NEUROPROTECTION AGAINST METHAMPHETAMINE INDUCED 
NEUROTOXICITY: APPLICATIONS FOR PARKINSON?S DISEASE 
 
Except where reference is made to the work of others, the work described in this 
dissertation is my own work or was done in collaboration with my advisory 
committee. This dissertation does not include proprietary or  
classified information. 
 
 
_________________________________ 
Bessy Thrash 
 
 
Certificate of Approval: 
 
 
_________________________________    _________________________________ 
Vishnu Suppiramaniam, Co-Chair      Muralikrishnan Dhanasekaran, Co-Chair 
Associate Professor        Assistant Professor 
Pharmacal Sciences        Pharmacal Sciences 
 
 
_________________________________         _________________________________ 
Daniel L. Parsons        William R. Ravis 
Professor         Professor 
Pharmacal Sciences        Pharmacal Sciences 
 
 
 
_________________________________ 
  George T. Flowers 
  Dean 
  Graduate School 
 
NEUROPROTECTION AGAINST METHAMPHETAMINE INDUCED 
NEUROTOXICITY: APPLICATIONS FOR PARKINSON?S DISEASE 
 
 
 
Bessy Thrash 
 
 
 
A Dissertation 
Submitted to 
the Graduate Faculty of 
Auburn University 
in Partial Fulfillment of the 
Requirements for the 
Degree of 
Doctor of Philosophy 
Auburn, Alabama 
May 9, 2009 
 
iii 
 
NEUROPROTECTION AGAINST METHAMPHETAMINE INDUCED 
NEUROTOXICITY: APPLICATIONS FOR PARKINSON?S DISEASE 
 
 
 
Bessy Thrash 
 
 
 
 
 
 
 
Permission is granted to Auburn University to make copies of this dissertation at its 
direction, upon request of individuals or institutions and at their expense. 
The author reserves all publication rights. 
 
 
 
 
 
 
_________________________________ 
                             Signature of Author 
 
 
 
_________________________________ 
                             Date of Graduation 
 
iv 
 
VITA 
 
 
Bessy Michelle Thrash, daughter of Michael Harmon Thrash and Kelly Nichols 
Thrash, was born on April 28, 1979. She is the third of four daughters born to Mr. and 
Mrs. Thrash, and was raised in the suburbs of Birmingham, Alabama. She attended the 
University of Alabama at Birmingham on a full academic scholarship, and earned her 
first baccalaureate degree in Criminal Justice and Chemistry in May of 2002. She earned 
her second baccalaureate degree in Biology in August of 2003 also from the University of 
Alabama at Birmingham. As an undergraduate, Ms. Thrash participated in the 
university?s interdisciplinary Honors Program and as a result graduated with full 
university honors. She remained at the University of Alabama at Birmingham, joining the 
Masters Program in Forensic Sciences. She earned a masters degree in Forensic Sciences 
in May 2005. She worked for the Alabama Department of Forensic Sciences from 2003 
to 2005. In August 2005 she joined the Auburn University doctoral program in Pharmacal 
Sciences, where she was funded as a graduate teaching and research assistant. Ms. Thrash 
has one son, John Carson Thrash, born on November 24, 1999. 
 
 
 
 
 
 
 
v 
 
DISSERTATION ABSTRACT 
NEUROPROTECTION AGAINST METHAMPHETAMINE INDUCED 
NEUROTOXICITY: APPLICATIONS FOR PARKINSON?S DISEASE 
Bessy Thrash 
Doctor of Philosophy, May 9, 2009 
(M.S.F.S University of Alabama at Birmingham, May 2005 
B.S. University of Alabama at Birmingham, August 2003 
B.S. University of Alabama at Birmingham, May 2002) 
 
173 Typed pages 
Directed by Muralikrishnan Dhanasekaran and Vishnu Suppiramaniam 
Parkinson?s disease is a progressive neurodegenerative disorder associated with 
selective degeneration of nigrostriatal dopaminergic neurons. It is the most common of 
the neurodegenerative movement disorders, affecting approximately 1% of the population 
over age 65. In Parkinson?s disease, the shortage of dopamine in the striatum causes 
various motor abnormalities and complete immobility usually occurs, despite treatment. 
Studies have implicated exposure to toxins like methamphetamine as contributors to the 
development of Parkinson?s disease. There is a significant degree of striatal dopamine 
depletion produced by methamphetamine, making the toxin useful in the creation of an 
animal model to study the disease. In this study a mouse model of Parkinson?s disease 
was created by administering two intraperitoneal injections of methamphetamine 
(10mg/kg) two hours apart. 
vi 
 
The neurotoxic effects of methamphetamine and neuroprotective effect of 
ramelteon, amantadine and salicylic acid were evaluated both in vitro and in vivo. Effects 
of reduced energy metabolism and oxidative stress were evaluated using biochemical 
assays. Changes in neurotransmitter levels (norepinepherine, serotonin and its metabolite, 
5-HIAA, dopamine and its metabolites, DOPAC and HVA) were measured using high 
pressure liquid chromatography (HPLC)-electrochemical detection. In addition, 
behavioral analysis was performed on the treated mice to evaluate the effect of 
methamphetamine on movement (catalepsy, akinesia, swim score, straub tail and tremor). 
Results of these studies revealed that methamphetamine caused significant generation of 
reactive oxygen species, significantly increased superoxide dismutase activity and 
significantly decreased Complex I activity both in vitro and in vivo. Methamphetamine 
caused significant dopamine depletion in the striatum of treated mice and caused 
significant alterations in movement behaviors as compared to controls. Ramelteon did not 
block the neurotoxic effects of methamphetamine. Amantadine (1mg/kg) blocked the 
neurotoxic effects of methamphetamine and salicylic acid (50 & 100mg/kg) also blocked 
the neurotoxic effects of methamphetamine, causing a reduced amount of dopamine 
depletion in the striatum. 
 
 
 
 
 
 
 
 
 
vii 
 
ACKNOWLEDGEMENTS 
I would like to thank God for giving me life that I might enjoy all things, for 
giving me the courage to follow my dreams and the wisdom and knowledge to see them 
through. Without Him, I am nothing.  
I would like to express gratitude for the guidance of my major advisors, Dr. 
Muralikrishnan Dhanasekaran and Dr. Vishnu Suppiramaniam. I would like to thank Dr. 
Daniel Parsons and Dr. William Ravis for their kindness and always encouraging words. 
I would like to thank Dr. Kevin Huggins for his review of this dissertation. I would also 
like to thank all of my former and current colleagues for their support and help. 
I offer sincere thanks to Mrs. Kandi Dawson for so willingly offering her 
friendship and help, and for making Auburn not seem so far away from home for me. I 
want to thank all of the friends that made up my pharmacy school ?family?. Thank you 
all for being my shoulders to cry on and the arms that held me up.  
I want to thank my parents for always believing in me and teaching me how to 
shoot for the stars; my three sisters for being the best friends anyone could ever have and 
for always being just a phone call away; and my nephews and niece for all the hugs and 
love they have given me over the years. Most of all, I want to thank my son, Carson, for 
his faith and pride in me, his love and encouragement, for little arms that were always 
thrown around me just when I needed it the most, and for being my inspiration.  
viii 
 
Style manual or journal used 
The Journal of Neuroscience 
 
Computer software used 
Microsoft Word 
Microsoft Excel 
Microsoft Power Point 
Adobe Photoshop 
Sigma stat. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
ix 
 
TABLE OF CONTENTS 
LIST OF FIGURES ......................................................................................................... xiv 
LIST OF TABLES ......................................................................................................... xviii 
1. INTRODUCTION ...........................................................................................................1 
2. REVIEW OF LITERATURE ..........................................................................................7 
2.1 Clinical and pathologic features of Parkinson?s disease ....................................7 
2.2 Etiology and treatment of Parkinson?s disease ..................................................8 
2.3 Methamphetamine............................................................................................11 
2.4 Methamphetamine Toxicity: Revelance for Parkinson?s disease ....................14 
2.5 Oxidative Stress in Parkinson?s disease: Relevance to Methamphetamine .....16 
2.6 Free Radical Damage .......................................................................................18 
2.7 Mitochondrial Dysfunction: Relevance to Parkinson?s disease.......................20 
2.8 Neurotoxins and Mitochondrial Dysfunction ..................................................22 
2.9 Excitotoxicity ...................................................................................................24 
2.10 Neuroinflammation ........................................................................................25 
2.11 Lipid Peroxidation and Protein Aggregation: Further Markers of 
Neurotoxicity .........................................................................................................26 
2.12 Apoptosis/Necrosis ........................................................................................27 
2.13 Possible Neuroprotective Treatments ............................................................28 
2.13.1 Ramelteon .......................................................................................28 
x 
 
2.13.2 Amantadine .....................................................................................31 
2.13.3 Salicylic Acid ..................................................................................33 
2.14 References ......................................................................................................37 
3. STATEMENT OF RESEARCH OBJECTIVES ...........................................................55 
4. NEUROTOXIC EFFECTS OF METHAMPHETAMINE ............................................56 
4.1 Overview ..........................................................................................................56 
4.2 Introduction ......................................................................................................57 
4.3 Materials and methods .....................................................................................60 
4.3.1 Animals .............................................................................................60 
4.3.2 Methamphetamine administration ....................................................60 
4.3.3 Behavioral Studies ............................................................................60 
4.3.4 Tissue Preparation for in vivo biochemical assay .............................62 
4.3.5 Measurement of Neurotransmitters ...................................................62 
4.3.6 Mouse Mitochondrial P2 Fraction for in vitro biochemical assay ....62 
4.3.7 Protein estimation  ............................................................................63 
4.3.8 Mitochondrial Complex I Activity....................................................63 
4.3.9 Mitochondrial Complex IV Activity .................................................64 
4.3.10 Assay of MAO activity ...................................................................64 
4.3.11 Assay of SOD activity ....................................................................64 
4.3.12 Assay of ROS production ...............................................................64 
4.3.13 Statistical Analysis ..........................................................................65 
4.4. Results  ............................................................................................................65 
xi 
 
4.4.1 Behavioral Analysis ..........................................................................65 
4.4.2 Effect on Neurotransmitters ..............................................................65 
4.4.3 In vivo Biochemical Analysis ...........................................................66 
4.4.4 In vitro Biochemical Analysis ..........................................................66 
4.5 Discussion ........................................................................................................67 
4.6 References ........................................................................................................71 
4.7. Figures.............................................................................................................77 
5. NEUROPROTECTION OF RAMELTEON AGAINST NEUROTOXIC AFFECTS 
OF METHAMPHETAMINE ............................................................................................86 
5.1 Overview ..........................................................................................................86 
5.2 Introduction ......................................................................................................87 
5.3 Materials and methods .....................................................................................89 
5.3.1 Animals .............................................................................................89 
5.3.2 Drug administration ..........................................................................90  
5.3.3 Behavioral Studies ............................................................................90 
5.3.4 Measurement of Neurotransmitters ...................................................92 
5.3.5 Tissue Preparation for in vivo biochemical assay .............................92 
5.3.6 Protein estimation .............................................................................92 
5.3.7 Mitochondrial Complex I Activity....................................................93 
5.3.8 Mitochondrial Complex IV Activity .................................................93 
5.3.9 Assay of MAO activity .....................................................................93 
5.3.10 Assay of ROS production ...............................................................94 
xii 
 
5.3.11 Statistical Analysis ..........................................................................94 
5.4 Results  .............................................................................................................94 
5.4.1 Behavioral Analysis ..........................................................................94 
5.4.2 Effect on Neurotransmitters ..............................................................95 
5.4.3 In vivo Biochemical Analysis ...........................................................95 
5.5 Discussion ........................................................................................................96 
5.6 References  .......................................................................................................99 
5.7 Figures ...........................................................................................................105 
6. NEUROPROTECTION OF AMANTADINE AGAINST METHAMPHETAMINE 
NEUROTOXICITY .........................................................................................................110 
6.1 Overview ........................................................................................................110 
6.2 Introduction ....................................................................................................111 
6.3 Materials and methods ...................................................................................113 
6.3.1 Animals ...........................................................................................113 
6.3.2 Drug administration ........................................................................113  
6.3.3 Behavioral Studies ..........................................................................114 
6.3.4 Measurement of Neurotransmitters .................................................115 
6.3.5 Tissue Preparation for in vivo biochemical assay ...........................115 
6.3.6 Protein estimation ...........................................................................116 
6.3.7 Mitochondrial Complex I Activity..................................................116 
6.3.8 Mitochondrial Complex IV Activity ...............................................116 
6.3.9 Assay of MAO activity ...................................................................117 
xiii 
 
6.3.10 Assay of ROS production .............................................................117 
6.3.11 Statistical Analysis ........................................................................117 
6.4 Results  ...........................................................................................................118 
6.4.1 Behavioral Analysis ........................................................................118 
6.4.2 Effect on Neurotransmitters ............................................................118 
6.4.3 In vivo Biochemical Analysis .........................................................119 
6.5 Discussion ......................................................................................................119 
6.6 References  .....................................................................................................122 
6.7 Figures ...........................................................................................................126 
7. NEUROPROTECTION OF SALICYLIC ACID AGAINST METHAMPHETAMINE 
NEUROTOXICITY .........................................................................................................131 
7.1 Overview ........................................................................................................131 
7.2 Introduction ....................................................................................................132 
7.3 Materials and methods ...................................................................................134 
7.3.1 Animals ...........................................................................................134 
7.3.2 Drug administration ........................................................................135 
7.3.3 Behavioral Studies ..........................................................................135 
7.3.4 Measurement of Neurotransmitters .................................................137 
7.3.5 Tissue Preparation for in vivo biochemical assay ...........................137 
7.3.6 Protein estimation ...........................................................................137 
7.3.7 Mitochondrial Complex I Activity..................................................138 
7.3.8 Mitochondrial Complex IV Activity ...............................................138 
xiv 
 
7.3.9 Assay of MAO activity ...................................................................138 
7.3.10 Assay of ROS production .............................................................139 
7.3.11 Statistical Analysis ........................................................................139 
7.4 Results  ...........................................................................................................139 
7.4.1 Behavioral Analysis ........................................................................139 
7.4.2 Effect on Neurotransmitters ............................................................140 
7.4.3 In vivo Biochemical Analysis .........................................................140 
7.5 Discussion ......................................................................................................141 
7.6 References  .....................................................................................................144 
7.7 Figures ...........................................................................................................149 
8. SUMMARY AND CONCLUSIONS  .........................................................................154 
 
 
 
 
 
 
 
 
 
 
 
xv 
 
LIST OF FIGURES 
2.1 Dopamine deficiency of the nigrostriatal system ..........................................................7 
2.2 Markers for Neurodegeneration  ..................................................................................10 
2.3 Methamphetamine chemical structure .........................................................................12 
2.4 Methamphetamine toxicity ..........................................................................................13 
2.5 Mitochondrial Dysfunction and Oxidative Stress ........................................................20 
2.6 Chemical Structure of Ramelteon ................................................................................29 
2.7 Chemical Structure of Amantadine ..............................................................................32 
2.8 Chemical Structure of Salicylic Acid ..........................................................................34 
4.1 Effect of methamphetamine on catalepsy ....................................................................77 
4.2 Effect of methamphetamine on akinesia ......................................................................77 
4.3 Effect of methamphetamine on swim time ..................................................................78 
4.4 Effect of methamphetamine on striatal dopamine depletion .......................................79 
4.5 Effect of methamphetamine on mitochondrial Complex I (in vivo) ............................80 
4.6 Effect of methamphetamine on mitochondrial Complex IV (in vivo) .........................80 
4.7 Effect of methamphetamine on mitochondrial MAO activity (in vivo) .......................81 
4.8 Effect of methamphetamine on mitochondrial SOD (in vivo) .....................................81 
4.9 Effect of methamphetamine on mitochondrial ROS (in vivo) .....................................82 
4.11 Effect of methamphetamine on mitochondrial Complex I (in vitro) .........................83 
4.12 Effect of methamphetamine on mitochondrial Complex IV (in vitro) ......................83 
xvi 
 
4.13 Effect of methamphetamine on mitochondrial MAO (in vitro) .................................84 
4.14 Effect of methamphetamine on mitochondrial SOD (in vitro) ..................................84 
4.15 Effect of methamphetamine on mitochondrial ROS (in vitro) ..................................85 
5.1 Effect of ramelteon on catalepsy ................................................................................105 
5.2 Effect of ramelteon on akinesia .................................................................................105 
5.3 Effect of ramelteon on swim time ..............................................................................106 
5.4 Effect of ramelteon on striatal dopamine depletion ...................................................107 
5.5 Effect of ramelteon on mitochondrial Complex I (in vivo) ........................................108 
5.6 Effect of ramelteon on mitochondrial Complex IV (in vivo) .....................................108 
5.7 Effect of ramelteon on mitochondrial MAO activity (in vivo) ..................................109 
5.8 Effect of ramelteon on mitochondrial ROS (in vivo) .................................................109 
6.1 Effect of amantadine on catalepsy .............................................................................126 
6.2 Effect of amantadine on akinesia ...............................................................................126 
6.3 Effect of amantadine on swim time ...........................................................................127 
6.4 Effect of amantadine on striatal dopamine depletion ................................................128 
6.5 Effect of amantadine on mitochondrial Complex I (in vivo) .....................................129 
6.6 Effect of amantadine on mitochondrial Complex IV (in vivo) ..................................129 
6.7 Effect of amantadine on mitochondrial MAO activity (in vivo) ................................130 
6.8 Effect of amantadine on mitochondrial ROS (in vivo) ..............................................130 
7.1 Effect of salicylic acid on catalepsy ...........................................................................149 
7.2 Effect of salicylic acid on akinesia ............................................................................149 
7.3 Effect of salicylic acid on swim time .........................................................................150 
xvii 
 
7.4 Effect of salicylic acid on striatal dopamine depletion ..............................................151 
7.5 Effect of salicylic acid on mitochondrial Complex I (in vivo) ...................................152 
7.6 Effect of salicylic acid on mitochondrial Complex IV (in vivo) ................................152 
7.7 Effect of salicylic acid on mitochondrial MAO activity (in vivo) .............................153 
7.8 Effect of salicylic acid on mitochondrial ROS (in vivo) ............................................153 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
xviii 
 
LIST OF TABLES 
4.1 Effects of methamphetamine on straub tail and tremor ...............................................78 
4.2 Effect of methamphetamine on striatal neurotransmitter and metabolite levels ..........79  
5.1 Effects of ramelteon on straub tail and tremor ...........................................................106 
5.2 Effect of ramelteon on striatal neurotransmitter and metabolite levels .....................107 
6.1 Effects of amantadine on straub tail and tremor ........................................................127 
6.2 Effect of amantadine on striatal neurotransmitter and metabolite levels ...................128 
5.1 Effects of salicylic acid on straub tail and tremor......................................................150 
5.2 Effect of salicylic acid on striatal neurotransmitter and metabolite levels ................151 
 
 
 
 
 
 
 
1 
 
1. INTRODUCTION 
Methamphetamine is a psycostimulant and sympathomimetic drug and is a 
powerful central nervous system (CNS) stimulant. The increased use and abuse of the 
drug has become a major problem in the United States. The recent increase in 
methamphetamine use, dependance and/or addicition has become a major health concern 
worldwide.  Despite the drug?s addictive properties, it is sometimes prescribed to treat 
attention-deficit hyperactivity disorder (ADHD), narcolepsy and obesity (Reynolds, 
1996). However, it is only used as a second line of treatment for these conditions, 
prescribed when amphetamine and methylphenidate cause the patient too many side 
effects. It is also used illegally for weight loss and to maintain alertness, focus, 
motivation, and mental clarity for extended periods of time, and for recreational 
purposes. Interestingly, there are medical histories of politician or leaders using 
methamphetamine intravenously by their personal physicians as a treatment for their 
depression and fatigue (Doyle, 2005). It is possible that their Parkinson-like symptoms 
which developed were related to the regular use of methamphetamine.   
Methamphetamine can cross the blood-brain barrier, triggering a release of 
monoamines (dopamine, norephinephrine and seratonin). It can also act as a dopamine 
reuptake inhibitor and in high concentrations as a monoamine oxidase inhibitor. The 
major dopaminergic pathways in the brain are nigrostriatal, mesolimbic, mesocortical and 
tuberinfundicular pathways. Methamphetamine can significantly stimulate the 
2 
 
mesolimbic reward and nigrostriatal pathway.  Stimulation of mesolimbic reward 
pathway by methamphetamine can cause euphoria and excitement, thus it is prone to 
abuse and addiction (McGregor et al., 2005). Methamphetamine affects biochemical 
mechanisms responsible for regulating heart rate, body temperature, blood pressure, 
appetite, attention, mood and responses associated with alertness or alarm conditions. 
Physical effects of the drug are similar to those seen in the fight-or-flight response 
induced by the release of epinephrine. This reaction includes increases heart rate and 
blood pressure, vasoconstriction, bronchodilation, and hyperglycemia. There is a 
decrease in appetite, a lowering of fatigue, and an increase in focus and mental alertness. 
Methamphetamine can be neurotoxic and has been shown to cause dopaminergic 
degeneration in rodents and primates (Davidson et al., 2001; Dluzen et al., 2001; Itzhak et 
al., 2002). High doses or chronic administration of methamphetamine produce losses in 
several markers of brain dopamine and serotonin neurons. Dopamine and serotonin 
concentrations, dopamine and seratonin uptake sites, and tyrosine and tryptophan 
hydroxylase activities are reduced after the administration of methamphetamine. It has 
been proposed that dopamine plays a role in methamphetamine induced neurotoxicity 
because experiments which reduce dopamine production or block the release of 
dopamine decrease the toxic effects of methamphetamine administration. Dopamine 
metabolism generates reactive oxygen species such as hydrogen peroxide. Hence, there is 
a strong possibiliy that the oxidative stress that occurs after taking methamphetamine can 
mediate its neurotoxicity (Yamamoto and Zhu, 1998). The long lasting dopamine 
depletion in the striatum mimics the neurodegenerative process seen in Parkinson?s 
3 
 
disease. This makes methamphetamine an ideal toxic candidate for producing an animal 
model for Parkinson?s disease. Such a model could be used to study the mechanism of 
dopaminergic neurodegeneration and for aiding therapeutic interventions in Parkinson?s 
disease (Lau et al., 2000; Gluck et al, 2003; Kita et al., 2003; LaVoie et al., 2004; 
Thomas et al., 2004; Riddle et al., 2006).  
Animal models are utilized in order to gain an understanding of the pathogenesis 
of Parkinson?s disease and the causes of the selective loss of dopaminergic neurons. 
Scientists want to mimic this pathogenesis as closely as possible. Therefore, these models 
are designed to reproduce the clinical and pathological features of Parkinson?s disease, 
such as the progressive loss of dopaminergic neurons, and can be very helpful in 
dissecting the many different molecular and biochemical pathways in the phenotype of 
Parkinson?s disease. As such, the methamphetamine animal model could help in gaining 
a better understanding of the neurodegeneration in the nigrostriatal system associated 
with Parkinson?s disease and could be used to investigate pharmacological agents for any 
neuroprotective or neurorestorative effects they may exhibit. This could lead to the 
development of much needed novel therapeutic agents and even to a proactive treatment 
strategy in Parkinson?s disease. With no proven neurorestorative or neuroprotective 
treatment strategies for this disease, these animal models are essential to discovering such 
treatments and to lowering the mortality rate of this disease. 
With this study we examined the neurotoxicity of methamphetamine, its role in 
dopamine depletion (implications for Parkinson?s disease) and possible neuroprotective 
treatments (ramelteon, amantadine and salicylic acid). Closing the mechanistic gap in 
4 
 
understanding could lead to proactive strategies to combat this disease.  A more complete 
understanding of the progressive and selective loss of the nigrostriatal dopaminergic 
neurons that occurs in Parkinson's disease could advance treatment of this disease in 
numerous ways. Discovery of a pharmacological agent to provide neuroprotection in the 
substantia nigra from oxidative stressand the scavenging of hydroxyl radicals, or the 
identification of a therapy that could protect dopaminergic cells from apoptosis induced 
by neurotoxins, could improve the duration and quality of life for all Parkinson?s disease 
sufferers. 
Parkinson?s disease causes a significant economic impact.  It is estimated that 
medical costs alone in Parkinson?s disease amount to around 25 billion dollars a year 
(Siderowf, 2003).  Examination of the neurotoxicity of methamphetamine, its role in 
dopamine depletion, and implications for Parkinson?s disease could aid in closing the 
mechanistic gap in understanding and could lead to proactive strategies to combat this 
disease. Abuse of drugs, like methamphetamine, also has a significant economic impact. 
The cost of drug abuse in America is estimated at around one trillion dollars a year 
(NIDA, 2002; Cadet et al., 2003). A better understanding of the mechanistic properties of 
this drug could also aid in strategies to combat addiction. Therefore, this study could have 
a two-fold beneficial effect for the American economy. 
 
 
 
 
5 
 
Refrences 
Cadet, J.L., Jayanthi, S., Deng, X. 2003. Speed kills: cellular and molecular basis of 
methamphetamine-induced nerve terminal degeneration and neuronal apoptosis. 
FASEB J. 17: 1775-1788. 
Davidson, C., Gow, A.J., Lee, T.H., Ellinwood, E.H. 2001. Methamphetamine 
neurotoxicity: necrotic and apoptotic mechanisms and relevance to human abuse 
and treatment. Brain Res Rev 36(1): 1-22.  
Dluzen, D.E., McDermott, J.L., Anderson, L.I. 2001. Tamoxifen diminishes 
methamphetamine-induced striatal dopamine depletion in intact female and male 
mice. J. Neourendocrinol. 13: 618-624. 
Doyle, D. 2005. Hitler's Medical Care. J Roy Coll Phys Edin. 35: 75-82. 
Gluck, M.R., Moy, L.Y., Jayatilleke, E., Hogan, K.A., Manzino, L., Sonsalla, P.K. 2001. 
Parallel increases in lipid and protein oxidative markers in several mouse brain 
regions after methamphetamine treatment. J. Neurochem. 79: 152-160. 
Itzhak, Y., Martin, J., Ali, S. 2002. Methamphetamine-induced dopaminergic 
neurotoxicity in mice: long-lasting sensitization to the locomotor stimulation and 
desensitization to the rewarding effects of methamphetamine. Prog Neuro-
psychoph. 26(6): 1177-1183.  
Kita, T., Wagner, G.C., Nakashima, T. 2003. Current research on methamphetamine-
induced oxidative stress in cultured mouse astrocytes. J. Pharmacol. Sci. 92: 178-
195. 
6 
 
Lau, J.W., Senok, S., Stadlin, A. 2000. Methamphetamine-induced oxidative stress in 
cultured mouse astrocytes. Ann. N. Y. Acad. Sci. 914: 146-156. 
LaVoie, M.J., Card, J.P., Hastings, T.G. 2004. Microglial activation precedes dopamine 
terminal pathology in methamphetamine-induced neurotoxicity. Exp. Neurol. 187: 
47-57. 
McGregor, C., Srisurapanont, M., Jittiwutikarn, J., Laobhripatr, S., Wongtan, T., White, 
J. 2005. The nature, time course and severity of methamphetamine withdrawal. 
Addiction. 100 (9): 1320-1329.  
NIDA Research Report. 2002. Methamphetamine abuse and addiction. 
http://www.nida.nih.gov/PDF/RRMetham.pdf. 
Riddle, E.L., Fleckenstein, A.E., Hanson, G.R. 2006. Mechanisms of methamphetamine-
induced dopaminergic neurotoxicity. AAPS J. 8: E413-E418. 
Rothman, et al. 2001. Amphetamine-Type Central Nervous System Potently than they 
Release Dopamine and Serotonin. Synapse. 39: 32-41.  
Siderowf, A., Stern, M. 2003. Update on Parkinson disease. Ann. Intern. Med. 138(8): 
651-658. 
Thomas, D.M., Walker, P.D., Benjamins, J.A., Geddes, T.J., Kuhn, D.M. 2004. 
Methamphetamine neurotoxicity in dopamine nerve endings of the striatum is 
associated with microglial activation. J. Pharmacol. Exp. Ther. 311: 1-7. 
Yamamoto, B., Zhu, W. 1998. The Effects of Methamphetamine on the Production of 
Free Radicals and Oxidative Stress. J Pharmacol Exp Ther. 287(1): 107-114.  
7 
 
2. REVIEW OF LITERATURE 
 
2.1 Clinical and pathologic features of Parkinson?s disease 
Parkinson?s disease is a progressive neurodegenerative disorder of the central 
nervous system. It is associated with a deficiency of striatal dopamine due to the selective 
degeneration of nigrostriatal dopaminergic neurons (Hornykiewicz, 1973). In Parkinson?s 
disease, most of the neurons located in the substantia nigra (figure 2.1) that produce 
dopamine have undergone neurodegeneration, causing a severe shortage of dopamine in 
the striatum (Caine and Langston, 1983). 
 
  
Figure 2.1. Dopamine deficiency of the nigrostriatal system: Dopamine neurons 
originate in the substantia nigra and terminate in the corpus striatum. 
8 
 
Dopamine from the substantia nigra activates neurons in the striatum that are 
important for initiation of movement. The loss of dopaminergic function leads to various 
motor symptoms including resting tremors, bradykinesia, muscular rigidity, and 
abnormalities in posture and gait (Kopin, 1992). Thus, one affected with Parkinson?s 
disease would be expected to exhibit some or all of the following motor deficits: 
slowness in initiation and execution of voluntary movements, increased muscle tone, 
increased resistance to movement, shuffling of feet, stooped posture and equilibrium and 
righting reflex (Sethy et al., 1997). There are also other symptoms related to the 
parasympathetic and sympathetic peripheral nervous system seen in Parkinson?s disease 
patients. In many cases of Parkinson?s disease, complete immobility eventually occurs 
despite treatment (Kopin, 1992; Marsden, 1994; Sethy et al., 1997). Parkinson?s disease 
is a slow-onset disease and the prevalence of Parkinson?s disease increases with age. 
There are estimates of up to 800 affected per 100,000 individuals age 65 or older (Savitt, 
2006). This makes it the most common of the neurodegenerative movement disorders, 
affecting approximately 1% of the population (Siderowf, 2003). The prolonged life span 
of the human population has brought about an increasing number of senior citizens in our 
society. The problems of neurodegenerative diseases have become a prevalent cause of 
disabilities in old age which poses an increasing problem.  
 
2.2 Etiology and treatment of Parkinson?s disease 
The etiology of Parkinson?s disease is not entirely clear. Though the specific 
cause of Parkinson?s disease is unknown, studies have shown that age, genetics and 
9 
 
environmental factors (figure 2.2) all contribute to the disease (Fahn and Cohen, 1992; 
Marsden, 1994; Sethy et al., 1997; Patel t al., 2006)). Parkinson?s disease can be sporadic 
and it also can be a familial form of disease. The genes that can cause the familial form of 
Parkinson?s disease are alpha-synuclein, parkin, DJ-1, PINK-1 and LRRK2.  
Environmental toxins have repeatedly been shown as contributors to the development of 
Parkinson?s disease (Ungerstedt, 1971; Davis et al., 1979; Burns et al., 1984; Di Monte et 
al., 2002; Sherer et al., 2002; Patel et al., 2006). These toxic substances have been 
suggested to contribute to Parkinson?s disease by producing loss of dopaminergic 
neurons of the nigrostriatal pathway.  Studies have demonstrated that exposures to the 
toxins can produce progressive, permanent and cumulative neurotoxicity of the 
nigrostriatal system. Among the environmental factors suspected to participate in the 
onset of Parkinson?s disease are the synthetic heroin compound MPTP (l-methyl-4-
phenyl tetrahydropyridinium) (Davis et al., 1979, Burns et al.,1984; Sherer et al., 2002, 
Di Monte et al., 2002), 6-hydroxydopamine (Ungerstedt, 1971) and the widely used and 
abused drug methamphetamine (Bondy et al., 2000), as well as environmental toxins like 
the herbicide paraquat (1, 1?-dimethyl-4, 4?-bipyridinium) (McCormack et al., 2005; 
Thiruchelvam et al., 2000a; Thiruchelvam et al., 2000b), and the fungicide maneb 
(manganese ethylene-bisdithiocarbamate) (Thiruchelvam et al., 2000a; Thiruchelvam et 
al., 2000b). Successful animal models for the study of Parkinson?s disease have been 
created using these toxins alone (Langston et al., 1983; Di Monte et al., 2002), or in 
combinations (Thiruchelvam et al., 2000a; Thiruchelvam et al., 2000b; Cory-Slechta et 
al., 2005). 
10 
 
  
Figure 2.2. Markers for Neurodegeneration: Oxidative stress, excitotoxicity, 
mitochondrial dysfunction, and inflammation are seen in the early physiology of 
Parkinson?s disease. Protein aggregation and cell death are seen in the later stages. 
 
The current treatment strategy for Parkinson?s disease remains primarily 
symptomatic, with no proven neuroprotective or neurorestorative treatments (Dawson 
and Dawson, 2003). Current treatments, including levodopa (a dopamine precursor), 
dopamine agonists, monoamine oxidase (MAO) inhibitors, anticholinergics and catechol 
O-methyltransferase (COMT) inhibitors, are used to alleviate symptoms and slow the 
progression of the disease, however such effects are temporary and these drugs often 
have debilitating side effects. Long-term drug therapy has been associated with motor 
complications that can be as disabling as the disease itself. There are also a few surgical 
11 
 
approaches that can be used as treatment. Howeve, despite advances in pharmacotherapy 
that have improved quality of life, the mortality rate among Parkinson?s disease sufferers 
remains largely unchanged. There is need for a proactive treatment strategy that could 
provide neuroprotection or neurorestoration. Since evidence has shown that neurotoxins 
play an important role in niagral degeneration, there is obviously a need to take a closer 
look at such toxins and utilize them in animal models to study possible neuroprotection in 
Parkinson?s disease. A mechanistic understanding of such toxicity could lead to proactive 
strategies to counteract the neurological damage.  
 
2.3 Methamphetamine 
Methamphetamine (N-methyl-1-phenyl-propan-2-amine-IUPAC; figure 2.3) is a 
white crystalline solid which is highly water soluble (1g/2ml) and has a melting point 
between 171 and 175 degrees Celsius. It is odorless, bitter tasting and slightly acidic in 
aqueous solution (Remington, 1985). A. Ogata, a Japanese chemist synthesized 
methamphetamine in 1919. His method of synthesis was based on the reduction of 
ephedrine.  Methamphetamine can be synthetically prepared by catalytic hydrogenation 
of ephedrine or pseudoephedrine which is then converted to hydrochloride. It can also be 
prepared by reducing the condensation product of benzylmethyl ketone and methylamine. 
Methamphetamine shares its structurally similarily with other CNS stimulants like 
amphetamine and methcathinone, which are also produced from ephedrine and 
pseudoephedrine by chemical reduction.  Methamphetamine is a synthetic stimulant and 
this is the major difference as compared to Cannabinoids and cocaine, which are obtained 
12 
 
from natural resources (canabinionids are dervided from Cannabis sativa and cocaine is 
derived from Erythroxylon coca). 
 
 
Figure 2.3. Methamphetamine chemical structure 
 
The methyl group present in the structure of methamphetamine makes the 
substance lipid soluble, thus easing transport across the blood brain barrier. It also 
increases the ressitance against enzymatic degradation by monoamine oxidase. 
Methamphetamine causes the dopamine, norepinephrine and serotonin transporters to 
reverse their direction of flow. This inversion leads to a release of these transmitters from 
the vesicles to the cytoplasm and from the cytoplasm to the synapse causing increased 
stimulation of post-synaptic receptors. Methamphetamine also indirectly prevents the 
monoamine reuptake of these neurotransmitters, causing them to remain in the synaptic 
cleft for a prolonged period (Rothman et al., 2001).  
Its effects on the body (figure 2.4) include acute central nervous system 
stimulation and cardiotoxicity, including induction of tachycardia, arrhythmias, 
hypertension and cardiovascular collapse. The drug has a high risk of dependency and 
abuse. Effects on the central nervous system include tremors, restlessness, agitation, 
insomnia, increased motor activity, headache, convulsions, and coma. Psychiatric side 
13 
 
effects include agitation, confusion, mood elevation, increased wakefulness, 
talkativeness, irritability and panic attacks. Chronic abuse can cause delusions and 
paranoia (Budavari, 1996).  
 
Figure 2.4. Methamphetamine toxicity 
 
A withdrawal syndrome occurs after abrupt cessation following chronic use 
(Budavari, 1996). Moreover, withdrawal from methamphetamine dependence is 
distinguished by protracted anhedonia, dysphoria and severe craving.  Withdrawal is also 
characterized by excessive sleeping, eating, and depression-like symptoms, often 
accompanied by anxiety and drug-craving (McGregor et al., 2005). Withdrawal 
symptoms of methamphetamine can be reduced by pharmacological or non-
pharmacological approaches.  Psychosocial behavioral therapy and Contingency reward 
14 
 
therapy have been successfully used in patients addicted to methamphetamine. Opioid 
receptor antagonists and antidepressants have been tested to reduce the severe withdrawal 
symptoms related to methamphetamine. Its widespread abuse has been a growing 
problem in recent years. It is a highly addictive stimulant with acute neurotoxic and 
powerful dopamine-depleting properties. Side effects of abuse of this drug include 
memory loss, aggression, psychotic symptoms and behavioral abnormalities, and 
potential heart and brain damage (NIDI Research Report, 2002).  
 
2.4 Methamphetamine Toxicity: Revelance for Parkinson?s disease 
Major markers seen in Parkinson?s disease are nigral neuronal loss and dopamine 
depletion in the striatum. Methamphetamine-induced neurotoxicity of the nigrostriatal 
dopaminergic system is commonly seen in lab studies (Asanuma et al., 2003). In 
experiments, repeated administration of the drug caused neurotoxicity in both rodents and 
non-human primates (Hashimoto et al., 2004; Zhang et al., 2006). It has been shown that 
methamphetamine treatment depletes dopamine as well as its metabolite, 3,4-
dihydroxyphenylacetic acid (DOPAC), in the striatum (Bondy et al., 2000). It has been 
further shown that repeated administration of methamphetamine reduces dopamine 
transporter (DAT) binding and, in extreme cases, causes cell apoptosis and persistent 
dopaminergic degeneration in the striatum (Davidson et al., 2001; Cadet et al., 2003; Kita 
et al., 2003). Examination of post-mortem brain tissue from methamphetamine users 
showed a significant decrease in dopamine and dopamine transporter density (Wilson et 
al. 1996).  
15 
 
Methamphetamine is highly lipid soluble and readily crosses the blood-brain 
barrier. It is a cationic lipophilic molecule that can diffuse into the mitochondria and be 
retained there. As such, it enters into the brain rapidly, interacts with transporters, and 
displaces dopamine in the vesicles releasing it into the synaptic cleft. Once there, the 
dopamine can be rapidly oxidized into quinones and semiquinones which are then 
converted through redox cycling to toxic radicals like superoxide, hydrogen peroxide and 
nitrogen radicals, creating oxidative stress (Halliwell, 1989; Stokes et al., 1999; Zhu et 
al., 2006). Oxidative stress causes an inhibition of mitochondrial ATP production, a 
major determinant of lethal cell injury. A recent study showed that cells can be protected 
from injury as long as ATP levels can be maintained. In this study, the use of fructose to 
maintain ATP increased cell survival. This study, therefore, indicates that the loss of ATP 
is a central mechanism of oxidative stress (Imberti et al., 1993). Mitochondrial DNA is 
sensitive to reactive oxygen species because mitochondria have only a limited arsenal of 
DNA repair processes. Therefore, mitochondrial DNA mutations would cause defects in 
the respiratory chain function leading to severe cellular damage. Free radicals can interact 
with sugars, proteins and lipids creating a number of modifications which could then lead 
to mitochondrial dysfunction, apoptosis and necrosis. The damage to the striatal terminals 
and the nigral cell bodies observed in methamphetamine users seems indicative of all of 
these toxic mechanisms, the same mechanisms indicated in Parkinson?s disease (Imberti 
et al., 1993; Jones and Vale, 2000). 
Toxicological studies on the brain tissue of human methamphetamine users have 
shown long term and possibly irreversible damage to dopaminergic neurons and loss of 
16 
 
striatal dopamine transporters even after as much as three years of abstinence from the 
drug (Cadet et al., 2003). Researchers have found dopaminergic toxicity in mice after a 
single dose of methamphetamine (25 mg/kg) (Hayashi et al., 2001) or after multiple 
doses of methamphetamine varying from 2.5 to 10 mg/kg given at 2 hour intervals 
(Sonsalla and Heillila, 1988; Chan et al., 1994; O?Callaghan and Miller, 1994). After 
such doses of methamphetamine there was seen a long lasting decrease in nigrostriatal 
dopamine similar to that seen in Parkinson?s disease (Sonsalla, et al., 1996). 
Though the exact mechanism of the neurotoxicity caused by methamphetamine 
use is unclear, mitochondrial dysfunction, as well as N-methyl-D-aspartic acid (NMDA) 
receptor-mediated excitotoxicity and neuroinflammation are all mechanisms commonly 
implicated in its toxicity. In addition, the long lasting damage to striatal neurons mimics 
that which is seen in the neurodegenerative process in Parkinson?s disease. This makes 
the methamphetamine animal model ideal to use for replication of Parkinson?s disease 
(Lau et al., 2000; Gluck et al, 2001; Kita et al., 2003; LaVoie et al., 2004; Thomas et al., 
2004; Riddle et al., 2006).  
 
2.5 Oxidative Stress in Parkinson?s disease: Relevance to Methamphetamine 
Oxidative stress (figure 2.5) has been strongly implicated in the pathogenesis of 
Parkinson?s disease. Brains from patients with Parkinson?s disease have shown elevated 
markers of oxidative damage, indicating that oxidative stress may play a key role in the 
disease (Hensley et al., 1998; Sherer et al., 2002). Oxidative species are important in 
cellular respiration and aid in the regulation of signal transduction pathways. However, 
17 
 
they can be converted into free radicals, containing unpaired electrons, which are 
collectively referred to as reactive oxygen species (ROS). Oxidative stress involves the 
excessive formation of reactive oxygen species, like the hydroxyl radicals (OH., OH-) 
and superoxide (O2.), among others, which are formed when molecular oxygen is reduced 
non-enzymatically (Fahn and Cohen, 1992; Jenner et al., 1992). The brain has been 
shown to contain low to moderate levels of enzymes such as catalase, superoxide 
dismutase and glutathione peroxidase that play an important role in the metabolism of 
reactive oxygen species (Cohen, 1988). Superoxide dismutase (SOD) converts the 
superoxide to hydrogen peroxide, and hydrogen peroxide is typically detoxified by 
glutathione peroxidase, and catalase. However, oxidative stress occurs when free radicals 
are in excess of these antioxidant defenses. Free radicals are capable of reacting with 
DNA, lipids and proteins to alter their structure and function (Turrens and Boveris, 1980; 
Hasegawa et al., 1990). These alterations would be evidenced by an accumulation of 
oxidized products which serve as markers of oxidation, such as aldehydes and 
isoprostanes from lipid peroxidation, protein carbonyls from protein oxidation and base 
adducts from DNA oxidation (Olanow, 1993).  
Because methamphetamine displaces the dopamine in the vesicles, large amounts 
of it are released into the synaptic cleft and cytosol. There is an established relationship 
between the oxidative process of dopamine itself and nigrostriatal degeneration. During 
the auto-oxidation, synthesis and metabolism of dopamine, hydrogen peroxide (H2O2) is 
produced as a byproduct by tyrosine hydroxylase (TSH) and monoamine oxidase (MAO) 
(Lotharius and O?Malley, 2000). The hydrogen peroxide, which can react 
18 
 
nonenzymatically to form highly reactive hydroxyl radicals, can then take part in a 
positive feedback loop causing further progression of oxidative damage (Jenner, 1998). 
This demonstrates that dopaminergic neurons exist in a constant state of oxidative stress, 
and this could also explain the selective toxicity on dopaminergic neurons observed in 
Parkinson?s disease. All current evidence suggests that oxidizing species exert a toxic 
effect that is significantly dangerous to the dopaminergic system, causing the death of 
dopaminergic neurons (Di Monte, 2001).  
 
2.6 Free Radical Damage 
The brain and nervous system are particularly vulnerable to free radical damage 
for a number of reasons. The membrane lipids in the brain contain high levels of 
polyunsaturated fatty acid side chains, which are prone to free radical attack, and are 
readily peroxidizable. The brain also consumes large quantities of total oxygen for its 
relatively small weight, contributing further to the formation of reactive oxygen species. 
It has been estimated that up to 2% of the oxygen consumed by healthy mitochondria is 
converted to superoxide, and this amount is higher in damaged and aged mitochondria. 
Evidence seems to suggest that the neurons in the substantia nigra are particularly prone 
to oxidative stress, which would explain the selective degeneration in this region 
(Halliwell, 1989). 
Another possible contributing factor to the neurotoxicity and selective 
neurodegeneration seen in the substantia nigra is the presence of iron in this area, which 
can also contribute to the production of reactive oxygen species. In the presence of 
19 
 
transition metals, hydrogen peroxide can be converted by the Fenton reaction, an iron 
mediated reaction that requires iron in the free ferrous form, to the highly reactive 
hydroxyl radical. Seen as further evidence that oxidative stress plays an important role in 
the development of Parkinson?s disease was the discovery of higher than normal levels of 
iron in the substantia nigra of Parkinson?s disease patients (Bharath et al., 2002) and 
lower than normal concentrations of reduced glutathione. Glutathione, which removes 
hydrogen peroxide, is present in lower levels in the brains of patients with Parkinson?s 
disease. Reduced glutathione is decreased in Parkinson?s disease substantia nigra by 
approximately 50% (Perry et al., 1986). Because of the deficiency in glutathione, there is 
higher than normal levels of hydrogen peroxide in the neurons. This hydrogen peroxide is 
further broken down into hydroxyl radicals by the Fenton reaction (Bharath et al., 2002; 
Foley and Riederer, 2000). Thus, the higher than normal iron levels in Parkinson?s 
disease patients is another strong indicator of the involvement of oxidative stress in the 
development of Parkinson?s disease. In addition, further evidence in favor of using 
methamphetamine to create an animal model for Parkinson?s disease is that the striatum 
has been shown to be the primary site of methamphetamine toxicity, just as it is the 
primary site of dopamine depletion in Parkinson?s disease (Kupperman et al., 1997), and 
by the finding of increased reactive oxygen species in methamphetamine intoxication 
(Huang et al., 1997; Yamamoto and Zhu, 1998; Kita et al., 1999).  
 
20 
 
 
Figure 2.5. Mitochondrial Dysfunction and Oxidative Stress 
 
2.7 Mitochondrial Dysfunction: Relevance to Parkinson?s Disease 
Mitochondrial dysfunction (figure 2.5) has long been thought to play a significant 
role in depletion of nigral neurons, leading to dopaminergic depletion. Evidence seems to 
suggest that the neurons in the substantia nigra are particularly prone to mitochondrial 
energy depletion, which would explain the selective degeneration in this region. The 
central dopaminergic system that plays such an important role in motor activity is 
comprised of a surprisingly small number of neurons. Because of this, it is especially 
vulnerable and even minor insults may lead to irreparable functional deficits (Fariello et 
al., 1988).  
The exact mode of cell death in the substantia nigra in Parkinson?s disease is 
unknown but early researchers suspected it involved some deficiency in mitochondrial 
21 
 
complex I (NADH:ubiquinone oxidoreductase). There is a tendency for free radicals to 
be more easily formed and less easily scavenged in the brain of Parkinson?s disease 
patients (Thiruchelvam et al., 2005). Along the mitochondrial electron transport chain are 
sites where electrons can ?leak? out and combine with molecular oxygen to form 
oxidizing species. Of particular relevance in Parkinson?s disease, mitochondrial complex 
I is one of the sites of electron leak along the electron transport chain (Ranjita et al., 
2002). An inhibition of complex I leads to an increased production of reactive oxygen 
species, resulting in a feed-forward cycle where complex I is further damaged and more 
reactive oxygen species are produced (Hensley et al., 1998; Sherer et al., 2002). 
Researchers observed a deficiency in complex I activity in Parkinson?s disease 
patients, including significantly reduced activity in the substantia nigra (Janetzky et al., 
1994) and platelets (Krige et al., 1992; Yoshino et al., 1992). In another study there was 
seen as much as a 30% decrease in complex I activity in the substantia nigra, striatum, 
skeletal muscle and platelets, with no detectable structural or mitochondrial DNA 
changes (Yoshino et al., 1992; Foley and Riederer, 2000). In addition to reduced complex 
I, mitochondria in patients with early, untreated Parkinson?s disease show decreased 
complex II (succinate-ubiquinone oxidoreductase)/ III (ubiquinol:cytochrome c 
oxidoreductase) activity (Shults 2005). Mitochondria play an important role in both cell 
survival and death, as the ATP produced there is critical in the mitochondrial respiratory 
chain providing protection to dopaminergic neurons (Tipton and Singer, 1993). Inhibition 
of mitochondrial respiration is sufficient to cause nigral cell death and is a common step 
22 
 
in the signal transduction pathways of apoptosis and necrosis inducing cell death (Hartley 
et al., 1994).  
 
2.8 Neurotoxins and Mitochondrial Dysfunction 
Animal studies with other neurotoxins like MPTP shed light on the role of 
mitochondrial dysfunction in Parkinson?s disease. MPTP is a precursor neurotoxin and its 
oxidized form, MPP+, generated by monoamine oxidase type B, is the active neurotoxin. 
The acute neurodegenerative action of this substance was discovered as a result of fatal 
poisonings in ?street heroin? users. After the neurotoxic effect of MPTP was found to 
induce parkinsonian symptoms in humans (Langston, 1985; Tetrud and Langston, 1989; 
Di Monte et al., 2002), laboratory studies showed that it also selectively damaged the 
nigrostriatal dopaminergic system in animals producing similar parkinsonian symptoms 
(Langston et al., 1983).  
The biochemical changes following MTPT induced toxicity reinforced 
researchers beliefs that Parkinson?s disease could involve a disorder of the mitochondrial 
electron transport chain (Strijks et al., 1997). MPTP inhibits mitochondrial functions by 
causing a blockade of complex I, interrupting the electron transport chain and leading to 
reduction in mitochondrial ATP production. The inhibition of mitochondrial complex I 
disrupts electron transport from NAHD dehydrogenase to ubiquinone (Schapira, 1995). 
Since the substantia nigra has the lowest concentration of ubiquinone within the brain, 
this is further evidence that this region is more susceptible to mitochondrial energy 
depletion (Strijks et al., 1997).  
23 
 
The theory that mitochondrial dysfunction plays a role in Parkinson?s disease has 
been strengthened by research demonstrating that chronic administration of rotenone, a 
complex I inhibitor, produced an animal model of Parkinson?s disease in rats (Betarbet et 
al., 2000). Further support for this theory came from studies showing that complex I 
defects from Parkinson?s disease platelets are transferable into mitochondrial deficient 
cell lines (Gu et al., 1998; Swerdlow et al., 1996). Also supporting the mitochondrial 
dysfunction theory, are recent studies on the antioxidant coenzyme Q10, which plays an 
important role in the mitochondrial electron transport chain as the electron acceptor for 
complex I. Levels of Q10 were found to be reduced in Parkinson?s disease patients. 
Coenzyme Q10 has provided protection against both MPTP (Akaneya et al., 1995) and 
rotenone (Sherer et al., 2003). Also, clinical studies have shown that Q10 can provide 
some protection for the nigrostriatal dopaminergic system and can slow the progressive 
disability of the disease (Shults, 2005).  
Methamphetamine is thought to cause mitochondrial dysfunction by disrupting 
the electron transfer chain. Because of its physical properties, it can diffuse into the 
mitochondria and the buildup of positively charged particles in the mitochondria 
interferes with the chemical gradient needed by the electron transport chain. This 
interferes both with the mitochondrial membrane and with ATP synthase which then 
causes the initiation of the apoptotic process (Chance and Williams, 1956, Davidson et 
al., 2001). A second proposed mechanism for methamphetamine induced mitochondrial 
dysfunction is by the displacement of Ca2+ molecules stored in the endoplasmic reticulum 
causing an influx into the mitochondria. The release of these molecules results in 
24 
 
activation of proaptotic factors like caspase-3 and cytochrome-c and disrupts cellular 
metabolism leading to necrosis (Davidson et al., 2001; Cadet et al., 2003). 
 
2.9 Excitotoxicity 
Excitotoxicity plays an important role in both Parkinson?s disease and 
methamphetamine toxicity. Excitotoxicity is caused when excessive glutamate is released 
to activate receptors of the NMDA subtype. Normal stimulation of these receptors has 
been shown to enhance dopamine release and synthesis within the striatum. These 
excitatory interactions are important for normal striatal function. Glutamate is the most 
abundant excitatory neurotransmitter; however, it becomes cytotoxic if released in 
excess, causing a calcium influx into the cell (Schwarcz and Meldrum, 1985). 
Methamphetamine causes a release of excess glutamate into the striatum via a direct 
pathway. Mitochondrial dysfunction, as seen in both Parkinson?s disease and 
methamphetamine toxicity, can lead to excitotoxicity, as a reduction in ATP would result 
in a loss of the ATP-dependent Mg-blockade of NMDA receptors, causing physiological 
concentrations of glutamate to mediate a calcium influx into the cell (Beal et al., 1993). 
The resulting rise in calcium increases nitric oxide synthase (NOS). This increases the 
production of nitric oxide (.NO) which reacts with the superoxide radical to form 
peroxynitrite and the hydroxyl radical, both powerful oxidizing agents (Dawson et al., 
1991). Thus, the end product of excitotoxicity is the formation of reactive oxygen species 
as well as reduced intracellular glutathione synthesis, two of the indicators that are often 
seen in Parkinson?s disease (Murphy et al., 1989). If mitochondrial dysfunction and 
25 
 
oxidative stress are caused by methamphatamine, it may also act to increase 
excitotoxicity. This has been seen in other toxins used to create aminal models for 
Parkinson?s disease. For example, it has been shown that excessive MPP+ concentrations 
promote excitotoxicity, by enhancing glutamate release (Carboni et al., 1990). 
Dopaminergic neurons in the substantia nigra are rich in glutamate receptors, and thus 
highly susceptible to excitotoxicity. This emphasizes the importance of a better 
understanding of the role of excitotoxicity in Parkinson?s disease.  
 
2.10 Neuroinflammation 
The neurodegeneration seen with acute administration of methamphetamine has 
been suggested to be associated with the induction of cyclooxygenase (COX) which 
causes a neuroinflammatory process resulting in deleterious events in the cell (Zhang et 
al.,2007). Similarly, there is increasing evidence that one contributor to the later 
development of Parkinson?s disease is inflammation of neuronal tissue, caused by 
exposure to microorganisms, toxicants, or environmental factors in early life. This 
inflammation involves the activation of brain immune cells, microglia. Activation of 
these cells can be beneficial in the protection of the cells, but they can also release 
neurotoxic cytokines which could have a deleterious effect and produce 
neurodegeneration (Liu et al., 2003). The substantia nigra has the highest density of 
microglia in the brain, and studies have shown that Parkinson?s disease patients have an 
even higher number of reactive glial cells than do patients without the disease (McGreer 
et al, 1988). Researchers have examined this as a risk factor for Parkinson?s disease for a 
26 
 
long time, and it has even been said that the influenza pandemic of 1918 caused an 
increase in the risk of developing the disease (Landrigan et al., 2005 ). Studies in animals 
have shown that dopaminergic neurodegeneration resulted from exposure to bacterial 
endotoxin lipopolysacchide while the animal was in utero (Gao et al., 2002). Evidence of 
the involvement of inflammation came from studies showing that animals exposed to 
rotenone showed enhanced neurodegeneration in the presence of glia. Further studies 
showed synergistic effects when rotenone was combined with lipopolysaccharide 
(Thiruchelvam et al., 2000b). This would support the use of anti-inflammatory 
therapeutic agents for Parkinson?s disease. 
 
2.11 Lipid Peroxidation and Protein Aggregation: Further Markers of Neurotoxicity 
Also significant in Parkinson?s disease, studies have shown increased levels of 
markers for lipid peroxidation (cholesterol lipid hydroperoxides) (Dexter et al., 1998; 
Dexter et al., 1994), markers for oxidative DNA damage (8-hydroxy-2-deoxyguanosine) 
(Alam et al., 1997a), and increased protein carbonyls (Alam et al., 1997b; Sanchez-
Ramos et al., 1994) in post mortem brain tissue of Parkinson?s disease patients. Reactive 
oxygen species cause lipid peroxidation by interacting with unsaturated lipids of 
membranes. This destroys organelles and causes cell death (Cicchetti et al., 2005). 
Further studies have demonstrated a link between oxidative damage and protein 
aggregates, which are characteristic features of Parkinson?s disease (Jenner, 2003). 
Evidence shows that oxidative damage impairs ubiquitination and degradation of proteins 
by the proteasome. This may aid in the aggregation of ?-synuclei. This is the main 
27 
 
protein forming eosinophilic inclusions known as Lewy bodies, a pathological hallmark 
of Parkinson?s disease. The formation of these Lewy bodies is a key determinant that 
differentiates this disorder from other neurodegenerative diseases (Manning-Bog et al., 
2002). An up-regulation and increased aggregation of ?-synuclein was also seen after 
administration of neurotoxins (Bus and Gibson 1984).  
 
2.12 Apoptosis/Necrosis 
There is some debate about the notion of whether cell death in Parkinson?s 
disease occurs by way of apoptosis or necrosis. These two pathways are not, however, 
mutually exclusive as apoptosis and necrosis can occur alone, in combination, or as 
sequential events. Both have been put forth as pathways for cell death in both Parkinson?s 
disease and methamphetamine toxicity. Apoptosis or programmed cell death is a gradual, 
progressive cell death. Necrosis, on the other hand, is a rapid form of cell death. 
Apoptosis is characterized by marked cell shrinkage, fragmentation of nuclear DNA by 
endonucleases, and chromatin condensation with the formation of nuclear vesicles, which 
are phagocytosed by nearby cells (Nijhawan et al., 2000; Zimmermanet al., 2001). 
Necrosis is characterized by a massive ionic influx, cellular swelling with disruption of 
subcellular organelles and rupture of plasma membrane. It does not, however, involve 
extensive damage to nuclear DNA. Oxidative stress and mitochondrial dysfunction have 
been shown to cause both necrotic and apoptotic cell death.  
Because of its central position in the energetics of the cell, the mitochondrion 
plays a central role in apoptosis. Mitochondria influence apoptosis by maintenance of 
28 
 
ATP levels, maintenance of the mitochondrial membrane potential, and the release of 
proapoptotic factors. Mitochondrial dysfunction leads to cessation of electron transport, 
which brings about an energy crisis followed by apoptosis/necrosis. The depletion of 
ATP initiates an apoptotic cell death mechanism, which may be initiated by exposure to 
one or more of the neurotoxins discussed in this paper (Langston et al., 1983). 
 
2.13 Possible Neuroprotective Treatments 
In summary, the previous sections provide literature support for the neurotoxic 
effects of methamphetamine and discuss the mechanisms implicated in causing damage 
to dopaminergic cells. Thus, methamphetamine?s use can increase the risk for 
nigrostriatal neurodegeneration significantly. The mechanistic similarity of 
methamphetamine toxicity to the dopaminergic neurodegeneration seen in Parkinson?s 
disease indicates the usefulness of this model to test pharmacological agents for 
neuroprotective properties against this neurodegeneration. The literature supports the idea 
that, in Parkinson?s disease, oxidative stress and mitochondrial dysfunction are primary 
mediators of excitotoxicity, neuroinflammation, apoptosis and necrosis (Nam et al., 
2005). Thus, if a pharmacological agent can disrupt these neurotoxic mechanisms, it 
could provide protection from the resulting neurodegeneration.  
 
2.13.1 Ramelteon 
Ramelteon, (S)-N-[2-(1,6,7,8-tetrahydro2H-indeno-[5,4-b]furan-8-yl)ethyl] 
propionamide, is an orally active sleep agent (for insomnia) and a hypnotic substance. It 
29 
 
has a molecular weight of 259.34 and an empirical formula of C16H21NO2. In its pure 
form, it is a white powder. It is freely soluble in organic solvents, such as methanol, 
ethanol, and dimethyl sulfoxide; soluble in 1-octanol and acetonitrile; and very slightly 
soluble in water and in aqueous buffers (Takeda, 2008a). Ramelteon (figure 2.6) is 
available commercially as Rozerem by Takeda Pharmaceuticals North America. The 8 
mg tablets are round, pale orange-yellow and film-coated, with ?TAK? and ?RAM-8? 
printed on one side. This is the first in a new class of sleep agents that selectively binds to 
MT1 and MT2 receptors versus binding to GABA A receptors, such as with other hypnotic 
drugs like zolpidem, eszopiclone, and zaleplon. Ramelteon is approved by the FDA for 
long-term use in the treatment of insomnia.  
 
 
Figure 2.6. Chemical Structure of Ramelteon 
 
Ramelteon is a melatonin receptor agonist with both high affinity for melatonin 
MT1 and MT2 receptors and selectivity over the MT3 receptor (Owen, 2006). This is 
believed to contribute to its sleep-promoting properties, as these receptors, acted upon by 
endogenous melatonin, are thought to be involved in the maintenance of the circadian 
rhythm underlying the normal sleep-wake cycle (Miyamoto et al., 2004).  The 
significance of ramelteon's lack of affinity for the MT3 receptor is not clear. The MT3 
30 
 
receptor appears almost exclusively in the gut and might not have any relationship to 
sleep or wakefulness.  
Ramelteon does not show any appreciable binding to GABAA receptors, which 
are associated with anxiolytic, myorelaxant, and amnesic effects (Wang et al., 2003). It 
also shows no appreciable affinity for receptors that bind neuropeptides, cytokines, 
serotonin, dopamine, noradrenaline, acetylcholine or opiates. The major metabolite of 
ramelteon, M-II, has weak affinity for the serotonin 5-HT2B receptor, but no appreciable 
affinity for other receptors or enzymes. All other known metabolites of ramelteon are 
inactive (Erman et al., 2006; Zammit et al., 2007). Thus, ramelteon has not been shown to 
produce dependence and has shown no potential for abuse, and the withdrawal and 
rebound insomnia that is typical with other GABA modulators is not present in 
ramelteon. It is currently the only non-scheduled prescription drug for the treatment of 
insomnia available in the United States (Kato et al., 2005; Takeda, 2008b). 
It is not well studied whether ramelteon has other functional similarities with 
melatonin, besides induction of sleep. Besides its primary function of regulating the sleep 
cycle, melatonin may exert a powerful antioxidant activity (Tan et al., 2007). Melatonin 
is an antioxidant that can easily cross cell membranes and the blood brain barrier 
(Hardeland, 2005).  Melatonin is a direct scavenger of OH, O2?, and NO (Poeggeler et al., 
1994). Unlike other antioxidants, melatonin does not undergo redox cycling, the ability of 
a molecule to undergo reduction and oxidation repeatedly. Redox cycling may allow 
other antioxidants (such as vitamin C) to regain their antioxidant properties. Melatonin, 
on the other hand, once oxidized, cannot be reduced to its former state because it forms 
31 
 
several stable end-products upon reacting with free radicals. Therefore, it has been 
referred to as a terminal (or suicidal) antioxidant (Tan et al., 2000). In animal models, 
melatonin has been demonstrated to prevent the damage to DNA by some carcinogens, 
stopping the mechanism by which they cause cancer (Karbownik et al., 2001). It also has 
been found to be effective in protecting against brain injury caused by reactive oxygen 
species release in experimental hypoxic brain damage in newborn rats (Tutunculer et al., 
2005). Melatonin's antioxidant activity may reduce neuronal damage caused in 
Parkinson's disease (Oaknin-Bendahan et al., 1995). This raises the interesting question 
of whether ramelteon might also have such beneficial effects. If it posesses the same free 
radical scavenging properties as melatonin (Reiter et al., 2001), it could prove to be a 
neuroprotective drug. 
 
2.13.2 Amantadine 
Amantadine (figure 2.7), 1-aminoadamantane, is an organic compound that is sold 
commercially as Amantadine hydrochloride, under the name Symmetrel, for use both as 
an antiviral and an antiparkinsonian drug. Its molecular weight is 187.71 with a 
molecular formula of C10H18NCl. Amantadine hydrochloride is a stable white or nearly 
white crystalline powder, freely soluble in water and soluble in alcohol and in chloroform 
(Sandoz, 2008).  
 
32 
 
 
Figure 2.7. Chemical Structure of Amantadine 
 
Amantadine was approved by the U.S. Food and Drug Administration in October 
1966 as a prophylactic agent against Asian influenza and eventually received approval for 
the treatment of Influenzavirus A in adults (Maugh, 1976; Maugh 1979). For the 
mechanism of its antiviral properties, amantadine interferes with a viral protein, M2 (an 
ion channel), which is required for the viral particle to become "uncoated" once it is taken 
inside the cell by endocytosis (Wang et al., 1993). In 1969 the drug was also discovered 
by accident to help reduce symptoms of Parkinson's disease. The mechanism for its 
antiviral and antiparkinsonian effects seems to be unrelated. The mechanism of its 
antiparkinsonian effect is poorly understood. The drug appears to induce release of 
dopamine from the nerve endings of the brain cells, together with stimulation of 
norepinephrine response. Furthermore, it appears to be a weak NMDA receptor 
antagonist and an anticholinergic (Blanpied et al., 2005). As an antiparkinsonian it can be 
used as monotherapy; or together with levodopa to treat levodopa-related motor 
fluctuations, or shortening of levodopa duration of clinical effect, and levodopa-related 
dyskinesias, such as choreiform movements associated with long-term levodopa use 
(Sandoz, 2008). 
33 
 
Amantadine also has several off label uses. It is frequently used to treat the 
characteristic fatigue often experienced by patients with multiple sclerosis (Cohen and 
Fisher, 1989). Additionally, there have been anecdotal reports that low-dose amantadine 
has been successfully used to treat ADHD (Hallowell and Ratey, 2005). Amantadine has 
also been shown to relieve SSRI-induced sexual dysfunction (Balogh et al., 1992; 
Shrivastava et al., 1995; Keller et al., 1997). It is amantadine?s effectiveness as an 
NMDA receptor antagonist that led to its inclusion in this study as possibly 
neuroprotective.  Since NMDA receptors are one of the most harmful factors in 
excitotoxicity, antagonists of the receptors have held much promise for the treatment of 
conditions that involve excitotoxicity, including traumatic brain injury, stroke, and 
neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and 
Huntington's disease (Maas, 2001). Excitotoxicity is commonly seen in Parkinson?s 
disease. The defective mitochondrial energy metabolism seen in Parkinson?s disease 
patients may predispose them to excessive glutamate-evoked calcium influx. This could 
cause toxicity to the cell (Beal, 1992; Blandini, 1996). MPTP-induced parkinsonism is 
attenuated by a blockade of NMDA receptors (Lange, 1993; Nash, 2000). Amantadine is 
the only NMDA receptor antagonist that is actively used to treat Parkinson?s disease. If it 
proved to be neuroprotective, with its therapeutic use already well established, it would 
prove cost effective as well as ground breaking.  
 
 
 
34 
 
2.13.3 Salicylic Acid 
Salicylic acid (figure 2.8), 2-Hydroxy-benzoic acid, is found in several plants, 
notably in wintergreen leaves and the bark of sweet birch. It was made synthetically by 
heating sodium phenolyate with carbon dioxide under pressure and microbial oxidation 
of naphthalene (Windholz, 1983). Its molecular weight is 138.1 with a molecular formula 
C7H6O3. It belongs to a class of drugs known as analgesics or antipyretics. Salicylic acid 
is a metabolite produced in the body following ingestion of Aspirin, acetylsalicylic acid, 
a commonly used nonsteroidal anti-inflammatory drug (Hardman, 1996). Salicylic acid is 
a white crystalline powder with a sweetish acrid taste. If prepared from natural methyl 
salicylate, it may have a faint mint like odour. It is available in forms of powder, 
ointments, cream, gel, liquids and plaster. Salicylic acid is soluble 1 in 460 to 550 of 
water, 1 in 15 of boiling water, 1 in 3 to 4 in alcohol, 1 in 3 in ether and 1 in 45 in 
chloroform (Reynolds, 1996).                
 
 
Figure 2.8. Chemical Structure of Salicylic Acid 
 
Aspirin was the first-discovered member of the class of drugs known as non-
steroidal anti-inflammatory drugs (NSAIDs), not all of which are salicylates, although 
they all have similar effects and most have inhibition of the enzyme cyclooxygenase as 
35 
 
their mechanism of action. Today, Aspirin is one of the most widely used medications in 
the world, with an estimated 40,000 metric tons of it being consumed each year. In 
countries where Aspirin is a registered trademark owned by Bayer, the generic term is 
acetylsalicylic acid (Warner and Mitchell, 2002). 
Medicines containing derivatives of salicylic acid, structurally similar to Aspirin, 
have been in medical use since ancient times. Salicylate-rich willow bark extract became 
recognized for its specific effects on fever, pain and inflammation in the mid-eighteenth 
century. By the nineteenth century pharmacists were experimenting with and prescribing 
a variety of chemicals related to salicylic acid, the active component of willow extract. A 
French chemist, Charles Frederic Gerhardt, was the first to prepare acetylsalicylic acid 
(named Aspirin in 1899) in 1853. In the course of his work on the synthesis and 
properties of various acid anhydrides, he mixed acetyl chloride with a sodium salt of 
salicylic acid (sodium salicylate). A vigorous reaction ensued, and the resulting melt soon 
solidified. In 1897, scientists at the drug and dye firm Bayer began investigating 
acetylsalicylic acid as a less-irritating replacement for standard common salicylate 
medicines. By 1899, Bayer had dubbed this drug Aspirin and was selling it around the 
world. Aspirin's popularity grew over the first half of the twentieth century, spurred by its 
effectiveness in the wake of the Spanish flu pandemic of 1918, and aspirin's profitability 
led to fierce competition and the proliferation of Aspirin brands and products, especially 
after the American patent held by Bayer expired in 1917 (Jeffreys, 2005).  
Aspirin suppresses the production of prostaglandins and thromboxanes. 
Prostaglandins are local hormones produced in the body and have diverse effects in the 
36 
 
body, including the transmission of pain information to the brain, modulation of the 
hypothalamic thermostat, and inflammation. Thromboxanes are responsible for the 
aggregation of platelets that form blood clots. Heart attacks are primarily caused by blood 
clots, and low doses of Aspirin are seen as an effective medical intervention for acute 
myocardial infarction. The major side-effect of this is that because the ability of blood to 
clot is reduced, excessive bleeding may result from the use of aspirin. Aspirin's ability to 
suppress the production of prostaglandins and thromboxanes is due to its irreversible 
inactivation of the cyclooxygenase (COX) enzyme. Cyclooxygenase is required for 
prostaglandin and thromboxane synthesis. Aspirin acts as an acetylating agent where an 
acetyl group is covalently attached to a serine residue in the active site of the COX 
enzyme. This makes aspirin different from other NSAIDs, such as ibuprofen, which are 
reversible inhibitors. There are at least two different types of cyclooxygenase: COX-1 
and COX-2. Aspirin irreversibly inhibits COX-1 and modifies the enzymatic activity of 
COX-2. Normally COX-2 produces prostanoids, most of which are pro-inflammatory. 
Aspirin-modified COX-2 produces lipoxins, most of which are anti-inflammatory (Vane, 
1971; Tohgi et al., 1992; Vane and Botting, 2003). 
Salicylic Acid is a known free radical scavenger (Kataoka et al., 1997). It has 
been shown to provide neuroprotection against MPTP induced neurotoxicity 
(Mohanakumar et al., 2000). Thus, it could provide much needed neuroprotection against 
oxidative stress which leads to neurodegeneration. With the ability of salicylic acid for 
free radical scavenging, it could play a much needed neuroprotetive role in not only 
Parkinson?s disease, but also in numerous other neurodegenerative disorders. 
37 
 
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55 
 
3. STATEMENT OF RESEARCH OBJECTIVES 
 
Overall, the conducted study seeks evidence that intraperitoneal 
methamphetamine administration in mice causes significantly reduced levels of 
dopamine in the brain, thus providing a relevant model for Parkinson?s disease. In 
addition, this research evaluates the neuroprotective mechanisms of ramelteon, 
amantadine and salicylic acid using the methamphetamine lesioned mice model of 
Parkinson?s disease. The long-term goal is to facilitate and improve the care of patients 
with Parkinson?s disease by developing a cost-effective anti-Parkinson?s drug that has the 
added benefit of a neuroprotective effect. This model could successfully further our 
understanding of the neurodegenerative process (i.e. dopamine depletion) that targets the 
nigrostriatal system. Studies on dopaminergic toxicants which are also widely used drugs 
of abuse could have added preventative effects by helping identify and increase 
awareness of the risks involved in such drug abuse. 
The hypothesis of this study is that methamphetamine will cause significant 
dopaminergic depletion and that the neuroprotective effects of ramelteon, amantadine and 
salicylic acid will directly correlate with augmented behavioral, neurochemical and 
biochemical paremeters in the brain of methamphetamine treated mice and will render 
neuroprotection by reducing neuronal dopamine depletion.  
 
56 
 
4. CHAPTER 1: NEUROTOXIC EFFECTS OF METHAMPHETAMINE 
 
4.1 Overview 
Methamphetamine can cause striatal dopamine depletion and could be useful in 
making an animal model to study Parkinson?s disease. Since the neurotoxic mechanism 
of methamphetamine is not well understood, this study was undertaken to gain greater 
mechanistic understanding of the drug. In this study, we examined the neurotoxic effects 
of methamphetamine. With regard to biochemical assays, we evaluated the effect of 
methamphetamine on mitochondrial reactive oxygen species (ROS) generation, 
monoamine oxidase (MAO) activity, superoxide dismutase (SOD) activity and complex I 
& IV activities.  Behavioral analysis evaluated the effect on movement (tremor, straub 
tail, catalepsy, akinesia and swim score). In addition, neurotransmitter levels were 
evaluated using high pressure liquid chromatography (HPLC)-electrochemical detection. 
Results showed that methamphetamine caused significant dopamine depletion in the 
striatum and also caused significant generation of reactive oxygen species and decreased 
complex I activity in the mitochondria. Behavioral analysis showed a significant effect on 
catalepsy, akinesia and reduced swim score. 
57 
 
4.2 Introduction 
Nigrostriatal dopaminergic neurons undergo selective degeneration in Parkinson?s 
disease leading to a shortage of dopamine in the striatum (Hornykiewicz, 1973; Caine 
and Langston, 1983).  Neurons in the striatum require dopamine to effectively control 
movement. Thus, the nigral dopaminergic neurodegeneration in Parkinson?s disease 
causes motor deficits like slowness in initiation and execution of voluntary movements, 
increased muscle tone, increased resistance to movement, equilibrium and righting reflex, 
shuffling of feet and stooped posture (Sethy et al., 1997). The dopamine depletion is also 
evidenced by various motor symptoms including resting tremors, bradykinesia, muscular 
rigidity, and abnormalities in posture and gait. Parkinson?s disease is a progressive 
disorder and complete immobility usually occurs, despite treatment (Kopin, 1992; 
Marsden, 1994; Sethy et al., 1997). Oxidative stress, mitochondrial dysfunction, 
apoptosis, neuroinflammation and N-methyl-D-aspartic acid (NMDA) receptor-mediated 
excitotoxicity are mechanisms commonly implicated in dopaminergic neurotoxicity (Lau 
et al., 2000; Dluzen et al., 2001; Gluck et al., 2001; Dawson and Dawson, 2003; Kita et 
al., 2003; Sherer et al., 2003; LaVoie et al., 2004; Thomas et al., 2004; Riddle et al., 
2006).  The above mentioned mechanisms are also indicated in Parkinson?s disease. It is 
well known that a significant degree of striatal dopamine depletion, similar to that seen in 
Parkinson?s disease, can be produced by endogenous and exogenous neurotoxins (Dluzen 
et al., 2001; Dawson and Dawson, 2003; Sherer et al., 2003). Studies have implicated 
exposure to toxins like 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP), 
paraquat, maneb, rotenone, manganese and iron as contributors to the development of 
58 
 
Parkinson?s disease (Dawson and Dawson, 2003; Sherer et al., 2003).  These toxins are 
useful in the creation of animal models to study the different aspects of Parkinson?s 
disease. With no proven neurorestorative or neuroprotective treatment strategies for this 
disease, these animal models are essential for discovering novel and efficient treatments 
that are proactive, not merely symptomatic, and to lowering the disease progression and 
mortality rate (Dawson and Dawson, 2003).  
Methamphetamine is a highly addictive drug that is widely abused in the United 
States (NIDA, 2002). Methamphetamine is also used clinically in the treatment of obesity 
and depression, but this psycho-stimulatory drug has highly addictive properties and 
neurotoxic effects (NIDA, 2002; McMillen, 1983).  It can cross the blood brain barrier 
and is possibly capable of causing damage to dopaminergic neurons.  Methamphetamine 
administration results in long lasting dopamine depletion in humans and animals 
(Davidson et al., 2001; Cadet et al., 2003; Kita et al., 2003).  Examination of post-mortem 
brain tissue from methamphetamine users showed a significant decrease in dopamine 
content and dopamine transporter density (Wilson et al., 1996).  Methamphetamine has 
been shown to induce striatal dopamine depletion in mice striatum, hence it can be ideal 
to study the disease progression and for evaluating neuroprotective drugs (Bondy et al., 
2000; Dluzen et al., 2001). It has been further shown that repeated administration of 
methamphetamine reduces dopamine transporter (DAT) binding and causes persistent 
dopaminergic degeneration in the striatum (Davidson et al., 2001; Cadet et al., 2003; Kita 
et al., 2003).   
59 
 
There is a lack of exact mechanistic understanding of the neurotoxicity of 
methamphetamine.  There are several possible mechanisms such as generation of reactive 
radicals, mitochondrial dysfunction and reduced energy metabolism and DNA damage 
leading to eventual cell death (Fibiger and Mogeer, 1971; Imam et al., 2001; Pubill et al., 
2005; Riddle et al., 2006). Mitochondria are vital organelles present in the mammalian 
cell that not only generate adenosine triphosphate but also perform such critical functions 
as hosting essential biosynthetic pathways, calcium buffering, and apoptotic signaling.  
The incomparable and excessive energy requirements of neurons require proper 
functioning and maintenance of mitochondria.  Mitochondrial abnormalities have been 
associated to several neurodegenerative diseases, but the evidence is particularly strong, 
and continuously accumulating, in Parkinson's disease and Alzheimer?s disease (Dawson 
and Dawson, 2003).  
In this study we examined in vitro effect of the methamphetamine using mouse 
brain mitochondria and for the in vivo experiments C57/Bl6 mice were used. We 
analyzed the effect of methamphetamine on various behavioral, biochemical and 
neurochemical parameters relating to nigral dopaminergic damage.  Methamphetamine 
like other dopaminergic neurotoxins induced significant behavioral changes similar to 
that seen in Parkinson?s disease, caused oxidative stress, mitochondrial dysfunction and 
induced significant dopamine depletion in the striatum. 
 
 
 
60 
 
4.3 Materials and Methods 
 
4.3.1 Animals 
Male C57/Bl6 mice (4-6 weeks old) weighing 20-30 g were purchased from 
Charles Rivers. They were housed in a temperature controlled room with a 12h day and 
night cycle with free access to food and water. They were housed for 2-4 days prior to 
experiments. All the experimental procedures were reviewed and approved by 
Institutional Animal Care and Use Committee (IACUC) at Auburn University. 
 
4.3.2 Methamphetamine administration 
C57BL/6 mice were separated into 3 groups (control, low dose methamphetamine 
& high dose methamphetamine) and treated with saline, methamphetamine (10mg/kg i.p., 
twice, 2hr apart) and methamphetamine (20mg/kg i.p., twice, 2hr apart), respectively. 
The animals were sacrificed 5 days after the last injection.  
 
4.3.3 Behavioral Studies 
Following methamphetamine administration, two examiners who were trained in 
evaluating different animal behaviors continuously observed mice. Tremor, straub tails, 
akinesia and catalepsy were monitored during this period following standard scoring 
procedures as described below. Animals were subjected to swim test on the 4th day after 
methamphetamine administration. 
61 
 
Straub tail: Straub tail was defined as elevation of tail to more than 45  with a 
curvature of the tail. The results are expressed as whether straub tail phenomenon was 
detected or not detected (Mohanakumar and Sood, 1986). 
Akinesia: Akinesia usually refers to the impaired ability to initiate movements. It 
was measured by noting the latency in second(s) of the animals to move all four limbs 
and the test was terminated if the latency exceeded 180s (Weihmuller et al., 1988; 
Muralikrishnan and Mohanakumar, 1998). 
Catalepsy: The term implies the inability of an animal to correct an externally 
imposed posture. Catalepsy was measured by placing the animals on a flat horizontal 
surface with both the hind limbs on a square wooden block (3 cm, high) and the latency 
in seconds was measured to move the hind limbs from the block to the ground. The 
animals were initially acclimatized on the wooden platform (Weihmuller et al., 1988; 
Murlikrishnan and Mohanakumar, 1998).  
Swim test: Swim test was carried out in water tubs (40 l x 25 w x 12 h, in cm). 
The depth of water was kept at 8 cm and the temperature was maintained at 27  2  C. 
The animals were acclimatized for 10 min one day prior to experimentation. Swim score 
scales were: 0 = hind part sinks with head floating, 1 = occasional swimming using hind 
limbs while floating on one side, 2 = occasional floating/swimming only, 3 = continuous 
swimming (Donnan et al., 1987). 
 
 
 
62 
 
4.3.4 Tissue Preparation for in vivo biochemical assays 
Animals were sacrificed by decapitation in the morning to avoid diurnal 
variations of the endogenous amines, enzymes, and other antioxidant molecules. The 
brain tissue was homogenized in 0.1 M phosphate buffer (pH 7.8), using a glass-Teflon 
homogenizer. Tissue homogenates were centrifuged at 10,000 g for 60 min at 4?C 
(Muralikrishnan and Mohanakumar, 1998; Muralikrishnan and Ebadi, 2001). 
 
4.3.5 Measurement of Neurotransmitters  
To determine dopamine depletion in the striatum, methamphetamine injected 
animals were sacrificed on the 5th day and the striata was dissected out and analyzed for 
DA content employing HPLC-electrochemistry. Dopamine and its metabolites were 
separated in high pressure liquid chromatography. An electrochemical detector was used 
to quantify dopamine and its metabolites. The C18 octadecyl silica column (8 cm X 4.6 
mm i.d.) was used for the separation of the monoamines. Brain tissue was sonicated in 
0.4 N HClO4. These samples were then centrifuged and the 20 ?l of supernatant used was 
injected in HPLC. The results were expressed as picomoles per milligram of tissue 
(Uthayathas et al., 2007). 
 
4.3.6 Mouse Mitochondrial P2 Fraction for in vitro biochemical assays 
Mitochondrial (P2) fraction was prepared from the mouse brain. The brain was 
homogenized in 0.1M phosphate buffer, pH 7.8, using a glass teflon homogenizer. Tissue 
homogenates were centrifuged at different rpm to get the mitochondrial P2 fraction. 
63 
 
Different concentrations of methamphetamine (1, 2.5, 5 & 10?M) were incubated for 60 
minutes with mitochondrial fraction obtained from the mouse brain. The in vitro effect of 
methamphetamine on complex I & IV activity, ROS generation, SOD activity and MAO 
activity in the mitochondria was evaluated using the following procedures: 
 
4.3.7 Protein estimation 
Protein was assayed using the coomassie plus protein assay reagent kit (Pierce, 
Rockford, IL). Bovine serum albumin (BSA) was used as a standard for protein 
measurement. 
 
4.3.8 Mitochondrial Complex I Activity  
Mitochondrial complex-I activity (NADH dehydrogenase activity) is based on the 
NADH oxidation. Oxidation of NADH by the NADH-dehydrogenase enzyme present in 
the tissue homogenate was measured spectrophotometrically at 340 nm.  The 
mitochondrial P2 suspension was added to the reaction mixture containing NADH, 
coenzyme Q0 and phosphate buffered saline, to analyze its effect on NADH oxidation by 
monitoring the decrease in absorbance at 340 nm.  A standard curve for NADH was 
obtained at 340 nm by using known quantities of NADH.  The change in absorbance 
indicates the amount of NADH oxidized.  The mitochondrial complex-I activity is 
expressed as the amount of NADH oxidized/mg protein (Wharton and Tzagotoff, 1967; 
Ramsay et al., 1986). 
  
64 
 
4.3.9 Mitochondrial Complex IV Activity  
Complex IV activity was based on the cytochrome-C oxidation. The 
mitochondrial P2 suspension was added to the reaction mixture containing cytochrome-C 
and phosphate buffered saline.  The oxidation was measured spectrophotometrically. The 
absorbance was measured at 550nm for 2 min and the enzyme activity was expressed as 
cytochrome-C oxidized/mg protein (Wharton and Tzagotoff, 1967; Ramsay et al., 1986).  
 
4.3.10 Assay of MAO activity 
Total MAO activity was based on the amount of 4-hydroxyquinoline formed by 
the oxidation of kynuramine (Morinan and Garratt, 1985).  4-hydroxyquinoline was 
measured fluorimetrically and the enzyme activity was expressed as 4-hyroxyquinoline 
formed/hour/mg protein (Muralikrishnan and Mohanakumar, 1998; Muralikrishnan and 
Ebadi, 2001). 
 
4.3.11 Assay of SOD activity 
SOD activity was analyzed following the method of Marklund and Marklund 
(1974). Pyrogallol oxidation was measured at 420nm. 
 
4.3.12 Assay of ROS production 
Conversion of nonfluorescent chloromethyl-DCF-DA (2?,7?- dichlorofluorescin 
diacetate) to fluorescent DCF was used to monitor ROS production 
spectrofluorometerically using an excitation wavelength of 492 nm and an emission 
65 
 
wavelength of 527 nm. The generation of ROS was measured and reported as relative 
fluorescence intensity (Dhanasekaran et al., 2008). 
 
4.3.13 Statistical Analysis 
Results are expressed as the means?SEM. The statistical significance was 
evaluated by the one-way analysis of variance (ANOVA) using SigmaStat version 3. 
Values of p less than or equal to 0.05 were considered significant. 
 
4.4 Results 
 
4.4.1 Behavioral Analysis 
Methamphetamine caused a significant motor impairment in the C57/BL6 mice. 
Intraperitoneal administration of methamphetamine (10 & 20 mg/kg) in mice induced 
significant dose dependant behavioral abnormalities including catalepsy (figure 4.1), 
akinesia (figure 4.2) and reduced swim time (figure 4.3) as compared to the controls. 
Straub tails and tremor were also observed (table 4.1). 
 
4.4.2 Effect of methamphetamine on neurotransmitters 
Administration of methamphetamine (10 & 20mg/kg) caused a significant 
reduction in the levels of dopamine in the striatum (figure 4.4). Methamphetamine (10 & 
20mg/kg) treated animals showed significant dopamine depletion (n=12, p<0.05) as 
compared to control animals. Methamphetamine also affected levels of other striatal 
66 
 
neurotransmitters (NE, 5HT, 5HIAA) and dopamine metabolites (DOPAC, HVA) as 
shown in table 4.2. 
 
4.4.3 In vivo biochemical analysis 
Intraperitoneal administration of methamphetamine (10 & 20mg/kg) in mice 
significantly decreased complex I activity (n=12, p<0.05; figure 4.5) while having no 
significant effect on complex IV activity (figure 4.6). Administration of 
methamphetamine had no significant effect on the total mitochondrial monoamine 
oxidase (MAO) activity (figure 4.7). However, the administration of methamphetamine 
(10 & 20mg/kg) increased superoxide dismutase (SOD) activity (n=12, p<0.05; figure 
4.8) and caused significant generation of reactive oxygen species (n=12, p<0.001; figure 
4.9).  
 
4.4.4 In vitro biochemical analysis 
Incubation of mouse mitochondrial P2 homogenate for 60 minutes with 
methamphetamine (1, 2.5, 5 & 10?M) significantly decreased complex I activity (n=6, 
p<0.05; figure 4.10) while having no significant effect on complex IV activity (figure 
4.11). Incubation with methamphetamine at the same concentration had no significant 
effect on the total mitochondrial monoamine oxidase (MAO) activity (figure 4.12). 
However, the incubation with methamphetamine (1, 2.5, 5 & 10?M) increased 
superoxide dismutase (SOD) activity (n=6, p<0.05; figure 4.13) and caused significant 
generation of reactive oxygen species (n = 6, p <0.001; figure 4.14).  
67 
 
4.5 Discussion 
Results of this study show that injection with methamphetamine caused a 
significant amount of dopamine depletion, as well as creating a striatal environment ripe 
for oxidative damage and mitochondrial dysfunction. Both the in vitro and in vivo results 
showed that methamphetamine caused significant generation of reactive oxygen species 
and increased superoxide dismutase activity. These results indicate an environment of 
oxidative stress. Methamphetamine also causes a significant decrease in complex I 
activity. Complex I mitochondrial dysfunction is specific to Parkinson?s disease. 
Similarly there are numerous markers in the nigrostriatal pathway to indicate the role of 
oxidative stress in nigral dopaminergic neurodegeneration. The neurochemical data 
showed that methamphetamine caused significant dopamine depletion in the striatum of 
greater than 50% for both the 10mg/kg and the 20mg/kg doses. Behavioral analysis 
showed a significant motor impairment (catalepsy, akinesia, straub tail, tremor and swim 
score). These motor abnormalities are comparable to those seen with dopaminergic 
neurodegeneration. 
Methamphetamine is a cationic and lipophilic compound capable of crossing the 
blood-brain barrier and acts as a potent stimulant. In the central nervous system, it 
displaces monoamines like dopamine, norepinephrine and serotonin from their vesicles. 
Once released, the dopamine is metabolized producing proxidant hydrogen peroxide and 
it can also be oxidized to reactive oxygen species. The combination of oxidative stress 
and mitochondrial complex I dysfunction leads to apoptosis and creates toxicity of striatal 
68 
 
nerve terminals. This leads to long lasting dopamine depletion (Stokes et al., 1999; Zhu et 
al., 2006). 
The brain and nervous system are particularly vulnerable to free radical damage 
for a number of reasons. The membrane lipids in the brain contain high levels of 
polyunsaturated fatty acid side chains, which are prone to free radical attack and are 
readily peroxidizable. The brain also consumes large quantities of total oxygen for its 
relatively small weight, contributing further to the formation of reactive oxygen species. 
It has been estimated that up to 2% of the oxygen consumed by healthy mitochondria is 
converted to superoxide, and this amount is higher in damaged and aged mitochondria. 
Oxidative species are important in cellular respiration and aid in the regulation of signal 
transduction pathways. However, they can be converted into free radicals, containing 
unpaired electrons, which are collectively referred to as reactive oxygen species (ROS). 
Oxidative stress involves the excessive formation of reactive oxygen species, like the 
hydroxyl radicals (OH., OH-) and superoxide (O2.), among others, which are formed 
when molecular oxygen is reduced non-enzymatically (Fahn and Cohen, 1992; Jenner et 
al., 1992). The brain has been shown to contain low to moderate levels of enzymes such 
as catalase, superoxide dismutase and glutathione peroxidase that play an important role 
in the metabolism of reactive oxygen species (Cohen, 1988). Superoxide dismutase 
(SOD) converts the superoxide to hydrogen peroxide, and hydrogen peroxide is typically 
detoxified by glutathione peroxidase and catalase. However, oxidative stress occurs when 
free radicals are in excess of these antioxidant defenses. Neurotoxins damaging the 
dopaminergic neurons have been shown to generate hydroxyl radicals and significantly 
69 
 
alter the antioxidant enzyme activities in the mouse brain (Muralikrishnan and 
Mohanakumar, 1998; Muralikrishnan and Ebadi, 2001).  Neurotoxins such as MPTP, 6-
OHDA, paraquat, maneb, iron and manganese have been shown to exhibit dopamine 
depletion in the mouse striatum.  
Oxidative stress causes an inhibition of mitochondrial ATP production, a major 
determinant of lethal cell injury. Mitochondrial DNA is sensitive to the toxicity caused by 
reactive oxygen species because mitochondria have only a limited arsenal of DNA repair 
processes. Therefore, mitochondrial DNA mutations would cause defects in the 
respiratory chain function leading to severe cellular damage. Free radicals can interact 
with sugars, proteins and lipids creating a number of modifications which could then lead 
to mitochondrial dysfunction, apoptosis and even necrosis. The damage to the striatal 
terminals and the nigral cell bodies observed in methamphetamine users is indicative of 
all of these toxic mechanisms, the same mechanisms indicated in Parkinson?s disease 
(Imberti et al., 1993; Jones and Vale, 2000).  
There are studies lending evidence to support the theory that oxidative stress and 
mitochondrial dysfunction play a key role in neurodegeneration. Recent studies were 
conducted on the antioxidant coenzyme Q10, which plays an important role in the 
mitochondrial electron transport chain as the electron acceptor for complex I. Levels of 
Q10 were found to be reduced in Parkinson?s disease patients. Coenzyme Q10 has 
provided protection against both MPTP (Akaneya et al., 1995) and rotenone (Sherer et 
al., 2003). Also, clinical studies have shown that Q10 can provide some protection for the 
70 
 
nigrostriatal dopaminergic system and can slow the progressive disability of the disease 
(Shults, 2005).  
Mitochondrial dysfunction has long been thought to play a significant role in 
depletion of nigral neurons, leading to dopaminergic depletion. Evidence seems to 
suggest that the neurons in the substantia nigra are particularly prone to oxidative stress, 
which would explain the selective degeneration in this region (Halliwell, 1998). The 
central dopaminergic system that plays such an important role in motor activity is 
comprised of a surprisingly small number of neurons. Because of this, it is especially 
vulnerable and even minor insults may lead to irreparable functional deficits (Fariello et 
al., 1988). Studies have implicated exposure to toxins like methamphetamine as 
contributors to the development of Parkinson?s disease. This study shows the significant 
degree of dopamine depletion produced by methamphetamine, evidenced by both 
behavioral experiments and HPLC data. It also offers biochemical evidence of oxidative 
stress and mitochondrial dysfunction. These results indicate the usefulness of 
methamphetamine in making an animal model to study Parkinson?s disease. The current 
treatment strategy for Parkinson?s disease remains primarily symptomatic, with no 
proven neuroprotective or neurorestorative treatments. An animal model with a Parkinson 
disease phenotype could be utilized to gain a better understanding of the 
neurodegeneration in the nigrostriatal system and to investigate pharmacological agents 
for any neuroprotective or neurorestorative effects they may exhibit.  
 
 
71 
 
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dopamine. J. Neurosci. Res. 55:659-665. 
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Methamphetamine neurotoxicity in dopamine nerve endings of the striatum is 
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Zhu, J.P.Q., Xu, W., Angulo, J.A. 2006. Methamphetamine induced cell death: selective 
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77 
 
4.7 Figures 
 
Figure 4.1. Effect of methamphetamine on catalepsy: Methamphetamine (10 & 20 
mg/kg) significantly increased catalepsy (n=12, p*<0.05) as compared to control animals. 
 
Figure 4.2. Effect of methamphetamine on akinesia: Methamphetamine (10 & 20 mg/kg) 
significantly increased akinesia (n=12, p*<0.05) as compared to control animals. 
78 
 
 
Figure 4.3. Effect of methamphetamine on swim score: Methamphetamine (10 & 20 
mg/kg) significantly decreased swim score (n=12, p*<0.05) as compared to control 
animals. Scored by immobility time. 
 
  average weight straub tail tremor 
Control 24.3?0.6 0 0 
Methamphetamine 10mg/kg 24.2?0.5 n=12 4.0?0.1 
Methamphetamine 20mg/kg 23.6?0.3 n=12 4.0?0.1 
 
Table 4.1. Effects of methamphetamine on straub tail and tremor: Methamphetamine (10 
& 20 mg/kg) administration induced straub tail and tremor. 
79 
 
 
Figure 4.4. Effect of methamphetamine on striatal dopamine content: Methamphetamine 
(10 & 20mg/kg) treated animals showed significant dopamine depletion (n=12, p*<.05) 
as compared to control animals. 
 
  
Control 
 
Methamphetamine 
 10mg/kg 
Methamphetamine 
 20mg/kg 
DOPAC 49.12?11.6 19.45?3.8 * 24.98?4.5 * 
HVA 24.73?1.7 18.10?1.7 * 14.60?1.7 * 
5-HT 2.99?1.6 2.69?0.2 4.28?0.7 
5-HIAA 11.37?1.7 9.86?0.5 24.76?3.1 * 
NE 15.21?0.9 14.49?0.9 12.91?0.9 * 
 
Table 4.2. Effect of Methamphetamine on striatal neurotransmitter and metabolite levels 
(expresed as average pmole/mg tissue ? SEM). * significant (n=12, p*<.05) as compared 
to control animals.  
80 
 
 
Figure 4.5. Effect of methamphetamine on mitochondrial Complex I activity (in vivo): 
Methamphetamine (10 & 20 mg/kg) significantly decreased complex I activity as 
compared to controls (n = 12, p*<0.05). Activity expressed as ?mol/min/mg protein. 
 
Figure 4.6. Effect of methamphetamine on mitochondrial Complex IV activity (in vivo): 
Methamphetamine (10 & 20 mg/kg) caused no significant change in complex IV activity 
(n=12). Activity expressed as ?mol/min/mg protein. 
81 
 
 
Figure 4.7. Effect of methamphetamine on mitochondrial MAO activity (in vivo): 
Methamphetamine (10 & 20 mg/kg) caused no significant change in MAO activity 
(n=12). Activity expressed as 4-OHQ/mg protein. 
 
Figure 4.8. Effect of methamphetamine on mitochondrial SOD (in vivo): 
Methamphetamine (10 & 20 mg/kg) significantly increased SOD activity (n = 12, 
p*<0.05) as compared to control animals. 
82 
 
 
Figure 4.9. Effect of methamphetamine on mitochondrial ROS (in vivo): 
Methamphetamine (10 & 20 mg/kg) caused significant generation of ROS (n = 12, 
p*<0.001) as compared to control animals. 
 
 
 
 
83 
 
 
Figure 4.10. Effect of methamphetamine on mitochondrial Complex I activity (in vitro): 
Methamphetamine (1, 2.5, 5 & 10?M) dose dependently decreased complex I activity, 
expressed as ?mol/min/mg protein (n = 6, p*<0.05) as compared to controls. 
 
Figure 4.11. Effect of methamphetamine on mitochondrial Complex IV activity (in vitro): 
Methamphetamine (1, 2.5, 5 & 10?M) had no significant effect on complex IV activity 
(n=6). Activity expressed as ?mol/min/mg protein. 
84 
 
 
Figure 4.12. Effect of methamphetamine on mitochondrial MAO activity (in vitro): 
Methamphetamine (1, 2.5, 5 & 10 ?M) had no significant effect on MAO activity (n=6). 
Activity expressed as 4-OHQ/mg protein. 
 
Figure 4.13. Effect of methamphetamine on mitochondrial SOD (in vitro): 
Methamphetamine (1, 2.5, 5 & 10 ?M) significantly and dose dependently increased 
SOD activity (n = 6, p*<0.05) as compared to controls. 
85 
 
 
Figure 4.14. Effect of methamphetamine on mitochondrial ROS (in vitro): 
Methamphetamine (2.5, 5 & 10 ?M) caused significant generation of reactive oxygen 
species (n = 6, p*<0.001) as compared to controls. H2O2 10?M served as a positive 
control.
86 
 
5. CHAPTER 2: NEUROPROTECTION OF RAMELTEON AGAINST 
NEUROTOXIC EFFECTS OF METHAMPHETAMINE 
 
5.1 Overview 
In this study we examined the neuroprotective effect of ramelteon against 
methamphetamine toxicity. Ramelteon is a novel melatonin receptor agonist that is used 
to treat insomnia. Melatonin itself is known to have neuroprotective effects such as 
antioxidant and antiapoptotic properties. If ramelteon possesses the same properties, it 
may reduce oxidative damage seen in neurodegenerative disorders like Parkinson's 
disease. Discovery of a neuroprotective pharmacological agent could lead to proactive 
strategies to prevent neurological damage. We examined the effect of ramelteon and 
methamphetamine on reactive oxygen species generation, superoxide dismutase, 
monoamine oxidaseactivity and complex I & IV activities. Behavioral analysis evaluated 
the effect on movement. Neurotransmitter levels were evaluated using HPLC-ECD. 
Results showed that ramelteon alone did not cause neurotoxicity to the cells. It showed 
no effect on behavior, biochemical or neurochemical parameters. Methamphetamine 
caused significant dopamine depletion in the striatum and also caused significant 
generation of reactive oxygen species and decreased complex I activity in the 
mitochondria. Behavioral analysis showed that methamphetamine had a significant effect 
on movement. Ramelteon did not block the neurotoxic effects of methamphetamine.  
87 
 
5.2 Introduction 
Ramelteon is a novel melatonin (MT) receptor agonist that binds to MT1 and 
MT2 receptors selectively over MT3 receptors. Ramelteon is approved by the FDA for 
long-term use in the treatment of insomnia and is available commercially as Rozerem by 
Takeda Pharmaceuticals North America.  Other drugs used in the treatment of insomnia 
are GABA modulators and have been shown to produce dependence, withdrawal and 
rebound insomnia. Ramelteon, however, has not been shown to produce dependence and 
has shown no potential for abuse. In addition, it does not appear to cause learning, 
memory or motor function impairment. It is currently the only non-scheduled prescription 
drug for the treatment of insomnia available in the United States (Kato et al., 2005). 
Ramelteon possess structural similarity with melatonin and hence shares lot of 
similar physiological effects. Melatonin has been established in research as a direct 
scavenger of reactive oxygen species (ROS), like OH, O2?, and NO (Poeggeler et al., 
1994). It also has been found to be effective in protecting against brain injury caused by 
ROS release in experimental hypoxic brain damage in newborn rats (Tutunculer et al., 
2005). Melatonin's antioxidant activity may reduce damage caused Parkinson's disease 
(Oaknin-Bendahan et al., 1995). If ramelteon possesses the same free radical scavenging 
properties as melatonin (Reiter et al., 2001) it could prove to be neuroprotective.  
Oxidative stress has been strongly implicated in the pathogenesis of Parkinson?s 
disease. Brains from patients with Parkinson?s disease have shown elevated markers of 
oxidative damage, indicating that oxidative stress may play a key role in the disease 
(Hensley et al., 1998; Sherer et al., 2002). Oxidative stress involves the excessive 
88 
 
formation of reactive oxygen species, like the hydroxyl radicals (OH., OH-) and 
superoxide (O2.), among others, which are formed when molecular oxygen is reduced 
non-enzymatically (Fahn and Cohen, 1992; Jenner et al., 1992). Oxidative stress occurs 
when free radicals are in excess of any antioxidant defense. There is a tendency for free 
radicals to be more easily formed and less easily scavenged in the brain of Parkinson?s 
disease patients (Thiruchelvam et al., 2005). Free radicals are capable of reacting with 
DNA, lipids, and proteins to alter their structure and function (Turrens and Boveris, 1980; 
Hasegawa et al., 1990). Melatonin's antioxidant activity may reduce neuronal damage 
caused in Parkinson's disease (Oaknin-Bendahan et al., 1995). 
One of the proposed mechanisms of this toxicity in Parkinson?s disease is 
oxidative stress. Oxidative stress causes an inhibition of mitochondrial ATP production, a 
major determinant of lethal cell injury. The damage to the striatal terminals and the nigral 
cell bodies observed in methamphetamine users is also indicative of oxidative stress, the 
same mechanisms indicated in Parkinson?s disease (Imberti et al., 1993; Jones and Vale, 
2000). Methamphetamine is a highly neurotoxic substance capable of causing damage to 
dopaminergic neurons. Its mechanism of toxicity is thought to include generation of 
reactive radicals which are capable of causing mitochondrial dysfunction and reduced 
energy metabolism and eventual cell death (Riddle et al., 2006; Fibiger and Mogeer, 
1971; Imam et al., 2001; Pubill et al., 2005).  
In Parkinson?s disease, most of the neurons located in the substantia nigra that 
produce dopamine undergo neurodegeneration, causing a severe shortage of dopamine in 
the striatum (Caine and Langston, 1983). Thus, one affected with Parkinson?s disease 
89 
 
would be expected to exhibit some or all of the following motor deficits: slowness in 
initiation and execution of voluntary movements, increased muscle tone, increased 
resistance to movement, shuffling of feet, stooped posture and equilibrium and righting 
reflex (Sethy et al., 1997). In many cases of Parkinson?s disease, complete immobility 
eventually occurs despite treatment (Marsden, 1994). Parkinson?s disease is a slow-onset 
disease and the prevalence of Parkinson?s disease increases with age. There are estimates 
of up to 800 affected per 100,000 individuals age 65 or older (Savitt, 2006). The current 
treatment strategy for Parkinson?s disease remains primarily symptomatic, with no 
proven neuroprotective or neurorestorative treatments (Dawson and Dawson, 2003).With 
this study we examined the neuroprotective effect of ramelteon against methamphetamine 
toxicity, with implications for Parkinson?s disease. Discovery of a neuroprotective 
pharmacological agent could lead to proactive strategies to counteract the neurological 
damage caused by the disease.  
 
5.3 Materials and Methods 
 
5.3.1 Animals 
Male C57/Bl6 mice (4-6 weeks old) weighing 20-30 g were purchased from 
Charles Rivers. They were housed in a temperature controlled room with a 12h day and 
night cycle with free access to food and water. They were housed for 2-4 days prior to 
experiments. All the experimental procedures were reviewed and approved by 
Institutional Animal Care and Use Committee (IACUC) at Auburn University. 
90 
 
5.3.2 Drug administration 
C57/Bl6 mice were separated into 5 groups (control, methamphetamine only, high 
dose ramelteon only, ramelteon high dose + methamphetamine and ramelteon low dose + 
methamphetamine). The groups were given i.p. injections once daily for 1 week with a 
mixture of polyethelyne glycol (PEG) and sterile water (control & methamphetamine 
groups), 30mg/kg ramelteon, dissolved in PEG and diluted to appropriate concentration 
with sterile water (ramelteon high dose), 30mg/kg ramelteon, dissolved in PEG and 
diluted to appropriate concentration with sterile water (ramelteon high dose + 
methamphetamine group),  and 3mg/kg ramelteon, dissolved in PEG and diluted to 
appropriate concentration with sterile water (ramelteon low dose + methamphetamine 
group). On day 7 the methamphetamine, ramelteon high dose + methamphetamine and 
ramelteon low dose + methamphetamine groups were injected with methamphetamine 
(10mg/kg i.p., twice, 2hr apart). The animals were sacrificed 5 days after the last 
injection.  
 
5.3.3 Behavioral Studies 
Following methamphetamine administration, two examiners who were trained in 
evaluating different animal behaviors continuously observed mice. Tremor, akinesia, 
catalepsy, and straub tails were monitored during this period following standard scoring 
procedures as described below. Animals were subjected to swim test on the 4th day after 
methamphetamine. 
91 
 
Straub tail: Straub tail was defined as elevation of tail to more than 45  with a 
curvature of the tail. The results are expressed as Straub tail phenomenon was detected or 
not detected (Mohanakumar and Sood, 1986). 
Akinesia: Akinesia usually refers to the impaired ability to initiate movements. It 
was measured by noting the latency in second(s) of the animals to move all four limbs 
and the test was terminated if the latency exceeded 180s (Weihmuller et al., 1988; 
Muralikrishnan and Mohanakumar, 1998). 
Catalepsy: The term implies the inability of an animal to correct an externally 
imposed posture. Catalepsy was measured by placing the animals on a flat horizontal 
surface with both the hind limbs on a square wooden block (3 cm, high) and the latency 
in seconds was measured to move the hind limbs from the block to the ground. The 
animals were initially acclimatized on the wooden platform (Weihmuller et al., 1988; 
Murlikrishnan and Mohanakumar, 1998).  
Swim test: Swim test was carried out in water tubs (40 l x 25 w x 12 h, in cm). 
The depth of water was kept at 8 cm and the temperature was maintained at 27  2  C. 
The animals were acclimatized for 10 min one day prior to experimentation. Swim score 
scales were: 0 = hind part sinks with head floating, 1 = occasional swimming using hind 
limbs while floating on one side, 2 = occasional floating/swimming only, 3 = continuous 
swimming (Donnan et al., 1987). 
 
 
 
92 
 
5.3.4 Measurement of Neurotransmitters  
To determine dopamine depletion in the striatum, methamphetamine injected 
animals were sacrificed on the 5th day and the striata was dissected out and analyzed for 
DA content employing HPLC-electrochemistry. Dopamine and its metabolites were 
separated in high pressure liquid chromatography. An electrochemical detector was used 
to quantify dopamine and its metabolites. The C18 octadecyl silica column (8 cm X 4.6 
mm i.d.) was used for the separation of the monoamines. Brain tissue was sonicated in 
0.4 N HClO4. These samples were then centrifuged and the 20 ?l of supernatant used was 
injected in HPLC. The results were expressed as picomoles per milligram of tissue 
(Uthayathas et al., 2007). 
 
5.3.5 Tissue Preparation for In vivo biochemical assays 
Animals were sacrificed by decapitation in the morning to avoid diurnal 
variations of the endogenous amines, enzymes, and other antioxidant molecules. The 
brain tissue was homogenized in 0.1 M phosphate buffer (pH 7.8), using a glass-Teflon 
homogenizer. Tissue homogenates were centrifuged at 10,000 g for 60 min at 4?C 
(Muralikrishnan and Mohanakumar, 1998; Muralikrishnan and Ebadi 2001). 
 
5.3.6 Protein estimation 
Protein was assayed using the coomassie plus protein assay reagent kit (Pierce, 
Rockford, IL). Bovine serum albumin (BSA) was used as a standard for protein 
measurement. 
93 
 
5.3.7 Mitochondrial Complex I Activity  
Mitochondrial complex-I activity (NADH dehydrogenase activity) is based on the 
NADH oxidation. Oxidation of NADH by the NADH-dehydrogenase enzyme present in 
the tissue homogenate was measured spectrophotometrically at 340 nm.  The 
mitochondrial P2 suspension was added to the reaction mixture containing NADH, 
coenzyme Q0 and phosphate buffered saline, to analyze its effect on NADH oxidation by 
monitoring the decrease in absorbance at 340 nm.  A standard curve for NADH was 
obtained at 340 nm by using known quantities of NADH.  The change in absorbance 
indicates the amount of NADH oxidized.  The mitochondrial complex-I activity is 
expressed as the amount of NADH oxidized/min/mg protein (Ramsay et al., 1987; 
Wharton and Tzagotoff, 1967). 
 
5.3.8 Mitochondrial Complex IV Activity  
Complex IV activity was based on the cytochrome-C oxidation. The 
mitochondrial P2 suspension was added to the reaction mixture containing cytochrome-C 
and phosphate buffered saline.  The oxidation was measured spectrophotometrically. The 
absorbance was measured at 550nm for 2 min and the enzyme activity was expressed as 
cytochrome-C oxidized/mg protein (Ramsay et al., 1987; Wharton and Tzagotoff, 1967). 
 
5.3.9 Assay of MAO activity 
Total MAO activity was based on the amount of 4-hydroxyquinoline formed by 
the oxidation of kynuramine (Morinan and Garratt, 1985).  4-hydroxyquinoline was 
94 
 
measured fluorimetrically and the enzyme activity was expressed as 4-hyroxyquinoline 
formed/hour/mg protein (Muralikrishnan and Mohanakumar, 1998; Muralikrishnan and 
Ebadi, 2001). 
 
5.3.10 Assay of ROS production 
Conversion of nonfluorescent chloromethyl-DCF-DA (2?,7?- dichlorofluorescin 
diacetate) to fluorescent DCF was used to monitor ROS production 
spectrofluorometerically using an excitation wavelength of 492 nm and an emission 
wavelength of 527 nm. The generation of ROS was measured and reported as relative 
fluorescence intensity (Dhanasekaran et al., 2008). 
 
5.3.11 Statistical Analysis 
Results are expressed as the means?SEM. The statistical significance was 
evaluated by the one-way analysis of variance (ANOVA) using SigmaStat version 3. 
Values of p less than or equal to 0.05 were considered significant. 
 
5.4 Results 
 
5.4.1 Behavioral Analysis 
Methamphetamine (10mg/kg) caused a significant motor impairment in the 
C57/BL6 mice. Intraperitoneal administration of ramelteon (3 & 30mg/kg) did not block 
methamphetamine induced behavioral abnormalities including catalepsy (figure 5.1), 
95 
 
akinesia (figure 5.2), reduced swim score (figure 5.3), straub tails or tremor (table 5.1) 
(n=6). Ramelteon (30mg/kg) alone did not cause any behavioral abnormalities (n=6). 
 
5.4.2 Effect of ramelteon and methamphetamine on neurotransmitters 
Administration of methamphetamine (10mg/kg) caused a significant reduction in 
the levels of dopamine in the striatum. Ramelteon (3 & 30mg/kg) did not block 
methamphetamine induced dopamine depletion (n=6; figure 5.4). Methamphetamine and 
ramelteon affected levels of other striatal neurotransmitters (NE, 5HT, 5HIAA) and 
dopamine metabolites (DOPAC, HVA). The effect of ramelteon and methamphetamine 
on these neurotransmitters is shown in table 5.2. Ramelteon (30mg/kg) alone did not 
cause a significant reduction in the amount of striatal dopamine (n=6). 
 
5.4.3 In vivo biochemical analysis 
Intraperitoneal administration of methamphetamine (10mg/kg) in mice 
significantly decreased complex I activity. Ramelteon did not block methamphetamine 
induced decrease in complex I activity (n = 6; figure 5.5). Neither ramelteon nor 
methamphetamine had a significant effect on complex IV activity (figure 5.6). Ramelteon 
and methamphetamine also had no significant effect on the total mitochondrial 
monoamine oxidase (MAO) activity (figure 5.7). Methamphetamine caused significant 
generation of reactive oxygen species and ramelteon also did not provide neuroprotection 
from this effect (n = 6; figure 5.8). Ramelteon (30mg/kg) alone did not cause any 
96 
 
significant generation of reactive oxygen species nor any significant decrease in 
mitochondrial complex I (n=6). 
 
5.5 Discussion 
Our previous research showed that methamphetamine?s mechanism of toxicity 
involves generation of reactive radicals leading to oxidative stress, mitochondrial 
disfunction, and damage to dopaminergic neurons. A combination of oxidative stress and 
mitochondrial dysfunction creates toxicity of dopaminergic neurons in the nigrostriatal 
tract. This leads to long lasting dopamine depletion (Stokes et al., 1999; Zhu et al., 2006).  
Oxidative stress causes an inhibition of mitochondrial ATP production, a major 
determinant of lethal cell injury. Mitochondrial DNA is sensitive to the toxicity caused by 
reactive oxygen species because mitochondria have only a limited arsenal of DNA repair 
processes. Therefore, mitochondrial DNA mutations would cause defects in the 
respiratory chain function leading to severe cellular damage. Free radicals can interact 
with sugars, proteins and lipids creating a number of modifications which could then lead 
to mitochondrial dysfunction, apoptosis and even necrosis. The damage to the striatal 
terminals and the nigral cell bodies observed in methamphetamine users is indicative of 
all of these toxic mechanisms, the same mechanisms indicated in Parkinson?s disease 
(Imberti et al., 1993; Jones and Vale, 2000). There are studies lending evidence to 
support the theory that oxidative stress and mitochondrial dysfunction play a key role in 
neurodegeneration. Research on antioxidants has shown that they could possibly be 
97 
 
neuroprotective against the neurodegeneration seen in Parkinson?s disease as well as 
other neurodegenerative diseases (Shults, 2005). 
It is not well studied whether ramelteon has other functional similarities with 
melatonin, besides induction of sleep. However, besides its primary function of 
regulating the sleep cycle, melatonin may exert a powerful antioxidant activity (Tan et 
al., 2007). Melatonin is an antioxidant that can easily cross cell membranes and the blood 
brain barrier (Hardeland, 2005).  Melatonin is a direct scavenger of free radicals 
(Poeggeler et al., 1994). Unlike other antioxidants, melatonin does not undergo redox 
cycling, the ability of a molecule to undergo reduction and oxidation repeatedly. Redox 
cycling may allow other antioxidants (such as vitamin C) to regain their antioxidant 
properties. Melatonin, on the other hand, once oxidized, cannot be reduced to its former 
state because it forms several stable end-products upon reacting with free radicals. 
Therefore, it has been referred to as a terminal (or suicidal) antioxidant (Tan et al., 2000).  
In animal models, melatonin has been demonstrated to prevent the damage to 
DNA by some carcinogens, stopping the mechanism by which they cause cancer 
(Karbownik et al., 2001). It also has been found to be effective in protecting against brain 
injury caused by reactive oxygen species release in experimental hypoxic brain damage 
in newborn rats (Tutunculer et al., 2005). Melatonin's antioxidant activity may reduce 
neuronal damage caused in Parkinson's disease (Oaknin-Bendahan et al., 1995). This 
raised the interesting question of whether ramelteon might also have such beneficial 
effects. Free radical scavenging properties, such as those seen with melatonin (Reiter et 
al., 2001), could add neuroprotective abilities to the drug. 
98 
 
Ramelteon alone did not induce any behavioral changes. Ramelteon (30mg/kg) 
did not induce tremor, akinesia, catalepsy or any other abnormality with regard to the 
movement or behavior. Ramelteon also had no effect on the body weight.  With regard to 
the biochemical activities, ramelteon had no effect of the reactive oxygen species. Unlike, 
many other drugs that affect the mitochondrial respiration, ramelteon had no effect on the 
complex-I or complex-IV activity. Monoamine oxidase is an important target in the 
treatment of Parkinson?s disease, however, ramelteon had no effect on this enzyme 
activity. Ramelteon also did not affect the dopamine content or dopaminergic turnover in 
the striatum. With regard to the effect of ramelteon against methamphetamine induced 
toxicity, results in this study show a significant amount of dopamine depletion due to 
administration of methamphetamine, as well of the creation of a striatal environment ripe 
for oxidative damage and mitochondrial dysfunction. However, the behavioral, HPLC-
ECD, and in vivo biochemical results showed that ramelteon did not provide significant 
neuroprotection from the insults of oxidative stress. Thus, ramelteon does not appear to 
exibit the antioxidant properties observed in melatonin. However, ramelteon alone did 
not cause any neurotoxicity to the cells. 
 
 
 
 
 
 
99 
 
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105 
 
5.7 Figures 
 
Figure 5.1. Effect of Ramelteon on catalepsy: Ramelteon (3 & 30mg/kg) did not block 
methamphetamine induced catalepsy (n=6). *significant (n=6, p*<.05) as compared to 
control animals. 
 
Figure 5.2. Effect of Ramelteon on akinesia: Ramelteon (3 & 30mg/kg) did not block 
methamphetamine induced akinesia (n=6). *significant (n=6, p*<.05) as compared to 
control animals. 
106 
 
 
 
Figure 5.3. Effect of Ramelteon on swim score: Ramelteon (3 & 30mg/kg) did not block 
methamphetamine induced decrease in swim score (n=6). Scored by immobility time. 
 
  average weight straub tail tremor 
Control 24.3?0.6 0 0 
Methamphetamine 10mg/kg 24.2?0.5 n=6 4.0?0.1 
Ramelteon 3mg/kg+ 
Methamphetamine 10mg/kg 24.3?1.3 n=6 4.0?0.1 
Ramelteon 30mg/kg+ 
Methamphetamine 10mg/kg 25.3?0.4 n=6 4.0?0.1 
Ramelteon 30mg/kg 25.5?0.5 0 0 
Table 5.1. Effect of Ramelteon on straub tail and tremor: Ramelteon alone did not induce 
tremor or straub tail. Ramelteon (3 & 30mg/kg) did not block/reduce methamphetamine 
induced straub tail and tremor (n=6). 
107 
 
 
Figure 5.4. Effect of Ramelteon on striatal dopamine content: Ramelteon (3 & 30mg/kg) 
did not block methamphetamine mediated dopamine depletion (n=6). *significant (n=6, 
p*<.05) as compared to control animals. asignificant (n=6, pa<.05) as compared to 
methamphetamine animals. 
 
  Control 
Methamphetamine 
 10mg/kg 
Ramelteon 
3mg/kg+ 
Methamphetamine 
10mg/kg 
Ramelteon 
30mg/kg+ 
Methamphetamine 
10mg/kg 
Ramelteon  
30mg/kg 
DOPAC 49.12?11.6 19.45?3.8 47.55?7.6 a 40.20?7.0 a 84.46?8.0 * a 
HVA 24.73?1.7 18.10?1.7 18.17?1.5 * 17.11?2.2 * 27.09?3.1 * a 
5-HT 2.10?1.6 2.69?0.2 2.66?0.2 2.53?0.1 3.48?0.3 
5-HIAA 11.37?1.7 9.86?0.5 47.55?7.6 * a 40.20?7.0 * a 84.47?8.0 * a 
NE 15.21?0.9 14.49?0.9 12.71?1.8 14.93?1.0 17.94?0.8 
 
Table 5.2. Effect of Ramelteon on striatal neurotransmitter and metabolite levels 
(expresed as average pmole/mg tissue ? SEM). *significant (n=6, p*<.05) as compared to 
control animals. asignificant (n=6, pa<.05) as compared to methamphetamine animals. 
108 
 
 
Figure 5.5. Effect of Ramelteon on mitochondrial Complex I activity (in vivo): 
Ramelteon (3 & 30mg/kg) did not block methamphetamine mediated decrease in 
complex I activity (n = 6). Activity expressed as ?mol/min/mg protein. *significant (n=6, 
p*<.05) as compared to control animals. 
 Figure 5.6. Effect of Ramelteon on mitochondrial Complex IV activity (in vivo): 
Ramelteon (3 & 30mg/kg) caused no significant change in complex IV activity (n=6). 
Activity expressed as ?mol/min/mg protein.  
109 
 
 
Figure 5.7. Effect of Ramelteon on mitochondrial MAO activity (in vivo): Ramelteon (3 
& 30mg/kg) caused no significant change in MAO activity (n=6). Activity expressed as 
4-OHQ/mg protein. 
 
Figure 5.8. Effect of Ramelteon on mitochondrial ROS (in vivo): Ramelteon (3 & 
30mg/kg) did not block methamphetamine mediated generation of ROS (n = 6). 
*significant (n=6, p*<.05) as compared to control animals. 
 
110 
 
6. CHAPTER 3: NEUROPROTECTION OF AMANTADINE AGAINST 
NEUROTOXIC EFFECTS OF METHAMPHETAMINE 
 
6.1 Overview 
Amantadine is a drug used to help reduce the symptoms of Parkinson's disease. It 
releases dopamine from the nerve terminals and it appears to be a weak NMDA receptor 
antagonist. Excitotoxicity is involved in the pathogenesis of Parkinson?s disease and 
NMDA receptors are thought to be one of the most harmful factors in excitotoxicity. 
Research has shown that excitotoxic effects can be blocked by NMDA receptor 
antagonists. Thus, amantadine could prove to be neuroprotective against the type of 
toxicity often seen in Parkinson?s dsease. We examined the effect of amantadine and 
methamphetamine on reactive oxygen species generation, monoamine oxidase activity 
and complex I & IV activities. Behavioral analysis evaluated the effect on movement. 
Neurotransmitter levels were evaluated using high pressure liquid chromatography 
(HPLC)-electrochemical detection. Results showed that methamphetamine caused 
significant dopamine depletion in the striatum and also caused significant generation of 
reactive oxygen species and decreased complex I activity in the mitochondria. 
Amantadine (1mg/kg) significantly blocked methamphetamine mediated dopamine 
depletion in the striatum and significantly blocked generation of reactive oxygen species. 
 
 
111 
 
6.2 Introduction 
Amantadine is a drug commonly used in the treatment of Parkinson's disease. 
Amantadine works, in part, by releasing dopamine from nigrostriatal neurons. However, 
it is amantadine?s effectiveness as an NMDA receptor antagonist that is of interest to this 
study (Blanpied et al., 2005). Since NMDA receptors are one of the most harmful factors 
in excitotoxicity, antagonists of the receptors have held much promise for the treatment 
of conditions that involve excitotoxicity, including traumatic brain injury, stroke, and 
neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and 
Huntington's disease (Maas, 2001).  
Excitotoxicity is a process whereby a prolonged or excessive activation of 
excitatory amino acid receptors leads to an irreversible process of cell death. The high 
level of oxidative stress in Parkinson?s disease sets off a chain of events leading to 
deficits in mitochondrial function, disruption of the electron transport chain and initiation 
of excitotoxicity. This leads to cell damage and death (Doble 1999). Excitatory 
interactions are important for normal striatal function. Glutamate is the most abundant 
excitatory neurotransmitter; however, it becomes cytotoxic if released in excess, causing 
a calcium influx into the cell (Schwarcz and Meldrum, 1985). Methamphetamine causes a 
release of excess glutamate into the striatum via a direct pathway. Also, mitochondrial 
dysfunction, as seen in both Parkinson?s disease and methamphetamine toxicity, can lead 
to excitotoxicity, as a reduction in ATP would result in a loss of the ATP-dependent Mg-
blockade of NMDA receptors, causing physiological concentrations of glutamate to 
mediate a calcium influx into the cell (Beal et al., 1993). The resulting rise in calcium 
 
112 
 
increases nitric oxide synthase (NOS). This increases the production of nitric oxide (.NO) 
which reacts with the superoxide radical to form peroxynitrite and the hydroxyl radical, 
both powerful oxidizing agents (Dawson et al., 1991). Thus, the end product of 
excitotoxicity is the formation of reactive oxygen species as well as reduced intracellular 
glutathione synthesis, two of the indicators that are often see in Parkinson?s disease 
(Murphy et al., 1989). 
Dopaminergic neurons in the substantia nigra are rich in glutamate receptors, and 
thus highly susceptible to excitotoxicity. Studies of excitotoxicity have implicated the 
NMDA receptor subtype as being the principal vehicle of excitotoxic damage, and 
research has shown that excitotoxic effects of glutamic acid can be blocked by NMDA 
receptor antagonists (Choi et al., 1988). In experiments using MPTP, NMDA receptor 
antagonists protected rats from the toxic effects of MPP1 (Turski et al., 1991). The 
NMDA receptor antagonist memantine is proposed to counteract cellular damage due to 
pathological activation of NMDA receptors by glutamate (Doraiswamy, 2002).  
Our earlier studies show that administration of methamphetamine to mice 
produces a loss of dopaminergic neurons and a syndrome that behaviorally, 
biochemically, and neurochemically resembles Parkinson?s disease. In this study, we use 
this model to study whether amantadine exhibits neuroprotection against the toxicity 
caused by methamphetamine. If amantadine proved to be a neuroprotectant substance, 
preventing the neuronal cell damage from excitotoxicity (Gualtieri et al., 1989), it could 
slow the progression of Parkinson?s disease. 
 
 
113 
 
6.3 Materials and Methods 
 
6.3.1 Animals 
Male C57/Bl6 mice (4-6 weeks old) weighing 20-30 g were purchased from 
Charles Rivers. They were housed in a temperature controlled room with a 12h day and 
night cycle with free access to food and water. They were housed for 2-4 days prior to 
experiments. All the experimental procedures were reviewed and approved by 
Institutional Animal Care and Use Committee (IACUC) at Auburn University. 
 
6.3.2 Drug administration 
C57/Bl6 mice were separated into 5 groups (control, methamphetamine only, high 
dose amantadine only, amantadine high dose + methamphetamine and amantadine low 
dose + methamphetamine). The groups were given i.p. injections once daily for one week 
with sterile water (control & methamphetamine groups), 10mg/kg amantadine, dissolved 
in sterile water (amantadine high dose), 10mg/kg amantadine, dissolved in sterile water 
(amantadine high dose + methamphetamine group), and 1mg/kg amantadine, dissolved in 
sterile water (amantadine low dose + methamphetamine group). On day 7 the 
methamphetamine, amantadine high dose + methamphetamine and amantadine low dose 
+ methamphetamine groups were injected with methamphetamine (10mg/kg i.p., twice, 
2hr apart). The animals were sacrificed 5 days after the last injection.  
 
 
 
 
114 
 
6.3.3 Behavioral Studies 
Following methamphetamine administration, two examiners who were trained in 
evaluating different animal behaviors continuously observed mice. Tremor, akinesia, 
catalepsy, and straub tails were monitored during this period following standard scoring 
procedures as described below. Animals were subjected to swim test on the 4th day after 
methamphetamine. 
Straub tail: Straub tail was defined as elevation of tail to more than 45  with a 
curvature of the tail. The results are expressed as Straub tail phenomenon was detected or 
not detected (Mohanakumar and Sood, 1986). 
Akinesia: Akinesia usually refers to the impaired ability to initiate movements. It 
was measured by noting the latency in second(s) of the animals to move all four limbs 
and the test was terminated if the latency exceeded 180s (Weihmuller et al., 1988; 
Muralikrishnan and Mohanakumar, 1998). 
Catalepsy: The term implies the inability of an animal to correct an externally 
imposed posture. Catalepsy was measured by placing the animals on a flat horizontal 
surface with both the hind limbs on a square wooden block (3 cm, high) and the latency 
in seconds was measured to move the hind limbs from the block to the ground. The 
animals were initially acclimatized on the wooden platform (Weihmuller et al., 1988; 
Murlikrishnan and Mohanakumar, 1998).  
Swim test: Swim test was carried out in water tubs (40 l x 25 w x 12 h, in cm). 
The depth of water was kept at 8 cm and the temperature was maintained at 27  2  C. 
The animals were acclimatized for 10 min one day prior to experimentation. Swim score 
scales were: 0 = hind part sinks with head floating, 1 = occasional swimming using hind 
 
115 
 
limbs while floating on one side, 2 = occasional floating/swimming only, 3 = continuous 
swimming (Donnan et al., 1987). 
 
6.3.4 Measurement of Neurotransmitters  
To determine dopamine depletion in the striatum, methamphetamine injected 
animals were sacrificed on the 5th day and the striata was dissected out and analyzed for 
DA content employing HPLC-electrochemistry. Dopamine and its metabolites were 
separated in high pressure liquid chromatography. An electrochemical detector was used 
to quantify dopamine and its metabolites. The C18 octadecyl silica column (8 cm X 4.6 
mm i.d.) was used for the separation of the monoamines. Brain tissue was sonicated in 
0.4 N HClO4. These samples were then centrifuged and the 20 ?l of supernatant used was 
injected in HPLC. The results were expressed as picomoles per milligram of tissue 
(Uthayathas et al., 2007). 
 
6.3.5 Tissue Preparation for In vivo biochemical assays 
Animals were sacrificed by decapitation in the morning to avoid diurnal 
variations of the endogenous amines, enzymes, and other antioxidant molecules. The 
brain tissue was homogenized in 0.1 M phosphate buffer (pH 7.8), using a glass-Teflon 
homogenizer. Tissue homogenates were centrifuged at 10,000 g for 60 min at 4?C 
(Muralikrishnan and Mohanakumar, 1998; Muralikrishnan and Ebadi 2001). 
 
 
 
116 
 
6.3.6 Protein estimation 
Protein was assayed using the coomassie plus protein assay reagent kit (Pierce, 
Rockford, IL). Bovine serum albumin (BSA) was used as a standard for protein 
measurement. 
 
6.3.7 Mitochondrial Complex I Activity  
Mitochondrial complex-I activity (NADH dehydrogenase activity) is based on the 
NADH oxidation. Oxidation of NADH by the NADH-dehydrogenase enzyme present in 
the tissue homogenate was measured spectrophotometrically at 340 nm.  The 
mitochondrial P2 suspension was added to the reaction mixture containing NADH, 
coenzyme Q0 and phosphate buffered saline, to analyze its effect on NADH oxidation by 
monitoring the decrease in absorbance at 340 nm.  A standard curve for NADH was 
obtained at 340 nm by using known quantities of NADH.  The change in absorbance 
indicates the amount of NADH oxidized.  The mitochondrial complex-I activity is 
expressed as the amount of NADH oxidized/min/mg protein (Ramsay et al., 1987; 
Wharton and Tzagotoff, 1967). 
 
6.3.8 Mitochondrial Complex IV Activity  
Complex IV activity was based on the cytochrome-C oxidation. The 
mitochondrial P2 suspension was added to the reaction mixture containing cytochrome-C 
and phosphate buffered saline.  The oxidation was measured spectrophotometrically. The 
 
117 
 
absorbance was measured at 550nm for 2 min and the enzyme activity was expressed as 
cytochrome-C oxidized/mg protein (Ramsay et al., 1987; Wharton and Tzagotoff, 1967). 
 
6.3.9 Assay of MAO activity 
Total MAO activity was based on the amount of 4-hydroxyquinoline formed by 
the oxidation of kynuramine (Morinan and Garratt, 1985).  4-hydroxyquinoline was 
measured fluorimetrically and the enzyme activity was expressed as 4-hyroxyquinoline 
formed/hour/mg protein (Muralikrishnan and Mohanakumar, 1998; Muralikrishnan and 
Ebadi, 2001). 
 
6.3.10 Assay of ROS production 
Conversion of nonfluorescent chloromethyl-DCF-DA (2?,7?- dichlorofluorescin 
diacetate) to fluorescent DCF was used to monitor ROS production 
spectrofluorometerically using an excitation wavelength of 492 nm and an emission 
wavelength of 527 nm. The generation of ROS was measured and reported as relative 
fluorescence intensity (Dhanasekaran et al., 2008). 
 
6.3.11 Statistical Analysis 
Results are expressed as the means?SEM. The statistical significance was 
evaluated by the one-way analysis of variance (ANOVA) using SigmaStat version 3. 
Values of p less than or equal to 0.05 were considered significant. 
 
 
118 
 
6.4 Results 
 
6.4.1 Behavioral Analysis 
Methamphetamine (10mg/kg) caused a significant motor impairment in the 
C57/BL6 mice. Intraperitoneal administration of amantadine (1 & 10mg/kg) showed no 
significant neuroprotection against methamphetamine induced behavioral abnormalities 
including catalepsy (figure 6.1), akinesia (figure 6.2), reduced swim time (figure 6.3), 
straub tails or tremor (table 6.1) (n=6). 
 
6.4.2 Effect of amantadine and methamphetamine on neurotransmitters 
Administration of methamphetamine (10mg/kg) caused a significant reduction in 
the levels of dopamine in the striatum. Amantadine (1mg/kg) significantly blocked 
methamphetamine induced dopamine depletion (n=6, p=<0.05), amantadine (10mg/kg) 
did not significantly block methamphetamine induced dopamine depletion (n=6) as 
compared to control animals (figure 6.4). Methamphetamine and amantadine also 
affected levels of other striatal neurotransmitters (NE, 5HT, 5HIAA) and dopamine 
metabolites (DOPAC, HVA). The effect of amantadine and methamphetamine on these 
neurotransmitters is shown in table 6.2. 
 
 
 
 
 
119 
 
6.4.3 In vivo biochemical analysis 
Intraperitoneal administration of methamphetamine (10mg/kg) in mice 
significantly decreased complex I activity. Amantadine did not block methamphetamine 
mediated decrease in complex I activity (n = 6; figure 6.5). Neither amantadine nor 
methamphetamine had a significant effect on complex IV activity (figure 6.6). 
Amantadine and methamphetamine also had no significant effect on the total 
mitochondrial monoamine oxidase (MAO) activity (figure 6.7). Methamphetamine 
caused significant generation of reactive oxygen species and amantadine (1mg/kg) did 
significantly block methamphetamine mediated generation of ROS (n = 6, p=<0.05). 
Amantadine (10mg/kg) did not block this effect (n = 6; figure 6.8)  
 
6.5 Discussion 
Methamphetamine displaces monoamines like dopamine from their vesicles. 
Once released, the dopamine can be oxidized to reactive oxygen species. The 
combination of oxidative stress and mitochondrial dysfunction creates toxicity of striatal 
nerve terminals (Stokes et al., 1999; Zhu et al., 2006).These reactive species can cause an 
inhibition of mitochondrial ATP production which can lead to excitotoxicity, as a 
reduction in ATP would result in a loss of the ATP-dependent Mg-blockade of NMDA 
receptors, causing physiological concentrations of glutamate to mediate a calcium influx 
into the cell (Beal et al., 1993). Overexposure of the neurons to glutamate is cytotoxic, 
and a major determinant of lethal cell injury (Reiter, 1998).  
 
120 
 
Glutamate is the most abundant excitatory neurotransmitter; however, it becomes 
cytotoxic if released in excess, causing a calcium influx into the cell (Schwarcz and 
Meldrum, 1985). Methamphetamine causes a release of excess glutamate into the 
striatum via a direct pathway. Also, because methamphetamine displaces the dopamine in 
the vesicles, large amounts of it are released into the synaptic cleft and cytosol. There is 
an established relationship between the oxidative process of dopamine itself and 
nigrostriatal degeneration. During the auto-oxidation, synthesis and metabolism of 
dopamine, hydrogen peroxide (H2O2) is produced as a byproduct by tyrosine hydroxylase 
(TSH) and monoamine oxidase (MAO) (Lotharius and O?Malley, 2000). The hydrogen 
peroxide, which can react nonenzymatically to form highly reactive hydroxyl radicals, 
can then take part in a positive feedback loop causing further progression of oxidative 
damage, reduced ATP production and excitotoxicity (Jenner, 1998). 
The central dopaminergic system that plays such an important role in motor 
activity is comprised of a surprisingly small number of neurons. Because of this, it is 
especially vulnerable and even minor insults may lead to irreparable functional deficits 
(Fariello et al., 1988). Dopaminergic neurons in the substantia nigra are rich in glutamate 
receptors, and thus highly susceptible to excitotoxicity. This emphasizes the importance 
of excitotoxicity as a target for possible neuroprotection in Parkinson?s disease. Our 
earlier studies show that administration of methamphetamine to mice produces a loss of 
dopaminergic neurons and a syndrome that behaviorally, biochemically, and 
neurochemically resembles Parkinson?s disease. In this study, we use this model to study 
whether amantadine exhibits neuroprotection against the toxicity caused by 
 
121 
 
methamphetamine. If amantadine proved to be a neuroprotectant substance, preventing 
the neuronal cell damage from excitotoxicity (Gualtieri et al., 1989), it could slow the 
progression of Parkinson?s disease. 
In this study, administration of methamphetamine caused a significant amount of 
dopamine depletion and a striatal environment ripe for oxidative damage, mitochondrial 
dysfunction and excitotoxicity. The neurochemical data obtained using HPLC-ECD 
showed that amantadine (1mg/kg) showed significant neuroprotection against 
methamphetamine induced dopamine depletion (n=6, p=<0.05). The in vivo biochemical 
results showed that methamphetamine caused significant generation of reactive oxygen 
species and that amantadine (1mg/kg) showed significant neuroprotection against 
methamphetamine induced generation of ROS (n=6, p=<0.05). The end product of 
excitotoxicity is the formation of reactive oxygen species (Murphy et al., 1989). 
Therefore, the reduction in ROS generated might indicate a reduction in excitotoxicity. 
The failure of the high dose to provide neuroprotection is likely due to the dopamine 
releasing effect of amantadine. Because dopamine itself can be oxidized into ROS, the 
higher dose of amantadine could be exacerbating the oxidative stress by releasing too 
much dopamine from the vesicles.  
Overall, these results indicate that amantadine is partially neuroprotective against 
neurotoxicity and dopamine depletion caused by methamphetamine administration. 
Despite advances in pharmacotherapy that have improved quality of life, the mortality 
rate among Parkinson?s disease sufferers remains largely unchanged. There is need for a 
proactive treatment strategy that could provide neuroprotection from the disease. 
 
122 
 
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Storey, E., Srivastava, R., Rosen, B.R., Hyman, B.T. 1993. Neurochemical and 
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Blanpied, T.A., Clarke, R.J., Johnson, J.W. 2005. Amantadine inhibits NMDA receptors 
by accelerating channel closure during channel block. J Neurosci. 25(13): 3312-
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Choi, D.W. 1988. Glutamate neurotoxicity and diseases of the nervous system. Neuron. 
1: 623?634.  
Dawson, V.L., Dawson, T.M., London, E.D., Bredt, D.S., Snyder, S.H. 1991. Nitric oxide 
mediates glutamate neurotoxicity in primary cortical cultures. Proc Natl Acad Sci 
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Doble, A. 1999. The Role of excitotoxicity in Neurodegenerative disease: implications 
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Donnan, G.A., Willis, G.L., Kaczmarczyk, S.J., Rowe, P. 1987. Motor function in the 1-
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Gualtieri, T., Chandler, M., Coons, T.B. 1989. Amantadine: a new clinical profile for 
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Jenner, P. 1998. Oxidative mechanisms in niagral cell death in Parkinson?s disease . Mov. 
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Maas, A.I. 2001. Neuroprotective agents in traumatic brain injury. Expert Opin Inv Drug. 
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Marklund, S., Marklund, G. 1974. Involvement of superoxide anion radical in the 
autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur. 
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Mohanakumar, K.P., Mohanty, S., Ganguly, D.K., 1995.Neonatal treatment with 5-HT 
antiserum alters 5-HT metabolismand function in adult rats. Neuroreport. 7: 238. 
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Muralikrishnan, D., Ebadi, M. 2001. SKF-38393, a dopamine receptor agonist, attenuates 
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Muralikrishnan, D., Mohanakumar, K.P. 1998. Neuroprotection by bromocriptine against 
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neurotoxicity in mice. 
FASEB J. 12: 905-912. 
Murphy, T.H., Miyamoto, M., Sastre, A., Schnaar, R.L., Coyle, J.T. 1989. Glutamate 
toxicity in a neuronal cell line involves inhibition of cystine transport leading to 
oxidative stress. Neuron. 2: 1547-1558. 
Ramsay, R.R., Salach, J.I., Dadgar, J., Singer, T.P. 1986. Inhibition of mitochondrial 
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Reiter, R.J. 1998. Oxidative damage in the central nervous system: protection by 
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Sandoz Inc. 2008. Amantadine Hydrochloride Drug Information Sheet. 
Schwarcz, R., Meldrum, B. 1985. Excitatory amino acid antagonists provide a therapeutic 
approach to neurological disorders. Lancet. 2:140-143. 
Stokes, A.H., Hastings, T.G., Vrana, K.E., 1999. Cytotoxic and genotoxic potential of 
dopamine. J. Neurosci. Res. 55:659-665.  
Turski, L., Bressler, K., Rettig, K. J., Loschmann, P. A. 1991. Protection of substantia 
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Uthayathas S, Karuppagounder SS, Tamer SI, Parameshwaran K, Degim T, 
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Weihmuller, F.B., Hadjiconstantinou, M.,  Bruno, J.P. 1988. Acute stress or neuroleptics 
elicit sensorimotor deficits in MPTP-treated mice. Neuroscience Letters. 85(1): 
137-142. 
Wharton, D.C., Tzagotoff, A. 1967. Cytochrome oxidase from beef heart mitochondria. 
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Zhu, J.P.Q., Xu, W., Angulo, J.A. 2006. Methamphetamine induced cell death: selective 
vulnurerability in neuronal subpopulations of the striatum in mice. 
Neuropharmacol. 140 (2): 607-622. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
126 
 
6.7 Figures 
 
Figure 6.1. Effect of Amantadine on catalepsy: Amantadine (1 & 10mg/kg) did not block 
methamphetamine induced catalepsy (n=6). *significant (n=6, p*<.05) as compared to 
control animals. 
 Figure 6.2. Effect of Amantadine on akinesia: Amantadine (1 & 10mg/kg) did not block 
methamphetamine induced akinesia (n=6). *significant (n=6, p*<.05) as compared to 
control animals. 
 
127 
 
 
Figure 6.3. Effect of Amantadine on swim score: Amantadine (1 & 10mg/kg) did not 
block methamphetamine induced decrease in swim score (n=6). Scored by immobility 
time. 
 
  average weight straub tail tremor 
Control 24.3?0.6 0 0 
Methamphetamine 10mg/kg 24.2?0.5 n=6 4.0?0.1 
Amantadine 1mg/kg + 
Methamphetamine 10mg/kg 23?0.16 n=6 4.0?0.1 
Amantadine 10mg/kg + 
Methamphetamine 10mg/kg 24.5?0.2 n=6 4.0?0.1 
Amantadine 10mg/kg 22.2?0.5 0 0 
Table 6.1. Effect of Amantadine on straub tail and tremor: Amantadine alone did not 
induce tremor or straub tail. Amantadine (1 & 10mg/kg) did not block/reduce 
methamphetamine induced straub tail and tremor (n=6). 
 
128 
 
 
Figure 6.4. Effect of Amantadine on striatal dopamine content: Amantadine (1mg/kg) 
significantly blocked methamphetamine mediated dopamine depletion and amantadine 
(10mg/kg) did not significantly block methamphetamine mediated dopamine depletion 
(n=6). *significant (n=6, p*<.05) as compared to control animals. asignificant (n=6, 
pa<.05) as compared to methamphetamine animals. 
 
Control 
Methamphetamine 
Amantadine 
1mg/kg+ 
Amantadine 
10mg/kg+ 
Methamphetamine 
10mg/kg 
Amantadine 
10mg/kg 
Methamphetamine 
10mg/kg 10mg/kg 
DOPAC 49.12?11.6 19.45?3.8 27.45?3.5 * a 22.01?3.4 * 61.23?8.6 * a 
HVA 24.73?1.7 18.10?1.7 16.35?0.7 * 16.16?0.7 * 20.67?1.3 * 
5-HT 2.10?1.6 2.69?0.2 2.92?0.4 2.99?0.3 3.97?0.3 * 
5-HIAA 11.37?1.7 9.86?0.5 6.60?0.5 * 7.39?0.4 * 25.11?0.6 * 
NE 15.21?0.9 14.49?0.9 16.57?0.6 15.30?0.8 10.59?0.4 * 
Table 6.2. Effect of Amantadine on striatal neurotransmitter and metabolite levels 
(expresed as average pmole/mg tissue ? SEM). *significant (n=6, p*<.05) as compared to 
control animals. asignificant (n=6, pa<.05) as compared to methamphetamine animals. 
 
129 
 
 Figure 6.5. Effect of Amantadine on mitochondrial Complex I activity (in vivo): 
Amantadine (1 & 10mg/kg) did not block methamphetamine mediated decrease in 
Complex I activity (n = 6). Activity expressed as ?mol/min/mg protein. *significant 
(n=6, p*<.05) as compared to control animals. 
 Figure 6.6. Effect of Amantadine on mitochondrial Complex IV activity (in vivo): 
Amantadine (1 & 10mg/kg) caused no significant change in complex IV activity (n=6). 
Activity expressed as ?mol/min/mg protein. 
 
130 
 
 
Figure 6.7. Effect of Amantadine on mitochondrial MAO activity (in vivo): Amantadine 
(1 & 10mg/kg) caused no significant change in MAO activity (n=6). Activity expressed 
as 4-OHQ/mg protein. 
 
Figure 6.8. Effect of Amantadine on mitochondrial ROS (in vivo): Amantadine (1mg/kg) 
blocked methamphetamine mediated generation of ROS; amantadine (10mg/kg) did not 
block methamphetamine mediated generation of ROS (n = 6). *significant (n=6, p*<.05) 
as compared to control animals. asignificant (n=6, pa<.05) as compared to 
methamphetamine animals. 
 
131 
 
7. CHAPTER 4: NEUROPROTECTION OF SALICYLIC ACID AGAINST 
NEUROTOXIC EFFECTS OF METHAMPHETAMINE 
 
7.1 Overview 
One of the leading causative theories in Parkinson?s disease involves damage to 
neuronal cells induced by oxidative stress, apoptosis, mitochondrial dysfunction and 
inflammation. Salicylic acid is a known free radical scavenger. Thus, it could provide 
much needed neuroprotection against oxidative stress leading to neurodegeneration. We 
examined the effect of sodium salicylate (a sodium salt of salicylic acid) and 
methamphetamine on reactive oxygen species (ROS) generation, monoamine oxidase 
(MAO) activity and complex I & IV activities. Behavioral analysis evaluated the effect 
on movement. Neurotransmitter levels were evaluated using HPLC-ECD. Results showed 
that methamphetamine caused significant dopamine depletion in the striatum and also 
caused significant generation of reactive oxygen species and decreased complex I activity 
in the mitochondria. Behavioral analysis showed that methamphetamine had a significant 
effect on movement. Sodium salicylate (50 & 100mg/kg) significantly blocked 
methamphetamine mediated dopamine depletion in the striatum. Sodium salicylate 
significantly blocked methamphetamine mediated generation of reactive oxygen species 
and decreased Complex I activity. In addition, sodium salicylate significantly blocked 
methamphetamine induced movement abnormalities. 
 
132 
 
7.2 Introduction 
Salicylic acid is a metabolite produced in the body following ingestion of aspirin, 
acetylsalicylic acid. It belongs to a class of drugs known as analgesics or antipyretics 
(Hardman, 1996). Aspirin was the first-discovered member of the class of drugs known 
as non-steroidal anti-inflammatory drugs (NSAIDs), which all have similar effects and 
most have inhibition of the enzyme cyclooxygenase as their mechanism of action. Today, 
aspirin is one of the most widely used medications in the world, with an estimated 40,000 
metric tons of it being consumed each year. In countries where Aspirin is a registered 
trademark owned by Bayer, the generic term is acetylsalicylic acid (Warner and Mitchell, 
2002). 
The etiology of Parkinson?s disease remains unclear. However, one of the leading 
theories involves damage to neuronal cells induced by oxidative stress, or an imbalance 
between the generation of free radicals and the body?s antioxidant defense systems 
(Cohen, 1998; Halliwell, 1989; Olanow, 1993). The high level of oxidative stress 
observed in Parkinson?s disease sets off a chain of events leading to deficits in 
mitochondrial function, disruption of the electron transport chain, neuroinflammation, 
excitotoxicity and eventual neuronal cell death (Doble 1999). 
In Parkinson?s disease, nigrostriatal dopaminergic neurons undergo selective 
degeneration, leading to a shortage of dopamine in the striatum (Hornykiewicz, 1973; 
Caine and Langston 1983). A possible contributing factor to this is the brain and nervous 
system?s particularly vulnerablity to free radical damage. It consumes a disproportionally 
large volume of oxygen for its small weight as compared to the rest of the body, 
 
133 
 
contributing to the formation of the highly reactive superoxide radical (Halliwell, 1989). 
Another possible contributing factor to the neurotoxicity and selective neurodegeneration 
seen in the substantia nigra is the presence of iron in this area, which can also contribute 
to the production of reactive oxygen species. In the presence of transition metals, 
hydrogen peroxide can be converted by the Fenton reaction, an iron mediated reaction 
that requires iron in the free ferrous form, to the highly reactive hydroxyl radical (Bharath 
et al., 2002). Oxidative stress causes an inhibition of mitochondrial ATP production, a 
major determinant of lethal cell injury. The damage to the striatal terminals and the nigral 
cell bodies observed in methamphetamine users is also indicative of oxidative stress, the 
same mechanisms indicated in Parkinson?s disease (Imberti et al., 1993; Jones and Vale, 
2000).  
Salicylic Acid is a known free radical scavenger (Kataoka et al., 1997). It has 
been shown to provide neuroprotection against MPTP induced neurotoxicity 
(Mohanakumar et al., 2000). Thus, it could provide much needed neuroprotection against 
oxidative stress leading to neurodegeneration. With the ability of salicylic acid for free 
radical scavenging, it could play a much needed neuroprotective role in not only 
Parkinson?s disease, but also in numerous other neurodegenerative disorders. 
Another possibly neuroprotective factor is salicylic acid?s anti-inflammatory 
mechanism. Researchers have examined neuroinflammation as a risk factor for 
Parkinson?s disease for a long time (Landrigan et al., 2005). There is increasing evidence 
that one contributor to the later development of Parkinson?s disease is inflammation of 
neuronal tissue. This inflammation involves the activation of brain immune cells, 
 
134 
 
microglia. Activation of these cells can be beneficial in the protection of the cells, but 
they can also release neurotoxic cytokines which could have a deleterious effect and 
produce neurodegeneration (Liu et al., 2003). The substantia nigra has the highest density 
of microglia in the brain, and studies have shown that Parkinson?s disease patients have 
an even higher number of reactive glial cells than do patients without the disease 
(McGreer et al, 1988). Evidence of the involvement of inflammation came from studies 
showing that animals exposed to rotenone showed enhanced neurodegeneration in the 
presence of glia. Further studies showed synergistic effects when rotenone was combined 
with lipopolysacchide (Thiruchelvam et al., 2000). This would support the use of anti-
inflammatory therapeutic agents in Parkinson?s disease. 
Our earlier studies show that administration of methamphetamine to mice 
produces a loss of dopamine in the striatum and a syndrome that behaviorally, 
biochemically, and neurochemically resembles Parkinson?s disease. In this study, we use 
this model to study whether salicylic acid exhibits neuroprotection against the toxicity 
caused by methamphetamine. If salicylic acid proved to be a neuroprotectant substance, 
preventing neuronal degeneration, it could slow the progression of Parkinson?s disease. 
 
7.3 Materials and Methods 
 
7.3.1 Animals 
Male C57/Bl6 mice (4-6 weeks old) weighing 20-30 g were purchased from 
Charles Rivers. They were housed in a temperature controlled room with a 12h day and 
 
135 
 
night cycle with free access to food and water. They were housed for 2-4 days prior to 
experiments. All the experimental procedures were reviewed and approved by 
Institutional Animal Care and Use Committee (IACUC) at Auburn University. 
 
7.3.2 Drug administration 
C57/Bl6 mice were separated into 5 groups (control, methamphetamine only, high 
dose sodium salicylate only, sodium salicylate high dose + methamphetamine and 
sodium salicylate low dose + methamphetamine). The groups were given i.p. injections 
once daily for one week with sterile water (control & methamphetamine groups), 
100mg/kg sodium salicylate, dissolved in sterile water (sodium salicylate high dose), 
100mg/kg sodium salicylate, dissolved in sterile water (sodium salicylate high dose + 
methamphetamine group), and 50mg/kg sodium salicylate, dissolved in sterile water 
(sodium salicylate low dose + methamphetamine group). On day 7 the 
methamphetamine, sodium salicylate high dose + methamphetamine and sodium 
salicylate low dose + methamphetamine groups were injected with methamphetamine 
(10mg/kg i.p., twice, 2hr apart). The animals were sacrificed 5 days after the last 
injection.  
 
7.3.3 Behavioral Studies 
Following methamphetamine administration, two examiners who were trained in 
evaluating different animal behaviors continuously observed mice. Tremor, akinesia, 
catalepsy, and straub tails were monitored during this period following standard scoring 
 
136 
 
procedures as described below. Animals were subjected to swim test on the 4th day after 
methamphetamine. 
Straub tail: Straub tail was defined as elevation of tail to more than 45  with a 
curvature of the tail. The results are expressed as Straub tail phenomenon was detected or 
not detected (Mohanakumar and Sood, 1986). 
Akinesia: Akinesia usually refers to the impaired ability to initiate movements. It 
was measured by noting the latency in second(s) of the animals to move all four limbs 
and the test was terminated if the latency exceeded 180s (Weihmuller et al., 1988; 
Muralikrishnan and Mohanakumar, 1998). 
Catalepsy: The term implies the inability of an animal to correct an externally 
imposed posture. Catalepsy was measured by placing the animals on a flat horizontal 
surface with both the hind limbs on a square wooden block (3 cm, high) and the latency 
in seconds was measured to move the hind limbs from the block to the ground. The 
animals were initially acclimatized on the wooden platform (Weihmuller et al., 1988; 
Murlikrishnan and Mohanakumar, 1998).  
Swim test: Swim test was carried out in water tubs (40 l x 25 w x 12 h, in cm). 
The depth of water was kept at 8 cm and the temperature was maintained at 27  2  C. 
The animals were acclimatized for 10 min one day prior to experimentation. Swim score 
scales were: 0 = hind part sinks with head floating, 1 = occasional swimming using hind 
limbs while floating on one side, 2 = occasional floating/swimming only, 3 = continuous 
swimming (Donnan et al., 1987). 
 
 
137 
 
7.3.4 Measurement of Neurotransmitters  
To determine dopamine depletion in the striatum, methamphetamine injected 
animals were sacrificed on the 5th day and the striata was dissected out and analyzed for 
DA content employing HPLC-electrochemistry. Dopamine and its metabolites were 
separated in high pressure liquid chromatography. An electrochemical detector was used 
to quantify dopamine and its metabolites. The C18 octadecyl silica column (8 cm X 4.6 
mm i.d.) was used for the separation of the monoamines. Brain tissue was sonicated in 
0.4 N HClO4. These samples were then centrifuged and the 20 ?l of supernatant used 
was injected in HPLC. The results were expressed as picomoles per milligram of tissue 
(Uthayathas et al., 2007). 
 
7.3.5 Tissue Preparation for In vivo biochemical assays 
Animals were sacrificed by decapitation in the morning to avoid diurnal 
variations of the endogenous amines, enzymes, and other antioxidant molecules. The 
brain tissue was homogenized in 0.1 M phosphate buffer (pH 7.8), using a glass-Teflon 
homogenizer. Tissue homogenates were centrifuged at 10,000 g for 60 min at 4?C 
(Muralikrishnan and Mohanakumar, 1998; Muralikrishnan and Ebadi 2001). 
 
7.3.6 Protein estimation 
Protein was assayed using the coomassie plus protein assay reagent kit (Pierce, 
Rockford, IL). Bovine serum albumin (BSA) was used as a standard for protein 
measurement. 
 
138 
 
7.3.7 Mitochondrial Complex I Activity  
Mitochondrial complex-I activity (NADH dehydrogenase activity) is based on the 
NADH oxidation. Oxidation of NADH by the NADH-dehydrogenase enzyme present in 
the tissue homogenate was measured spectrophotometrically at 340 nm.  The 
mitochondrial P2 suspension was added to the reaction mixture containing NADH, 
coenzyme Q0 and phosphate buffered saline, to analyze its effect on NADH oxidation by 
monitoring the decrease in absorbance at 340 nm.  A standard curve for NADH was 
obtained at 340 nm by using known quantities of NADH.  The change in absorbance 
indicates the amount of NADH oxidized.  The mitochondrial complex-I activity is 
expressed as the amount of NADH oxidized/min/mg protein (Ramsay et al., 1987; 
Wharton and Tzagotoff, 1967). 
 
7.3.8 Mitochondrial Complex IV Activity  
Complex IV activity was based on the cytochrome-C oxidation. The 
mitochondrial P2 suspension was added to the reaction mixture containing cytochrome-C 
and phosphate buffered saline.  The oxidation was measured spectrophotometrically. The 
absorbance was measured at 550nm for 2 min and the enzyme activity was expressed as 
cytochrome-C oxidized/mg protein(Ramsay et al., 1987; Wharton and Tzagotoff, 1967). 
 
7.3.9 Assay of MAO activity 
Total MAO activity was based on the amount of 4-hydroxyquinoline formed by 
the oxidation of kynuramine (Morinan and Garratt, 1985).  4-hydroxyquinoline was 
 
139 
 
measured fluorimetrically and the enzyme activity was expressed as 4-hyroxyquinoline 
formed/hour/mg protein (Muralikrishnan and Mohanakumar, 1998; Muralikrishnan and 
Ebadi, 2001). 
 
7.3.11 Assay of ROS production 
Conversion of nonfluorescent chloromethyl-DCF-DA (2?,7?- dichlorofluorescin 
diacetate) to fluorescent DCF was used to monitor ROS production 
spectrofluorometerically using an excitation wavelength of 492 nm and an emission 
wavelength of 527 nm. The generation of ROS was measured and reported as relative 
fluorescence intensity (Dhanasekaran et al., 2008). 
 
7.3.12 Statistical Analysis 
Results are expressed as the means?SEM. The statistical significance was 
evaluated by the one-way analysis of variance (ANOVA) using SigmaStat version 3. 
Values of p less than or equal to 0.05 were considered significant. 
 
7.4 Results 
 
7.4.1 Behavioral Analysis 
Methamphetamine (10mg/kg) caused a significant motor impairment in the 
C57/BL6 mice. Intraperitoneal administration of sodium salicylate (50 & 100mg/kg) 
significantly blocked methamphetamine induced behavioral abnormalities including 
 
140 
 
catalepsy (figure 7.1), akinesia (figure 7.2), reduced swim time (figure 7.3), straub tails or 
tremor (table 7.1) (n=6, p<0.05). 
 
7.4.2 Effect of salicylic acid and methamphetamine on neurotransmitters 
Administration of methamphetamine (10mg/kg) caused a significant reduction in 
the levels of dopamine in the striatum. Sodium salicylate (50 &100mg/kg) significantly 
blocked methamphetamine induced dopamine depletion (n=6, p=<0.05), as compared to 
control animals (figure 7.4). Methamphetamine and sodium salicylate also affected levels 
of other striatal neurotransmitters (NE, 5HT, 5HIAA) and dopamine metabolites 
(DOPAC, HVA). The effect of sodium salicylate and methamphetamine on these 
neurotransmitters is shown in table 7.2. 
 
7.4.3 In vivo biochemical analysis 
Intraperitoneal administration of methamphetamine (10mg/kg) in mice 
significantly decreased complex I activity. Sodium salicylate (50 & 100mg/kg) did not 
block methamphetamine induced decrease in complex I activity (n = 6; figure 7.5). 
Neither sodium salicylate nor methamphetamine had a significant effect on complex IV 
activity (figure 7.6). Sodium salicylate and methamphetamine also had no significant 
effect on the total mitochondrial monoamine oxidase (MAO) activity (figure 7.7). 
Methamphetamine caused significant generation of reactive oxygen species and sodium 
salicylate (50 & 100mg/kg) did significantly block this effect (n = 6, p=<0.05) (figure 
7.8).  
 
141 
 
7.5 Discussion 
Salicylic acid irreversibly inactivates of the cyclooxygenase (COX) enzyme. This 
irreversible inactivation makes it different from other NSAIDs, such as ibuprofen, which 
are reversible inhibitors. The inhibition of the COX enzyme is responsible for its anti-
inflammatory properties (Vane, 1971; Tohgi et al., 1992; Vane and Botting, 2003). 
However, the neuroprotective properties of salicylic acid are thought to be more because 
of its role as a scavenger of hydroxyl radicals than its COX-inhibitory action (Ferger et 
al., 1999; Mohanakumar et al., 2000). 
Because methamphetamine displaces dopamine in the vesicles, large amounts of 
it are released into the synaptic cleft and cytosol. Once released, the dopamine can be 
oxidized to reactive oxygen species. These reactive species can cause an inhibition of 
mitochondrial ATP production. The combination of oxidative stress and mitochondrial 
dysfunction creates toxicity of striatal nerve terminals (Stokes et al., 1999; Zhu et al., 
2006). The central dopaminergic system that plays such an important role in motor 
activity is comprised of a surprisingly small number of neurons. Because of this, it is 
especially vulnerable and even minor insults may lead to irreparable functional deficits 
(Fariello et al., 1988).  
Salicylic acid has been shown to prevent the MPTP induced reduction in 
dopamine levels. It inhibits the action of this neurotoxin on the mitochondrial electron 
transport chain, a common source of free radicals in the cell. In addition, salicylic acid 
has been shown to block the neurotoxic effects of MPTP on the enzymatic defence 
 
142 
 
system of the brain such as superoxide dismutase, glutathione peroxidase and catalase 
(Ferger et al., 1999).  
The reduction in glutathione levels induced by MPTP is significantly inhibited by 
salicylic acid. These results imply that salicylic acid is not only capable of scavenging 
free radicals, but also of enhancing the cell?s own mechanisms against toxicity. In a 
MPTP mouse model of Parkinson?s disease, salicylic acid blocked toxin-induced 
glutathione and dopamine depletion. This indicates its role as a hydroxyl radical 
scavenger and its neuroprotective properties. In addition, this study showed, for the first 
time, that salicylic acid pre-treatment significantly improved motor impairments (akinesia 
and catalepsy) caused by MPTP administration (Mohanakumar et al., 2000). 
Our earlier studies show that administration of methamphetamine to mice 
produces a loss of dopamine in the striatum and a syndrome that behaviorally, 
biochemically, and neurochemically resembles Parkinson?s disease. In this study, we use 
this model to study whether salicylic acid exhibits neuroprotection against the toxicity 
caused by methamphetamine. Our results showed that administration of 
methamphetamine caused a significant amount of dopamine depletion and a striatal 
environment ripe for oxidative stress, mitochondrial dysfunction and neuronal 
degeneration. The neurochemical data obtained using HPLC-ECD showed that sodium 
salicylate (50 & 100mg/kg) provided significant neuroprotection against 
methamphetamine induced dopamine depletion (n=6, p=<0.05). The in vivo biochemical 
results showed that methamphetamine caused significant generation of reactive oxygen 
species and that sodium salicylate (50 & 100mg/kg) showed significant neuroprotection 
 
143 
 
against methamphetamine induced generation of ROS (n=6, p=<0.05). Overall, these 
results indicate that salicylic acid is neuroprotective against neurotoxicity and dopamine 
depletion caused by methamphetamine administration. Despite advances in 
pharmacotherapy that have improved quality of life, the mortality rate among Parkinson?s 
disease sufferers remains largely unchanged. If salicylic acid proved to be a 
neuroprotectant substance, preventing neuronal cell damage it could slow the progression 
of Parkinson?s disease. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
144 
 
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7.7 Figures 
 
Figure 7.1. Effect of Salicylic acid on catalepsy: Sodium salicylate (50 & 100mg/kg) 
blocked methamphetamine induced catalepsy. *significant (n=6, p*<.05) as compared to 
control animals. asignificant (n=6, pa<.05) as compared to methamphetamine animals. 
 
Figure 7.2. Effect of Salicylic acid on akinesia: Sodium salicylate (50 & 100mg/kg) 
blocked methamphetamine induced akinesia. *significant (n=6, p*<.05) as compared to 
control animals. asignificant (n=6, pa<.05) as compared to methamphetamine animals. 
 
150 
 
 
Figure 7.3. Effect of Salicylic acid on swim score: Sodium salicylate (50 & 100mg/kg) 
blocked methamphetamine induced decrease in swim score (n=6). Scored by immobility 
time. 
 
  average weight straub tail tremor 
Control 24.3?0.6 0 0 
Methamphetamine 10mg/kg 24.2?0.5 n=6 4.0?0.1 
Sodium salicylate 50mg/kg + 
Methamphetamine 10mg/kg 22.4?0.3 n=4 2.0?0.1 
Sodium salicylate 100mg/kg + 
Methamphetamine 10mg/kg 23.9?0.3 n=1 1.5?0.1 
Sodium salicylate 100mg/kg 23.6?0.4 0 0 
Table 7.1. Effect of Salicylic acid on straub tail and tremor: Sodium salicylate alone did 
not induce tremor or straub tail. Sodium salicylate (50 & 100mg/kg) blocked/reduced 
methamphetamine induced straub tail and tremor (n=6). 
 
151 
 
 
Figure 7.4. Effect of Salicylic acid on striatal dopamine content: Sodium salicylate (50 & 
100mg/kg) blocked methamphetamine mediated dopamine depletion. *significant (n=6, 
p*<.05) as compared to control animals. asignificant (n=6, pa<.05) as compared to 
methamphetamine animals. 
 
Control 
Methamphetamine 
Sodium salicylate 
50mg/kg+ 
Sodium salicylate 
100mg/kg+ 
Methamphetamine 
10mg/kg 
Sodium 
salicylate 
10mg/kg 
Methamphetamine 
10mg/kg 100mg/kg 
DOPAC 49.12?11.6 19.45?3.8 14.51?0.1 * 12.84?0.7 * 14.85?1.5 * 
HVA 24.73?1.7 18.10?1.7 25.82?1.9 a 22.87?1.6 a 19.23?2.0 a 
5-HT 2.99?1.6 2.69?0.2 2.31?0.3 1.90?0.2 * a 1.75?0.4 * a 
5-HIAA 11.37?1.7 9.86?0.5 9.82?0.8 9.22?0.6 7.59?0.7* a 
NE 15.21?0.9 14.49?0.9 16.93?1.6 24.09?0.6 21.30?1.2 * 
Table 7.2. Effect of Salicylic acid on striatal neurotransmitter and metabolite levels 
(expresed as average average pmole/mg tissue ? SEM). *significant (n=6, p*<.05) as 
compared to control animals. asignificant (n=6, pa<.05) as compared to 
methamphetamine animals. 
 
152 
 
 
Figure 7.6. Effect of Salicylic acid on mitochondrial Complex I activity (in vivo): Sodium 
salicylate (50 & 100mg/kg) did not block methamphetamine mediated decrease in 
complex I activity (n = 6). Activity expressed as ?mol/min/mg protein. *significant (n=6, 
p*<.05) as compared to control animals. 
 
Figure 7.7. Effect of Salicylic acid on mitochondrial Complex IV activity (in vivo): 
Sodium salicylate (50 & 100mg/kg) caused no significant change in complex IV activity 
(n=6). Activity expressed as ?mol/min/mg protein. 
 
153 
 
 
Figure 7.8. Effect of Salicylic acid on mitochondrial MAO activity (in vivo): Sodium 
salicylate (50 & 100mg/kg) caused no significant change in MAO activity (n=6). Activity 
expressed as 4-OHQ/mg protein. 
 
Figure 7.10. Effect of Salicylic acid on mitochondrial ROS (in vivo): Sodium salicylate 
(50 & 100mg/kg) blocked methamphetamine mediated generation of ROS. *significant 
(n=6, p*<.05) as compared to control animals. asignificant (n=6, pa<.05) as compared to 
methamphetamine animals. 
 
154 
 
8. SUMMARY AND CONCLUSIONS 
Previous studies have implicated exposure to toxins as contributors to the 
development of Parkinson?s disease. Our research provided evidence that 
methamphetamine is significantly toxic to dopaminergic neurons.This study shows the 
significant loss of dopaminergic function induced by methamphetamine as evidenced by 
both behavioral and biochemical experiments and with neurochemical data. It also offers 
evidence of oxidative stress and mitochondrial dysfunction. This indicates that 
administration of methamphetamine to mice produces a dopamine depletion in the 
striatum and a syndrome that behaviorally, biochemically, and neurochemically 
resembles Parkinson?s disease. These results indicate the usefulness of methamphetamine 
in making an animal model to study Parkinson?s disease. 
We used this model to ascertain if ramelteon, amantadine and salicylic acid 
exhibit neuroprotection against the toxicity caused by methamphetamine. In both 
methamphetamine toxicity and in Parkinson?s disease, the high level of oxidative stress 
leads to deficits in mitochondrial function, disruption of the electron transport chain, 
initiation of excitotoxicity and neuroinflammation. All of these mechanisms are sufficient 
to cause apoptosis and/or necrosis of neuronal cells. Therefore, each mechanism could be 
considered a target for therapeutic intervention. Discovery of a neuroprotective 
 
155 
 
pharmacological agent could lead to proactive strategies to counteract the neurological 
damage caused by Parkinson?s disease.  
 Overall, these results indicate that amantadine is partially neuroprotective but 
salicylic acid fully protected against methamphetamine-induced dopamine depletion. As 
previously stated, our long-term goal in this study is to facilitate and improve the care of 
patients with Parkinson?s disease by developing cost cost-effective anti-Parkinson?s 
drugs with the added benefit of a neuroprotective effect. There is a great need for 
neuroprotective and neurorestorative treatments for this disease. 
Neuroprotection was observed in mice that were pretreated with sodium salicilate 
or amantadine before the administration of methamphetamine. Both drugs augmented 
dopaminergic neuronal function in the brain of methamphetamine treated mice and 
reduced striatal dopamine depletion. This provides strong supporting evidence for the use 
or continued use of these drugs as therapeutic agents to provide relief and protective 
benefits for Parkinson?s disease.