hypersusceptibility to pentylenetetrazole-induced seizure in diazepam-withdrawn

mice. Eur J Pharmacol 1998, 344 (1), 27-30.

83. Przegalinski, E.; Baran, L.; Siwanowicz, J., The role of nitric oxide in the kainate-

induced seizures in mice. Neurosci Lett 1994, 170 (1), 74-6.

84. Kaputlu, I.; Uzbay, T., L-NAME inhibits pentylenetetrazole and strychnine-

induced seizures in mice. Brain Res 1997, 753 (1), 98-101.

147

85. Osonoe, K.; Mori, N.; Suzuki, K.; Osonoe, M., Antiepileptic effects of inhibitors

of nitric oxide synthase examined in pentylenetetrazol-induced seizures in rats. Brain Res

1994, 663 (2), 338-40.

86. Tsuda, M.; Suzuki, T.; Misawa, M., Aggravation of DMCM-induced seizure by

nitric oxide synthase inhibitors in mice. Life Sci 1997, 60 (23), PL339-43.

87. Kirkby, R. D.; Carroll, D. M.; Grossman, A. B.; Subramaniam, S., Factors

determining proconvulsant and anticonvulsant effects of inhibitors of nitric oxide

synthase in rodents. Epilepsy Res 1996, 24 (2), 91-100.

88. Kashihara, K.; Sakai, K.; Marui, K.; Shohmori, T., Kainic acid may enhance

hippocampal NO generation of awake rats in a seizure stage-related fashion. Neurosci

Res 1998, 32 (3), 189-94.

89. Takei, Y.; Takashima, S.; Ohyu, J.; Takami, T.; Miyajima, T.; Hoshika, A.,

Effects of nitric oxide synthase inhibition on the cerebral circulation and brain damage

during kainic acid-induced seizures in newborn rabbits. Brain Dev 1999, 21 (4), 253-9.

90. Przegalinski, E.; Baran, L.; Siwanowicz, J., The role of nitric oxide in chemically-

and electrically-induced seizures in mice. Neurosci Lett 1996, 217 (2-3), 145-8.

91. Borowicz, K. K.; Starownik, R.; Kleinrok, Z.; Czuczwar, S. J., The influence of

L-NG-nitroarginine methyl ester, an inhibitor of nitric oxide synthase, upon the

anticonvulsive activity of conventional antiepileptic drugs against maximal electroshock

in mice. J Neural Transm 1998, 105 (1), 1-12.

148

92. Urbanska, E. M.; Drelewska, E.; Borowicz, K. K.; Blaszczak, P.; Kleinrok, Z.;

Czuczwar, S. J., NG-nitro-L-arginine, a nitric oxide synthase inhibitor, and seizure

susceptibility in four seizure models in mice. J Neural Transm 1996, 103 (10), 1145-52.

93. Del-Bel, E. A.; Oliveira, P. R.; Oliveira, J. A.; Mishra, P. K.; Jobe, P. C.; Garcia-

Cairasco, N., Anticonvulsant and proconvulsant roles of nitric oxide in experimental

epilepsy models. Braz J Med Biol Res 1997, 30 (8), 971-9.

94. Rigaud-Monnet, A. S.; Heron, A.; Seylaz, J.; Pinard, E., Effect of inhibiting NO

synthesis on hippocampal extracellular glutamate concentration in seizures induced by

kainic acid. Brain Res 1995, 673 (2), 297-303.

95. Van Leeuwen, R.; De Vries, R.; Dzoljic, M. R., 7-Nitro indazole, an inhibitor of

neuronal nitric oxide synthase, attenuates pilocarpine-induced seizures. Eur J Pharmacol

1995, 287 (2), 211-3.

96. Alexander, C. B.; Ellmore, T. M.; Kokate, T. G.; Kirkby, R. D., Further studies on

anti- and proconvulsant effects of inhibitors of nitric oxide synthase in rodents. Eur J

Pharmacol 1998, 344 (1), 15-25.

97. Proctor, M. R.; Fornai, F.; Afshar, J. K.; Gale, K., The role of nitric oxide in

focally-evoked limbic seizures. Neuroscience 1997, 76 (4), 1231-6.

98. Liang, L. P.; Ho, Y. S.; Patel, M., Mitochondrial superoxide production in

kainate-induced hippocampal damage. Neuroscience 2000, 101 (3), 563-70.

99. Sashindranath, M.; McLean, K. J.; Trounce, I. A.; Cotton, R. G.; Cook, M. J.,

Early hippocampal oxidative stress is a direct consequence of seizures in the rapid

electrical amygdala kindling model. Epilepsy Res 2010, 90 (3), 285-94.

149

100. Patel, M., Mitochondrial dysfunction and oxidative stress: cause and consequence

of epileptic seizures. Free Radic Biol Med 2004, 37 (12), 1951-62.

101. Kudin, A. P.; Kudina, T. A.; Seyfried, J.; Vielhaber, S.; Beck, H.; Elger, C. E.;

Kunz, W. S., Seizure-dependent modulation of mitochondrial oxidative phosphorylation

in rat hippocampus. Eur J Neurosci 2002, 15 (7), 1105-14.

102. Patel, M.; Liang, L. P.; Hou, H.; Williams, B. B.; Kmiec, M.; Swartz, H. M.;

Fessel, J. P.; Roberts, L. J., 2nd, Seizure-induced formation of isofurans: novel products

of lipid peroxidation whose formation is positively modulated by oxygen tension. J

Neurochem 2008, 104 (1), 264-70.

103. Rowley, S.; Patel, M., Mitochondrial involvement and oxidative stress in

temporal lobe epilepsy. Free Radic Biol Med 2013, 62, 121-31.

104. Seegers, U.; Potschka, H.; Loscher, W., Transient increase of P-glycoprotein

expression in endothelium and parenchyma of limbic brain regions in the kainate model

of temporal lobe epilepsy. Epilepsy Res 2002, 51 (3), 257-68.

105. Nagatomo, I.; Akasaki, Y.; Uchida, M.; Kuchiiwa, S.; Nakagawa, S.; Takigawa,

M., Influence of dietary zinc on convulsive seizures and hippocampal NADPH

diaphorase-positive neurons in seizure susceptible EL mouse. Brain Res 1998, 789 (2),

213-20.

106. Chuang, Y. C.; Chen, S. D.; Lin, T. K.; Liou, C. W.; Chang, W. N.; Chan, S. H.;

Chang, A. Y., Upregulation of nitric oxide synthase II contributes to apoptotic cell death

in the hippocampal CA3 subfield via a cytochrome c/caspase-3 signaling cascade

150

following induction of experimental temporal lobe status epilepticus in the rat.

Neuropharmacology 2007, 52 (5), 1263-73.

107. Cendes, F., Febrile seizures and mesial temporal sclerosis. Curr Opin Neurol

2004, 17 (2), 161-4.

108. Engel, J., Jr.; Pitkanen, A.; Loeb, J. A.; Dudek, F. E.; Bertram, E. H., 3rd; Cole,

A. J.; Moshe, S. L.; Wiebe, S.; Jensen, F. E.; Mody, I.; Nehlig, A.; Vezzani, A., Epilepsy

biomarkers. Epilepsia 2013, 54 Suppl 4, 61-9.

109. Brovkovych, V.; Patton, S.; Brovkovych, S.; Kiechle, F.; Huk, I.; Malinski, T., In

situ measurement of nitric oxide, superoxide and peroxynitrite during endotoxemia. J

Physiol Pharmacol 1997, 48 (4), 633-44.

110. Malinski, T.; Taha, Z.; Grunfeld, S.; Burewicz, A.; Tomboulian, P.; Kiechle, F.,

Measurements of Nitric-Oxide in Biological-Materials Using a Porphyrinic Microsensor.

Analytica Chimica Acta 1993, 279 (1), 135-140.

111. Malinski, T.; Huk, I., Measurement of nitric oxide in single cells and tissue using

a porphyrinic microsensor. Curr Protoc Neurosci 2001, Chapter 7, Unit7 14.

112. Martin Feelisch, J. S. S., The Oxyhemoglobin Assay. John Wiley & Sons Ltd:

1996; p 455-478.

113. Xue, J.; Ying, X.; Chen, J.; Xian, Y.; Jin, L., Amperometric ultramicrosensors for

peroxynitrite detection and its application toward single myocardial cells. Anal Chem

2000, 72 (21), 5313-21.

114. Koppenol, W. H.; Kissner, R.; Beckman, J. S., Syntheses of peroxynitrite: to go

with the flow or on solid grounds? Methods Enzymol 1996, 269, 296-302.

151

115. Reed, J. W.; Ho, H. H.; Jolly, W. L., Chemical Syntheses with a Quenched Flow

Reactor - Hydroxytrihydroborate and Peroxynitrite. Journal of the American Chemical

Society 1974, 96 (4), 1248-1249.

116. Turski, W. A.; Cavalheiro, E. A.; Schwarz, M.; Czuczwar, S. J.; Kleinrok, Z.;

Turski, L., Limbic seizures produced by pilocarpine in rats: behavioural,

electroencephalographic and neuropathological study. Behav Brain Res 1983, 9 (3), 315-

35.

117. Reiter, C. D.; Teng, R. J.; Beckman, J. S., Superoxide reacts with nitric oxide to

nitrate tyrosine at physiological pH via peroxynitrite. J Biol Chem 2000, 275 (42), 32460-

6.

118. Zhao, R.; Zhang, X. Y.; Yang, J.; Weng, C. C.; Jiang, L. L.; Zhang, J. W.; Shu, X.

Q.; Ji, Y. H., Anticonvulsant effect of BmK IT2, a sodium channel-specific neurotoxin, in

rat models of epilepsy. Br J Pharmacol 2008, 154 (5), 1116-24.

119. Brennan, A. M.; Suh, S. W.; Won, S. J.; Narasimhan, P.; Kauppinen, T. M.; Lee,

H.; Edling, Y.; Chan, P. H.; Swanson, R. A., NADPH oxidase is the primary source of

superoxide induced by NMDA receptor activation. Nat Neurosci 2009, 12 (7), 857-63.

120. Bedard, K.; Krause, K. H., The NOX family of ROS-generating NADPH

oxidases: physiology and pathophysiology. Physiol Rev 2007, 87 (1), 245-313.

121. Vallet, P.; Charnay, Y.; Steger, K.; Ogier-Denis, E.; Kovari, E.; Herrmann, F.;

Michel, J. P.; Szanto, I., Neuronal expression of the NADPH oxidase NOX4, and its

regulation in mouse experimental brain ischemia. Neuroscience 2005, 132 (2), 233-8.

152

122. Hwang, J.; Ing, M. H.; Salazar, A.; Lassegue, B.; Griendling, K.; Navab, M.;

Sevanian, A.; Hsiai, T. K., Pulsatile versus oscillatory shear stress regulates NADPH

oxidase subunit expression: implication for native LDL oxidation. Circ Res 2003, 93

(12), 1225-32.

123. Pedruzzi, E.; Guichard, C.; Ollivier, V.; Driss, F.; Fay, M.; Prunet, C.; Marie, J.

C.; Pouzet, C.; Samadi, M.; Elbim, C.; O'Dowd, Y.; Bens, M.; Vandewalle, A.;

Gougerot-Pocidalo, M. A.; Lizard, G.; Ogier-Denis, E., NAD(P)H oxidase Nox-4

mediates 7-ketocholesterol-induced endoplasmic reticulum stress and apoptosis in human

aortic smooth muscle cells. Mol Cell Biol 2004, 24 (24), 10703-17.

124. Noh, K. M.; Koh, J. Y., Induction and activation by zinc of NADPH oxidase in

cultured cortical neurons and astrocytes. J Neurosci 2000, 20 (23), RC111.

125. Stielow, C.; Catar, R. A.; Muller, G.; Wingler, K.; Scheurer, P.; Schmidt, H. H.;

Morawietz, H., Novel Nox inhibitor of oxLDL-induced reactive oxygen species

formation in human endothelial cells. Biochem Biophys Res Commun 2006, 344 (1), 200-

5.

126. ten Freyhaus, H.; Huntgeburth, M.; Wingler, K.; Schnitker, J.; Baumer, A. T.;

Vantler, M.; Bekhite, M. M.; Wartenberg, M.; Sauer, H.; Rosenkranz, S., Novel Nox

inhibitor VAS2870 attenuates PDGF-dependent smooth muscle cell chemotaxis, but not

proliferation. Cardiovasc Res 2006, 71 (2), 331-41.

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