effects of trpm2 inhibition in neuroprotection following
TRANSCRIPT
Effects of TRPM2 Inhibition in Neuroprotection following Neonatal Hypoxic-Ischemic Brain Injury
by
Feiya Li
A thesis submitted in conformity with the requirements for the degree of Master of Science
Department of Physiology University of Toronto
© Copyright by Feiya Li 2017
ii
Effects of TRPM2 Inhibition in Neuroprotection following
Neonatal Hypoxic-Ischemic Brain Injury
Feiya Li
Master of Science
Department of Physiology
University of Toronto
2017
Abstract
Neonatal hypoxic-ischemic (HI) brain injury is a major cause of acute mortality and
chronic neurological morbidity in infants and children. Studies indicate that transient
receptor potential melastatin 2 or TRPM2 (a non-selective cation channel with high
permeability to calcium that can be activated by intracellular adenosine diphosphoribose
[ADPR] and H2O2) can mediate neuronal death following acute ischemic insults in adult
mice as well as HI brain injury in neonatal mice. My study tested the effect of a newly
described TRPM2 channel inhibitor AG490 by using a H2O2-induced neuronal cell death
in vitro model and mouse HI brain injury in vivo model. I found that the inhibition of
TRPM2 channels by AG490 demonstrates a neuroprotective effect both in vitro and in
vivo. The neuroprotective effect of AG490 following post-injury treatment suggests the
potential clinical implications of this drug, including the possible prevention of HI related
neurological complications such as hypoxic-ischemic encephalopathy.
iii
Acknowledgements
I would first like to express my profound gratitude to my supervisor, Dr. Hong-Shuo Sun,
for taking me as a student in September 2015 and giving me, an international student, this
opportunity to work in such a wonderful and collaborative lab. Over the past 2 years, he
has given me so much advice and trained me to improve my research capabilities as well
as my language skills. I would also like to express my gratitude to my mentor, Dr. Zhong-
Ping Feng, for her critical advice and guidance throughout the program. Dr. Feng knows
students well and reads my minds, and her strong scientific sense and logical thinking gave
me so much good advice in my project design as well as scientific presentation skills.
During the past 2 years of graduate study, I grew up fast and learnt a lot. When I looked
back to my first draft of proposal, which almost got fully re-written by Dr. Sun, and thought
back to my first presentation practice on the lab meeting, I clearly noticed that how much
time and effort my supervisor Dr. Sun and my mentor Dr. Feng had put on me. I would
like to sincerely express my gratefulness for their patience and supports. I would also like
to thank my supervisory committee members, Dr. Tianru Jin and Dr. Shuzo Sugita, for
their good comments and insightful advice and being supportive throughout my training
process.
I would like to thank all of my lab mates: Vivian Szeto, Ekaterina Turlova, Haitao Wang,
Raymond Wong, Nancy Dong, Sammen Huang, Ahmed Abussaud, Ji-Sun Kim, Shuzhen
Zhu, Meihua Bao, Qing Li, Joseph Leung, for their help whenever I needed during my
graduate study process. Especially thank to Haitao, who was the first one I met in lab and
taught me everything including techniques and background knowledges and gave
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suggestions in my project design. Also, specially thank to Vivian, who was always patient
and gave me courage whenever I was feeling either a bit lost because of the culture
differences, or struggling with my projects.
I also want to thank all of my close friends I met in Knox College (Fan Xia, Linyang Yu,
Dongzi Li, Siqi Liu, Qing Yuan…) and everyone in FIP committee (Kiru, Ankur,
Hanna…), for making my life in University of Toronto more colorful and full of laughter.
Special thank to Dongzi Li and Qing Yuan, for their understanding, accompany and
encouragements throughout my preparation of this thesis and the thesis defense.
Finally, I would like to express my gratitude to my family: my parents, grandparents, my
uncle and aunt, for providing me with unfailing supports and continuous encouragements
throughout my years of study. This accomplishment would not have been possible without
them.
v
Table of Contents
Abstract ............................................................................................................................... ii
Acknowledgements ............................................................................................................ iii
Table of Contents ................................................................................................................ v
List of Abbreviations ....................................................................................................... viii
List of Figures ................................................................................................................... xii
Chapter 1 Introduction ....................................................................................................... 1
1 Neonatal Hypoxic-Ischemic Brain Injury .................................................................... 1
1.1 Incidence and global impact ................................................................................. 1
1.2 Standard diagnostic criteria for neonatal HI ......................................................... 2
1.3 Current therapy ..................................................................................................... 3
2 Failure in Targeting Glutamate Receptors as Pharmacological Targets ...................... 4
3 Targeting Non-Glutamate Channels ............................................................................ 7
3.1 Sodium-calcium exchangers ................................................................................. 7
3.2 Hemichannels ........................................................................................................ 8
3.3 Volume-regulated anion channels......................................................................... 9
3.4 Acid-sensing ion channels (ASICs) ...................................................................... 9
3.5 Transient receptor potential melastatin (TRPM) subfamily ............................... 10
4 Transient Receptor Potential Channels (TRPs) ......................................................... 12
5 TRPM2 Channel ........................................................................................................ 12
5.1 TRPM2 protein structure, transmembrane topology and distribution ................ 12
5.2 TRPM2 biophysical properties and gating mechanism ...................................... 16
5.3 The physiological and pathophysiological role of TRPM2 channels ................. 17
6 Pharmacological Interactions ..................................................................................... 20
6.1 Flufenamic acid (FFA) ........................................................................................ 20
6.2 Anti-fungal agents (clotrimazole and econazole) ............................................... 20
6.3 2-APB ................................................................................................................. 21
6.4 Divalent heavy metal cations .............................................................................. 21
6.5 AG490 ................................................................................................................. 21
Chapter 2 Rationale and Hypothesis ................................................................................ 23
vi
Rationale ....................................................................................................................... 23
Hypothesis..................................................................................................................... 23
Chapter 3 Aims and Experimental Design....................................................................... 24
Aims: ............................................................................................................................. 24
Experimental Design Outline:....................................................................................... 24
Chapter 4 Materials and Methods .................................................................................... 25
1 Ethics Approval ......................................................................................................... 25
2 Animals ...................................................................................................................... 25
3 Reagents ..................................................................................................................... 25
4 Cell Culture ............................................................................................................ 25
5 In vitro H2O2-induced Neuronal Cell Death Model ................................................... 26
6 Cell Viability Assay ................................................................................................... 26
7 Electrophysiology (Whole Cell Patch Clamp) ........................................................... 27
8 Drug Administration .................................................................................................. 27
9 In vivo Hypoxic-Ischemic Mouse Model .................................................................. 28
10 Infarct Volume Measurement, Whole Brain Imaging and Histological Assessments
....................................................................................................................................... 29
10.1 TTC staining/Infarct volume measurement ...................................................... 29
10.2 Whole brain imaging/Nissl staining.................................................................. 29
11 Neurobehavioral Assessments ................................................................................. 30
11.1 Geotaxis reflex .................................................................................................. 30
11.2 Cliff avoidance test ........................................................................................... 30
11.3 Grip test ............................................................................................................. 31
11.4 Passive avoidance test ....................................................................................... 31
12 Immunohistochemistry and Confocal Imaging ........................................................ 32
13 Western Blot ............................................................................................................ 32
14 Statistics and Data Analysis ..................................................................................... 33
Chapter 5 Results ............................................................................................................. 34
1. The level of TRPM2 mRNA expression in the hippocampus and cortex increases
consistently with the developmental age ...................................................................... 34
2 AG490 as a pharmacological inhibitor of the TRPM2 channel ................................. 36
3 AG490 protects neurons from H2O2-induced cell injury in vitro .............................. 38
vii
4 The Effect of AG490 Pre-treatment on Hypoxic-Ischemic Brain Injury in vivo. ..... 40
4.1 Pre-treatment with TRPM2 inhibitor AG490 reduced the brain infarct volume
of hypoxic-ischemic brain injury in vivo. ................................................................. 41
4.2 Pre-treatment with TRPM2 inhibitor AG490 reduced brain damage following
hypoxic-ischemic brain injury. ................................................................................. 44
4.3 Pre-treatment with TRPM2 inhibitor AG490 promotes recovery after HI
challenge ................................................................................................................... 46
4.4 Pre-treatment with TRPM2 inhibitor AG490 improves short-term
neurobehavioral performance after HI ...................................................................... 47
4.5 Pre-treatment with TRPM2 inhibitor AG490 also improves long-term
neurobehavioral performance after HI ...................................................................... 49
4.6 Pre-treatment with TRPM2 inhibitor AG490 reduces reactive astrocyte
activation ................................................................................................................... 51
4.7 Pre-treatment with TRPM2 inhibitor AG490 may reduce HI brain damage
through Akt mediated signaling pathways ................................................................ 53
5 The Effect of AG490 Post-treatment on Hypoxic-Ischemic Brain Injury in vivo. .... 54
5.1 AG490 *post-treatment 1 (30 mg/kg, i.p.) reduced brain infarct volume of
hypoxic-ischemic brain injury in vivo. ..................................................................... 54
5.2 AG490 *post-treatment 1 (30 mg/kg, i.p.) reduced brain damage following
hypoxic-ischemic brain injury .................................................................................. 54
5.3 AG490 *post-treatment 1 (30 mg/kg, i.p.) improves neurobehavioral
performance and general recovery after HI .............................................................. 57
6 AG490 *post-treatment 2 (30mg/kg, i.p., immediately after HI induction)
demonstrate a trend towards neuroprotection following HI brain injury ..................... 59
Discussion ......................................................................................................................... 60
1. Connection between clinics and the current study .................................................... 60
2. Summary of major findings ...................................................................................... 61
3. Significance of the current study .............................................................................. 62
4. Differences between neonatal HI brain injury and adult stroke ............................... 62
5. Proposed mechanism of neonatal HI brain injury..................................................... 63
6. Pitfalls in the current study and proposed future directions ..................................... 65
REFERENCES ................................................................................................................. 68
viii
List of Abbreviations
ADPR Adenosine disphosphate ribose
Akt Protein kinase B
AMP Adenosine monophosphate
AMPA α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
receptor
ASICs Acid-sensing ion channels
ATP Adenosine triphosphate
BDNF Brain-derived neurotrophic factor
cAMP Cyclic adenosine monophosphate
cADPR Cyclic adenosine diphosphate ribose
Ca2+ Calcium ion
CaM Calmodulin
Cl- Chloride ion
CNS Central nervous system
CTL Control
Cx43 Connexin 43
DMSO Dimethyl sulfoxide
EEG Electroencephalography
E16 Embryonic day 16
FFA Flufenamic acid
GAPDH Glyceraldehyde 3-phosphata
GFAP Glial fibrillary acidic protein
ix
GPCRs G-protein-coupled receptors
GSK-3α Glycogen synthase kinase 3 alpha
GSK-3β Glycogen synthase kinase 3 beta
HEK293 Human embryonic kidney 293
HI Hypoxic-Ischemic
HIE Hypoxic-Ischemic Encephalopathy
H2O2 Hydrogen Peroxide
ICC Immunocytochemistry
IHC Immunohistochemistry
i.p. Intraperitoneal
IP3 Inositol 1,4,5-trisphosphate
i.v. Intravenous
IV Current-voltage
JAK2 Janus kinase 2
KATP ATP-sensitive potassium channel
K+ Potassium ion
KO Knockout
MAPK Mitogen-activated protein kinases
MCAO Middle cerebral artery occlusion
MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide)
MRI Magnetic resonance imaging
Na+ Sodium ion
NAD Nicotinamide adenine dinucleotide
x
NCX Na(+)/Ca(2+) exchanger/Sodium-calcium exchanger
NMDA N-methyl-D-aspartate receptor
NSAIDs Non-steroidal anti-inflammatory drugs
NUDT9-H Nudix (Nucleoside Diphosphate Linked Moiety X)-Type
Motif 9
OGD Oxygen glucose deprivation
PARG Poly (ADP-ribose) glycohydrolase
PARP Poly (ADP-ribose) polymerase
PCR Polymerase chain reaction
pH Potential of Hydrogen
PI3K Phosphoinositide 3-kinase
PIP2 Phosphatidylinositol 4,5-bisphosphate
P7/P14 Postnatal day 7/14
RCT Randomized controlled trials
ROS Reactive oxygen species
siRNA Small interfering RNA
S5/S6 Segment 5/ Segment 6
TRPs Transient receptor potential ion channel superfamily
TRPA Transient receptor potential ankyrin
TRPC Transient receptor potential canonical
TRPM Transient receptor potential melastatin
TRPV Transient receptor potential vanilloid
TRPP Transient receptor potential polycystin
TRPML Transient receptor potential mucolipin
xi
TRPM2 Transient receptor potential melastatin 2
TTC Tetrazolium chloride
VRACs Volume-regulated anion channels
WT Wildtype
xii
List of Figures
Figure 1. Classical glutamate receptors (NMDA and AMPA receptor) model of neuronal
cell death …………………………………………………………………………….........5
Figure 2. Potential mechanisms involved in excitotoxicity following ischemic stress.......9
Figure 3. Family tree of TRP channel superfamily………………………………………11
Figure 4. TRPM2 protein structure and variants…………………………………….......13
Figure 5. Proposed mechanisms of TRPM2 channel activation by H2O2 and involvement
TRPM2 channel activity in physiological and pathophysiological processes...................16
Figure 6. TRPM2 channel inhibitors.................................................................................19
Figure 7. An outline of the project experimental design...................................................21
Figure 8. TRPM2 expression in the cortex and hippocampus increases with
development.......................................................................................................................31
Figure 9. AG490 efficiently inhibited H2O2-induced TRPM2 current on TRPM2
overexpression HEK293 cells............................................................................................33
Figure 10. TRPM2 inhibitor AG490 pre-treatment reduced neuronal cell death following
H2O2-induced cell injury....................................................................................................35
Figure 11. Timeline of neonatal hypoxic-ischemic injury and experimental
procedures..........................................................................................................................36
Figure 12. Lower dose of AG490 (15 mg/kg) did not show effect on
neuroprotection..................................................................................................................38
Figure 13. Pretreatment of TRPM2 currents inhibitor AG490 (30 mg/kg) reduced brain
infarct volume of hypoxic-ischemic brain injury in vivo...................................................39
Figure 14. TRPM2 inhibitor AG490 reduced brain damage following hypoxic-ischemic
brain injury.........................................................................................................................41
xiii
Figure 15. AG490 pre-treatment (20 min before HI injury) improves general health
recovery after HI challenge...............................................................................................42
Figure 16. AG490 pre-treatment (20 min before HI injury) improves neurobehavioral
performance after HI challenge.........................................................................................44
Figure 17. Long – term behavioral assessment of functional recovery in the pre-treatment
paradigm following hypoxic-ischemic injury....................................................................46
Figure 18. AG490 pre-treatment restores neuronal cell numbers and reduces reactive
astrocyte activation............................................................................................................53
Figure 19. Biochemical assessment of signaling pathways affected by hypoxic-ischemic
insult on neonatal brain in a pre-treatment paradigm........................................................54
Figure 20. *Post-treatment 1 (30 mg/kg, i.p.) reduced brain infarct volume of hypoxic-
ischemic brain injury in vivo..............................................................................................56
Figure 21. TRPM2 inhibitor AG490 reduced brain damage following hypoxic-ischemic
brain injury.........................................................................................................................57
Figure 22. AG490 post-treatment 1 (immediately after ischemic injury) improves general
health and neurobehavioral performance after HI challenge.............................................59
Figure 23. *Post-treatment 2 (30 mg/kg, i.p.) shows a trend of neuroprotective effect
following hypoxic-ischemic brain injury...........................................................................60
Figure 24. Propose mechanism for inhibitory effect of AG490........................................65
1
Chapter 1 Introduction
1 Neonatal Hypoxic-Ischemic Brain Injury
When a term neonate’s (defined as 36 gestational weeks or later1-3) brain does not receive sufficient
oxygen (hypoxia) and blood (ischemia), neonatal hypoxic-ischemic encephalopathy may result (or
HIE for short4). Neonatal encephalopathy (HIE) can result from diverse conditions, with hypoxic-
ischemic brain injury (HI brain injury) being the most common. Therefore, the terms HI brain
injury and HIE are usually used synonymously. HI brain injury is a condition which can cause
significant mortality and long-term morbidity. The injury can be a clinical consequence of
perinatal, birth and/or neonatal asphyxia4.
1.1 Incidence and global impact
Clinically, most HI brain injury cases are due to birth asphyxia which causes 840,000 or 23 % of
all neonatal deaths worldwide5, 6. The severity and length of oxygen and blood deprivation affects
whether HI brain injury occurs and how severe it is. Due to different levels of severity, patients
suffer from symptoms ranging from transient behavioral abnormalities, occasional periods of
apnea and seizure-related symptoms to even death from cardiorespiratory failure5. 50-80 % of the
survivors will suffer from severe developmental or cognitive delays, motor impairments and
learning disabilities5, 7. Symptoms may worsen as the child continues to develop. The lifetime costs
to the health care system have been estimated to be as high as $1.5 million per person in Canada5.
2
1.2 Standard diagnostic criteria for neonatal HI
Based on biomedical markers that correlate to clinical outcomes, there is an established set of
predictors for neonatal HI brain injury1, 7, 8:
i) Apgar Score
At birth, doctors and nurses carefully examine the newborn's condition and give a
number rating from 0 to 10. This number is called an Apgar score. The Apgar rates skin
color, heart rate, muscle tone, reflexes and breathing effort. An Apgar score of less than
or equal to 5 (at 5 and 10 minutes after birth) clearly confers an increased relative risk
for HI brain injury9-11.
ii) Fetal Umbilical Artery pH
Fetal Umbilical Artery pH less than 7.0 and/or a base deficit worse than or equal to
minus 12 mmol/L from cord/arterial/venous/capillary blood gas obtained within 60
minutes of birth increase the probability of neonatal HI brain injury2, 10, 12.
iii) MRI
Magnetic resonance imaging (MRI) is the neuroimaging modality that best defines the
nature and extent of neonatal HI brain injury. Distinct patterns of MRI aberrations are
recognized in term neonates due to HI brain injury. Recently, electroencephalography
(EEG) has also shown helpful information for predicting the clinical outcome of HI
brain injury, though some studies reported that EEG is not as reliable as MRI13.
iv) Presence of Multisystem Organ Failure and Abnormal Neurological Signs
Dysfunction of multiple organs also results in a higher risk for neonatal HI brain
injury9. The dysfunctional organ systems include hematologic abnormalities, cardiac
dysfunction, metabolic derangements and gastrointestinal injury, or a combination of
3
any of above. Abnormal neurological signs are supplementary predictors of HI brain
injury. For example, hypotonic muscles or lack of a sucking reflex may also indicate a
probability of HI brain injury8.
1.3 Current therapy
There is currently only one available licensed treatment for HI: hypothermia14-16.
Hypothermia should be offered to term infants with moderate or severe HI brain injury within 6
hours of birth. 6 large randomized controlled trials (RCT) of hypothermia for neonatal HI brain
injury have been published, with all involved neonates being ≥36 weeks of gestation (termed
infants). The target temperature was 33°C to 34°C, with the cooling duration being 72 hrs14, 15, 17.
Rewarming was processed slowly, with an increase of 0.5°C per hour14, 15. Neonates underwent a
set of neurological examinations during the process as well as at the end of cooling. Some clinical
trials have demonstrated that either head cooling or whole body cooling reduces mortality or
disability either in all the infants or within a certain group of infants between 18 to 24 months of
age14-16, 18.
Even though hypothermia has shown impressive clinical outcomes and is currently well
established as a standard treatment for neonates suffering from moderate to severe HI brain injury,
it is stated to be “partially effective”19, 20. Therefore, there is an urgent need for novel therapeutic
opportunities beyond this current form of treatment.
4
2 Failure in Targeting Glutamate Receptors as Pharmacological
Targets
Although the exact pathophysiology of HI brain injury is not completely understood, it is
commonly accepted that a lack of sufficient blood flow in conjunction with decreased blood
oxygen content leads to loss of normal cerebral autoregulation and diffuse brain injury21, 22.
Glutamate is an important excitatory neurotransmitter at excitatory synapses in the CNS22-24. Based
on the theory of excitotoxicity, it has long been accepted that a lack of blood flow can lead to high
concentrations of glutamate release and eventually HI brain injury25. Therefore, glutamate
receptors have been extensively investigated as potential therapeutic targets for neuroprotection.
The biochemical cascade of the theory of excitotoxicity is summarized as follows22: Lack of
cerebral blood flow triggered energy failure and neuronal depolarization which then released large
amounts of glutamate into the extracellular space. Excessive glutamate in the extracellular space
overactivated NMDA (N-methyl-d-aspartic acid) and AMPA (dl-α-amino-3-hydroxy-5-methyl-4-
isoxazole propionic acid) glutamate receptors, leading to an increased influx of calcium.
Subsequently, intracellular calcium overload can be observed within neurons., which is thought to
induce neuronal cell death. Popular strategies against excitotoxicity have targeted the
pharmacological blockage of NMDA receptors. Whereas several compounds including
dezocilipine maleate (MK-801), aptiganel hydrochloride (Cerestat), dexthrometorphan (DMX)
and CGS 19755 (Selfotel) demonstrated promising neuroprotective effects in rodent models21, 26,
all clinical trials aimed at using NMDA and AMPA glutamate receptors as pharmacological targets
failed to yield the expected protective outcomes22, 27.
One potential explanation for this failure is that the injured brain may attempt to recruit
endogenous recovery mechanisms22, 28. While glutamate signals may be truly neurotoxic to
5
neurons, this may suggest that some aspects of the signaling process may still have beneficial
effects. It is already known that synaptic NMDA receptors and extrasynaptic NMDA receptors
may have opposite effects following activation21, 29. The activation of synaptic receptors could
promote cell survival while the activation of extrasynaptic receptors can downregulate BDNF and
ultimately lead to cell death22, 30. Another potential limitation of this standard NMDA-AMPA
model is that it does not consider the roles of cells other than neurons, for example, astrocytes and
oligodendrocytes23, 31, 32. As astrocytes and oligodendrocytes also express NMDA and AMPA
receptors23, 33, they too are vulnerable to excessive glutamate and play important roles in glutamate
regulation. Additionally, glutamate is an essential neurotransmitter that is necessary for important
physiological processes. Therefore, the blockage of glutamate receptors during the treatment of
ischemic injury may lead to unwanted side effects. Studies have shown that the hypofunction of
NMDA receptors may be partially responsible for the memory loss associated with
aging34. Schizophrenia has also been reported in association with NMDA receptor dysfunction35.
6
Figure 1. Classical glutamate receptor (NMDA and AMPA receptor) model of neuronal cell
death. This classical glutamate receptor driven model indicates the potential roles of NMDA and
AMPA receptors involved in inducing neuronal cell death through excessive extracellular
glutamate. Overactivation of these two channels leads to intracellular calcium imbalance,
consequently inducing several pathways that eventually lead to neuronal cell death. This figure
was modified from Elaine Besancon et al., 2008.
7
Taken together, the traditional model of excitotoxicity emphasized treatment at the level of
glutamate receptor channels to curtail such events. However, the failure of targeting glutamate
receptors in all clinical trials indicated that new therapeutic targets needed to be identified for HI
brain injury.
3 Targeting Non-Glutamate Channels
Several non-glutamate ion channels, including transient receptor potential channels, acid-sensing
channels22, hemichannels36, volume-regulated anion channels37 and sodium-calcium exchangers
(NCX)38, 39 etc. have been implicated and thus identified as potential novel therapeutic targets for
ischemic brain injury.
3.1 Sodium-calcium exchangers
The Na(+)/Ca(2+) exchangers (NCX) are bi-directional transmembrane proteins that express
widely in the brain38. Under normal physiological conditions, the NCXs exchange one calcium ion
out of the cell with three sodium ions going into the cell38. Under pathophysiological conditions
like ischemia, the activity of NCXs can be reversed38, 40-42. Instead of transporting calcium ions
out of cells, they may alternatively transport them into cells. Since dysregulation of sodium and
calcium homeostasis is a fundamental hallmark following ischemic brain injury, the role of NCXs
under ischemia has been studied both in vitro and in vivo41-44. However, the conclusions remain
controversial. Some studies have shown that using NCXs blockers may reduce brain infarction in
in vivo stroke models43-45, while other reports brought up conflicting results that inhibition of
NCXs can lead to even worse infarction outcomes22, 42. These variations may in part be due to the
8
diverse responses of NCXs to differences in the severity of ischemic brain injury22. Under mild
ischemic brain injury conditions, the NCXs operate in transporting calcium ions out of cells.
Therefore, blockage of NCXs reduces calcium extrusion and ends up worsening calcium-mediated
cell injury45. On the other hand, severe ischemic brain injury conditions involving an overload of
intracellular sodium leads to the reverse where NCXs conduct calcium into the cells41, 46. Hence,
the blockage of NCXs under these severe ischemic brain injury conditions may potentially be
neuroprotective. Therefore, the targeting of these differential responses and the resulting beneficial
effects needs to be further elucidated.
3.2 Hemichannels
Hemichannels are proteins that are involved in forming gap junctions. One gap junction channel
is composed of two hemichannels, and each hemichannel consists of a hexamer from the connexins
transmembrane protein family47. The gating of gap junction channels is regulated by the
phosphorylation status of connexin proteins47, 48. Under normal conditions, the two hemichannel
components stay in a closed state while forming an open state for gap junction channels. Under
ischemic conditions, the active opening of hemichannels leads to a reduction in the opening of gap
junction channels which consequently results in decreased cell-cell communication36, 48, 49.
Research on hemichannels has elucidated their clear involvement in the response to ischemia brain
injury. However, their precise role remains elusive and controversial. Some studies have shown
that knocking out connexin 43 (Cx43) in mice results in an increased level of brain infarct volume
and apoptosis following stroke47, 50-54. In contrast, other studies report that under specific
conditions, gap junctions exacerbate ischemic brain injury by spreading cytotoxic substances into
cells22, 50, 55. Further validation on the role of hemichannels is warranted before we can truly assess
them as therapeutic targets for stroke and brain injury.
9
3.3 Volume-regulated anion channels
Chloride (Cl-) permeates the cell membrane through several types of Cl– channels. An important
class of Cl– channels is the volume-regulated anion channel (VRAC). VRACs are responsible for
mediating the swelling-induced Cl– current37, 56, which also plays essential role in the regulatory
mechanism in cells for balancing cell volume during osmotic perturbations57-59. Under normal
physiological conditions, VRACs stay in a closed state58-60. Under pathophysiological conditions
like ischemia, VRACs are abnormally overactivated58. Once overactivated, subsequent
pathological mechanisms can be triggered, including the inhibition of NCXs due to ATP
depletion22. VRACs inhibitors have shown to provide a neuroprotective effect following stroke in
rats61-64, which supports the hypothesis that the activation of VRACs can be neurotoxic. DCPIB,
a VRACs specific inhibitor, was recently shown to have a neuroprotective effect in several rodent
models of hypoxic-ischemic brain injury62.
3.4 Acid-sensing ion channels (ASICs)
Acidosis, which worsens neurotoxicity, is a featured outcome that always follows ischemia65, 66.
Acid-sensing ion channel 1a (ASIC1a) is highly expressed in the brain and is believed to be
involved in acidosis induced brain injury65, 67. During ischemic conditions, the accumulation of
lactic acid rapidly decreases the pH level of the brain to 6.2 or even lower, and subsequently
activates ASICs68, 69. In vitro and in vivo studies have shown that the blockage of ASICs results in
neuroprotective effects. In particular, an in vitro study showed that ASIC currents and ASIC
desensitization could be amplified following oxygen-glucose deprivation (OGD), which increased
the length of calcium influx70. Other in vivo studies have shown that knockouts of ASIC1 in mice
protected animals from acidosis-reduced brain injury, and the administration of ASIC blockers
10
reduced brain infarct volume following MCAO brain injury68, 71. Hence, evidence suggests ASICs
as potential therapeutic targets for the inhibition of ischemic neuronal death.
3.5 Transient receptor potential melastatin (TRPM) subfamily
The transient receptor potential melastatin (TRPM) protein family is one of 6 subfamilies among
the TRP channels superfamily72. Two members of the family, TRPM7 and TRPM2, have been
implicated in mediating neuronal cell death73-78. In vitro pharmacological blockage79 and in vivo
siRNA suppression of TRPM7 both resulted in neuroprotective effects80. Recently, in vivo studies
have investigated the role of TRPM2 channels in hypoxic-ischemic brain injury and the results
showed that knockouts of TRPM2 channels in rodent models have neuroprotective effects81-83. The
underlying mechanism of TRPM family-mediated ischemic brain injury still remains unknown.
Since TRPM proteins are calcium permeable ion channels, dysregulation of calcium levels and
overload of intracellular calcium levels are the most acceptable mechanisms78, 83, 84. However,
TRPM ion channels as TRPM7 are also permeable to other ions such as zinc85, which has also
been illustrated to play a role during the hypoxic ischemic cascade. In this study, our focus is on
the role of TRPM2 channels during neonatal hypoxic ischemic brain injury.
11
Figure 2. Potential mechanisms involved in excitotoxicity following ischemic stress. The left
half of the figure shows the traditional glutamate driven model of excitotoxicity. The right half of
the figure shows that more and more evidence nowadays suggests that despite the traditional
model, non-glutamate driven channels including NCXs, hemichannels, VRACs, ASICs, TRPs
may play important roles in mediating the excitotoxicity following ischemic stress. This figure
was modified from Elaine Besancon et al., 2008.
12
4 Transient Receptor Potential Channels (TRPs)
As mentioned above, the TRPM channel subfamily is one of 6 subfamilies among the TRP channel
superfamily. The TRP channel was first identified as a protein in Drosophila melanogaster86. TRP
channels are non-selective cation channels that can be grouped into 6 families named TRPC
(canonical), TRPM (melastatin), TRPV (vanilloid), TRPP (polycystin), TRPML (mucolipin) and
TRPA (ankyrin) 72, 86, 87. There are 28 mammalian TRP channels that have been identified to date88.
All TRP channels consist of six transmembrane domains arranged in a tetrameric structure, and
they are widely expressed in various cell types in the body including neurons87, 88.
5 TRPM2 Channel
5.1 TRPM2 protein structure, transmembrane topology and
distribution
TRPM2 is a calcium-mediated nonselective cation channel. TRPM2 channels are expressed in
many tissues including the brain (high expression), lung, liver and heart89, 90. TRPM2 also
expresses in various cell types, including neurons, microglial cells, immune cells and pancreatic
β-cells78, 89.
TRPM2 proteins are encoded by TRPM2 genes. In rodents, the TRPM2 gene consists of 34 exons
and spans around 61 kb. In human, the TRPM2 gene consists of 32 exons and spans about 90 kb,
with the location of the gene being on chromosome 21q22.390. There is an additional exon located
at the 5’ terminus with a CgG island in the human TRPM gene. The full length transcript of
TRPM2 is approximately 6.5 kb and encodes TRPM2 protein comprising of 1503 amino acids
13
Figure 3. The TRP channel superfamily. The TRP channel superfamily comprises of 6
subfamilies including TRPC (canonical), TRPM (melastatin), TRPV (vanilloid), TRPP
(polycystin), TRPML (mucolipin) and TRPA (ankyrin).
14
with a molecular weight of 170 kDa90. In addition to full length TRPM2 transcripts, four splice
variants of TRPM2 have been identified: TRPM2-ΔN, TRPM2-ΔC, TRPM2 –S and TRPM2-
SSF89-91. Consistent with their names, TRPM2-ΔN is loss of amino acids 538–557 in the N-
terminus; TRPM2-ΔC is loss of amino acids 1292–1325 in the C-terminus, particularly the CAP
domain of the NUDT9-H domain; TRPM2-S (short) is loss of the entire C terminus including the
channel pore; TRPM2-SSF (striatum short form) is loss of the first 214 amino acids of the N-
terminal and has been found to uniquely express within the striatum89-91.
The TRPM2 protein structure consists of six transmembrane segments (S1-S6) with a pore loop
region located between S5 and S690. To form a channel, TRPM proteins typically assemble into
homotetramers with both N- and C- termini flanking the intracellular sides90. At the N-terminus,
there are 4 homologous regions and a calmodulin (CaM) binding domain, which is a region that
plays an important role in regulating the channel activation property90. At the C-terminus, there is
a TRP box as well as a coiled-coil domain, both of which are assumed to be essential for TRPM2
homogenous tetrameric assembly90.
15
Figure 4. TRPM2 protein structure and variants. The upper panel of the figure shows a
representative structure of the TRPM2 channel and its topology. The lower panel of the figure
shows different forms of TRPM2, including the full-length long form TRPM2 (TRPM2-FL),
TRPM2 cleavage of N terminus K538-Q557 (TRPM2-ΔN), TRPM2 cleavage of C terminus T-
1292-L1325 (TRPM2-ΔC), TRPM2 short striatum variant that has 214 residues missing from the
C terminus (TRPM2-SSF) and TRPM2 cleavage of the entire C terminus (TRPM2-S). This figure
was modified from Lin-Hua Jiang et al., 2010.
16
5.2 TRPM2 biophysical properties and gating mechanism
The TRPM2 channel exhibits a linear I/V curve, which suggests that channel activity is
independent from voltage-gating87, 92. Instead, TRPM2 is a ligand-gated channel that can be
activated by several intracellular and extracellular features, among which ADPr and hydrogen
peroxide are the most potent activators89, 90.
Hydrogen peroxide can directly and indirectly activate TRPM2 channels. The ability of H2O2 to
activate TRPM2 channels has attracted significant scientific interest. Studies have used the H2O2
driven mechanism for explaining the pathological processes that are mediated by elevation of the
oxidative microenvironment79, 93. Such pathological processes include hypoxic ischemic (HI) brain
injury, diabetes, inflammation and other neurodegenerative disorders like bipolar diseases.94, 95
Endogenously, H2O2 is initially generated from mitochondria following oxidative
phosphorylation96. Exogenously, generation of H2O2 is induced as a result of responding to
external factors such as certain drugs, heavy metals, visible light or even heat, in consistence with
other reactive oxygen species (ROS) like hydroxyl radicals (OH.)97-99. Overgeneration of ROS
results in dramatic damage to biological molecules such as DNA and proteins, or any molecule
that is involved in the chain reaction cascade producing cellular damage and disease.
In terms of activating the TRPM2 channel indirectly, hydrogen peroxide activates the TRPM2
channel through regulation of the metabolic pathway that produces ADPR100. ADPR can bind to
TRPM2 at the active site in the NUDT9-H region at the C-terminus90, 100. The extracellular
stimulation of hydrogen peroxide leads to an intracellular increase in hydrolase activity, which
thereby hydrolyzes more NAD+ and cADPR to produce additional ADPR90. Another source of
ADPR is the action combination of poly (ADPR) polymerases (PARPs, PARP enzymes) and poly
(ADPR) glycohydrolases (PARG enzymes)89, 90, 101. This source indirectly generates ADPR
17
through the formation and hydrolysis of poly-ADPR when it is overactivated in response to DNA
damage89, 90, 100, 101. At the TRPM2 N-terminus, calcium can bind to the CaM-binding motif, which
is another mechanism of gating that is independent of ADPR and hydrogen peroxide89, 90, 102.
5.3 The physiological and pathophysiological role of TRPM2
channels
As mentioned above, TRPM2 has been identified in several different cell types including neurons,
immune cells and pancreatic β-cells. Therefore, it is not surprising that TRPM2 is associated with
ailments such as CNS diseases and type II diabetes78, 103, 104. However, the precise mechanisms of
these pathologies still require further investigation. In this case, oxidative stress78, 105-107 and
amyloid beta108-110 mediated pathological activation of TRPM2 channels seem the most likely
mechanisms. TRPM2 is highly permeable to calcium, and can mobilize calcium ions from both
the extracellular and intracellular spaces. Hence, its biological significance is strongly associated
with the intracellular calcium level. Under normal physiological conditions, the calcium-mediated
activity of the TRPM2 channel has been reported to be involved in several physiological processes,
including inflammation111, synaptic transmission112, microglial activation113 and insulin
secretion114. Under pathophysiological conditions, the abnormal overactivation of the TRPM2
channel may lead to intracellular calcium overload, which can subsequently lead to various
diseases.
In the CNS, TRPM2 is most abundantly expressed in the brain where it has been implicated in
triggering numerous physiological and pathophysiological processes. For example, TRPM2 is
involved in mediating neuronal cell death that can consequently lead to CNS diseases including
stroke, Alzheimer’s disease and bipolar disorder. One study has shown that patients with bipolar
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disorders have relatively higher levels of basal intracellular calcium ions and that the TRPM2 gene
sites are located within chromosome region 21q22.3, conferring increased susceptibility to this
pathology95. Another study showed that knocking out TRPM2 reduced ischemic brain damage in
MCAO model of adult mice83. Our lab also recently demonstrated that TRPM2 knockouts provide
a neuronal protective effect following HI brain injury in neonatal mice115. Together, these previous
studies indicate that TRPM2 is a promising therapeutic target for the treatment of HI brain injury.
Therefore, my project will examine the effects of TRPM2 inhibition on neuroprotection that may
lead to potential drug development for neonatal hypoxic-ischemic brain injury.
In addition to the CNS, TRPM2 has also been identified in pancreatic β-cells114. Activation of
TRPM2 channels has also been linked to insulin secretion and H2O2-induced apoptosis of insulin-
secreting cells, implicating a potential role of TRPM2 in diabetes. A recent study using the TRPM2
knockout mouse model revealed the involvement of the channel in insulin secretion from
pancreatic β-cells. In this case, TRPM2 knockout mice demonstrated relatively higher basal blood
glucose levels in comparison to WT mice, while plasma insulin levels remained similar114.
TRPM2 has also been identified in some cell types in the immune system, including macrophages,
neutrophils and lymphocytes, suggesting a possible association with inflammatory diseases90, 116.
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Figure 5. Proposed mechanisms of TRPM2 channel activation by H2O2 and involvement
TRPM2 channel activity in physiological and pathophysiological processes. H2O2 can activate
the opening of TRPM2 channels directly and indirectly. Activation of the TRPM2 channel leads
to extracellular calcium influx, which may further facilitate the channel opening. Influx of calcium
leads to intracellular calcium imbalance, which suggests the involvement of TRPM2 channels in
physiological processes such as insulin release, cytokine production, increased endothelial
permeability and cell death. Therefore, the actions of TRPM2 may contribute to pathologies or
disease conditions. +: activation. This figure was modified from Lin-Hua Jiang et al., 2010.
20
6 Pharmacological Interactions
As there is significant interest in the exploration of TRPM2 as a potential target for
neurodegenerative diseases, the pharmacology of TRPM2 has also started to receive considerable
attention in the field of research. Through patch clamp electrophysiological techniques and the
availability of HEK293 cells, different TRPM2 channel inhibitors have been studied. Thus far,
none have demonstrated a desirable specificity with compounds that have been reported to have
an inhibitory effect on the TRPM2 channel also affecting other TRP channels89.
6.1 Flufenamic acid (FFA)
Flufenamic acid (FFA) was the first TRPM2 blocker identified117. It belongs to class of non-
steroidal anti-inflammatory drugs (NSAIDs). Such fenamates are capable of producing anti-
inflammatory effects in the CNS. Studies have been conducted on TRPM2-overexpression
HEK293 cells, where FFA evoked a pH-dependent inhibition of ADPR- or H2O2-induced cation
currents117. However, the inhibitory effect of FFA is not limited to the TRPM2 channel; it can
similarly affect other channels in the TRP family including TRPM4, TRPM5, TRPC3 and
TRPC590, 117. Furthermore, it also has an activation effect on TRPC6 and transient receptor
potential ankyrin 1 (TRPA1)90, 117. Therefore, fenamates such as FFA are hardly satisfactory tools
in clarifying the role of the TRPM2 channel.
6.2 Anti-fungal agents (clotrimazole and econazole)
Anti-fungal compounds block TRPM2 channels activated by ADPR in TRPM2-overexpressing
HEK293 cells, but the inhibitory effect is irreversible89, 118.
21
6.3 2-APB
2-APB was first identified as an inositol 1,4,5-trisphosphate(IP3) receptor antagonist. It has also
been reported to exert an inhibitory effect on certain TRPC and TRPM channels while having an
activation effect on some TRPV channels24, 117, 119.
6.4 Divalent heavy metal cations
Some heavy metal ions, La3+ and Gd3+ for instance, are known to have an inhibitory effect on most
TRP channels, though TRPM2 seems to be an exception. A recent study shows that divalent copper
(Cu2 + ) may be a potent TRPM2 channel blocker98.
6.5 AG490
Recently, AG490 was identified to have an inhibitory effect on the TRPM2 channel120. AG490
was shown to almost completely block H2O2-induced intracellular Ca2+ increase and significantly
reduce H2O2-induced TRPM2 currents120. While H2O2 can also activate the TRPA1 channel,
AG490 has no significant effect on the H2O2-induced Ca2+ influx mediated by TRPA1 channels.
ADPR is another endogenous activator for TRPM2. However, in a patch clamp study on TRPM2-
overexpressing HEK293 cells, AG490 only inhibited H2O2-induced but not ADPR- induced
TRPM2 inward current120. Structurally, AG490 belongs to the tyrphostin family and was
synthesized in the early 1990s. Since AG490 was initially found to be a JAK2 inhibitor, the same
study examined whether the inhibitory effect of AG490 on TRPM2 activity was dependent on the
inhibition of JAK2. The study tested the effects of other JAK2 inhibitors, none of which had an
effect on H2O2-induced Ca2+ increase by TRPM2 channels. Thus, the results suggested that the
inhibitory effect of AG490 on TRPM2 activation is independent from JAK2 inhibition120. Hence,
AG490 may manifest its inhibitory effect by scavenging intracellular hydroxyl radicals.
22
Figure 6. TRPM2 channel inhibitors. Shown above are the chemical structures of compounds
that have been indicated to have an inhibitory effect on TRPM2 channels. FFA, anti-fungal agents
(clotrimazole and econazole) and 2-APB are the classical TRPM2 inhibitors. However, studies
have shown their inhibitory effects to be unsatisfactory. AG490 is a newly discovered TRPM2
inhibitor which inhibits activation by efficiently blocking TRPM2 currents.
23
Chapter 2 Rationale and Hypothesis
Rationale: HI brain injury is a severe public health issue with no effective pharmacological
method of prevention thus far. It has been previously confirmed that TRPM2 plays a
neuroprotective role in adult cerebral ischemia using TRPM2 KO mice83. Our lab has also recently
shown that knocking out TRPM2 provided a neuroprotective effect following HI brain injury in
neonates115. Therefore, I proposed to test the effects of a TRPM2 channel inhibitor in HI brain
damage in relation to potential drug development for HI brain injury.
AG490 is a recently identified TRPM2 current inhibitor that efficiently blocks H2O2 induced
TRPM2 current in HEK cells120. AG490 was initially discovered as a JAK2 inhibitor but could
inhibit TRPM2 in a JAK2 independent manner120. In this case, AG490 may act as a ROS scavenger
and reduce the concentration of hydrogen peroxide: . It was previously
reported that silencing TRPM2 using siRNA reduced H2O2-induced neuronal cell death in vitro79.
It is also reported that following hypoxic-ischemic injury, there is an accumulation of H2O2 in the
neonatal brain at postnatal day 7 (neonatal mice brains) which is not been seen in the postnatal day
42 mouse brain (adult mice brains)121. Thus, it is expected that TRPM2 may play a greater role in
neonates compared to adult, and AG490 could be a suitable pharmacological tool for my project
Hypothesis: I hypothesize that inhibition of TRPM2 channels by TRPM2 inhibitor AG490
provides neuroprotection following hypoxic-ischemic brain injury in neonates.
24
Chapter 3 Aims and Experimental Design
Aims:
AIM1: Investigate the effect of TRPM2 inhibition on H2O2-induced neuronal cell death in vitro.
AIM2: Investigate the effect of TRPM2 inhibition on neonatal hypoxic-ischemic brain injury in
vivo.
Experimental Design Outline:
Figure 7. An outline of the project experimental design.
25
Chapter 4 Materials and Methods
1 Ethics Approval
All protocols were carried out strictly accordant to Canadian Council on Animal Care (CCAC
protocol) guidelines. Protocols were approved by the local Animal Care Committee (Office of
Research Ethics, University of Toronto). All of the experiments have been reported using the
ARRIVE guidelines.
2 Animals
In considering the day of birth as postnatal day 0 (P0), pups used in this study were P7. Timed-
pregnant CD-1 mice were purchased from Charles River Laboratories (Sherbrooke, QB, Canada).
Mice were housed under temperature condition of 20 ± 1°C and a 12 hrs light/dark cycle with free
access to a standard laboratory chow diet and water.
3 Reagents
TRPM2 inhibitor AG490 (CAS #82749-70-0) was purchased from Tocris (BRS, UK). 30 %
Hydrogen Peroxide (HC4060-500ML) was purchased from Biobasic (Amherst, NY, USA). Cresyl
violet, 2,3,5- triphenyl- 2H- tetrazolium chloride (T8877, TTC) and Dimethyl sulfoxide (D2650,
DMSO) were purchased from Sigma- Aldrich (St. Louis, MO, USA).
4 Cell Culture
TRPM2 overexpression HEK293 were cultured as follows: doxycycline-inducible HEK293 cells
with stable expression of TRPM2/ pCDNA4 were cultured with DMEM supplemented with 10 %
FBS, 1 % antibiotic-antimyocotic, blasticidin (5μg/ml, Sigma-Aldrich, MO, USA) and zeocin
26
(0.4mg/ml, Invitrogen, USA). TRPM2 expression was induced by adding 1 μg/ml doxycycline
(1μg/ml, Sigma-Aldrich, USA) to the culture at least 24 hrs before experiments.
Embryonic primary cortex neurons were cultured from E16 CD-1 mice. Dissected cortexes were
digested with 0.025 % trypsin/EDTA at 37°C for 15 min. Cell density was determined using an
Improved Neubauer hemocytometer, and 1.0× 104 cells were plated on poly-D-lysine coated glass
coverslips (12 mm no. 1 German Glass, Bellco cat. no. 1943-10012, Sigma-Aldrich, USA). The
cells were kept in 5 % CO2 at 37°C in culture medium (Neurobasal medium supplemented with
1.8 % B27, 0.25 % Glutamax, and 1 % antibiotic-antimyocotic).
5 In vitro H2O2-induced Neuronal Cell Death Model
The H2O2-induced cell death in vitro model was carried out using CD-1 E16 embryos. Dilutions
of H2O2 were made from a 30 % stock solution into culture medium prior to each experiment.
Exposures to H2O2 were performed by simple addition of a specific volume of H2O2 diluted in
culture medium at x100 directly to each well in a 96-well plate. After exposure to H2O2, the plate
was kept in 5 % CO2 at 37°C for 24 hrs before biochemical measurements were performed.
6 Cell Viability Assay
Cell viability was assessed by MTT assay. MTT, a yellow tetrazole, will be reduced by NAD(P)H-
dependent cellular oxidoreductase enzymes to insoluble formazan with a purple color when
incubating with culture medium. The ratio of yellow MTT to purple formazan indicates the amount
of viable cells. Cells were plated on 96-well culture plates with density of 5×104 cells/ml. Cells
were treated by adding various concentrations of AG490 and incubated with AG490 for 24 hrs.
MTT (0.5 mg/ml MTT in PBS) was diluted with culture medium with a dilution ratio of 1:10 and
added to each well. After 3 hrs of incubation, the medium was removed from each well and 100
27
μl DMSO was added. The absorbance at 490 nm was measured in a microplate reader (Syngery
H1, Biotek, USA). Cell viability was expressed as a percentage of the control.
Cell death assessment was carried out by using propidium iodide (PI) staining. Propidium iodide
is a fluorescent intercalating agent that can only cross the membrane when the cells are necrotic,
therefore, it can be used to stain cells and differentiate cells with different living status (either
necrotic or healthy). Propidium iodide (PI, 1 μg/ml) was added in living cultures. After 24 hrs, the
fluorescent intensity was measured by the Synergy HT Multi-Mode Micro Plate Reader.
7 Electrophysiology (Whole Cell Patch Clamp)
Whole-cell patch-clamp experiments were carried out using an Axopatch 700B (Axon
Instruments, Inc.) to examine the effect of AG490 on TRPM2 current in TRPM2 over-expression
HEK293 cells. TRPM2 over-expression HEK293 cells were induced with 1 μg/ml doxycycline at
least 24 hrs before whole cell patch clamping. Currents were recorded using a 400 ms voltage ramp
protocol (-100 to +100 mV) with an interval of 5 s at 2 kHz and digitized at 5 kHz. Pipette solution
containedv(in mM): 145 cesium methanesulfonate, 8 NaCl, 10 EGTA, and 10 HEPES, pH adjusted
to 7.2 with CsOH. Bath solution contained (in mM) 140 NaCl, 5 KCl, 2 CaCl2, 20 HEPES, and 10
glucose (pH adjusted to 7.4 with NaOH). After filling with pipette solution, patch pipette resistance
was between 5-9 megaohms. pClamp 9.2 software was used for data generation and Clampfit 9.2
was used for data analysis. All experiments were carried out at room temperature.
8 Drug Administration
In vitro administration: Various concentrations of AG490 were prepared by dissolving the
compound in B27-free culture medium. AG490 was added to the culture media prior to exposure
to H2O2.
28
In vivo administration: Pups with body weights of 5 g were randomly assigned into: sham control
group (Sham), HI + vehicle (Vehicle, 5 % DMSO and 5 % Tween-80 in 0.9 % saline) or HI +
AG490 (AG490, 30 mg/kg). AG490 was dissolved in 5 % DMSO and 5 % Tween-80 (P-8074) in
0.9 % saline for the final concentration of 30 mg/kg. AG490 or vehicle control was administered
to the pups 20 min prior to ischemia induction for pre-treatment as well as right after ischemia
induction for post-treatment 1 and immediately after hypoxia for post-treatment 2. The compound
was administered intraperitoneally (i.p.) in a volume of 20 μl/g (injection ratio to body weight).
9 In vivo Hypoxic-Ischemic Mouse Model
Mouse hypoxic-ischemic (HI) model was performed according to a well described protocol with
modifications77, 80, 122. Postnatal day 7 (P7) mice were anesthetized with isoflurane (3.0 % for
induction and 1.5 % for maintenance). The whole process contained two main parts: ischemia and
hypoxia. Ischemia was carried out by isolation of the right common carotid artery and then ligation
with a bipolar electrocoagulation device (Vetroson V-10 Bi-polar electrosurgical unit, Summit Hill
Laboratories, Tinton Falls, NJ, USA). The remaining ligated artery was cut using microscissors.
Pups were then returned to their dam and allowed to recover for 1.5 hrs. After recovery, the
hypoxia process was then achieved by placing the pups in a 37°C chamber (A-Chamber A-15274
with ProOx 110 Oxygen Controller/E-720 Sensor, Biospherix, NY, USA) perfused with a gas
mixture of 7.5 % oxygen and 92.5 % nitrogen for 60 min. A homoeothermic blanket control unit
(K-017484 Harvard Apparatus, MA, USA) was used to monitor the chamber temperature. Animals
in sham groups only undergo exposure of common carotid artery under anesthesia but not ligation
and hypoxia.
29
10 Infarct Volume Measurement, Whole Brain Imaging and
Histological Assessments
10.1 TTC staining/Infarct volume measurement
24 hrs after the HI, brain tissues were collected and coronally sectioned into four ~1 mm slices.
These slices were stained with 2,3,5-triphenyltetrazolium chloride (TTC), a redox indicator
(indicate cellular respiration for differentiating between metabolically active and inactive tissues),
to visualize the infarct area. Slices were stained with 1.5 % TTC and placed in a dark incubator
maintained at 37 oC for 20 min. The infarct areas were traced using image analysis software
(ImageJ). Infarct volume will be calculated by summing the representative areas in all brain
sections and multiplying by the slice thickness. After correcting for edema, the infarct volumes
will be calculated as follows: Corrected infarct volume (CIV), (%) = [contralateral hemisphere
volume - (ipsilateral hemisphere volume - infarct volume)] /contralateral hemisphere volume *
100 %.
10.2 Whole brain imaging/Nissl staining
7 days after the HI, whole brain tissues were collected and imaged to reveal morphological changes
between the groups. At this stage, the infarct areas in the brains underwent liquefactive necrosis
and the severity of the brain damage would be quantified. These brains were subsequently sliced
into ~100 µm coronal sections and stained with 1 % Cresyl violet (Nissl) to indicate histological
brain damage. The infarct areas were traced using image analysis software (ImageJ). Infarct
volume will be calculated as follows: infarct volume (IV), (%) = infarct volume/contralateral
hemisphere volume * 100 %.
30
11 Neurobehavioral Assessments
Short term neurobehavioral tests (geotaxic reflex, cliff aversion and grip test) were carried out to
assess the recovery outcomes of the HI on P1, P3 and P7 days after HI. These reflexes were chosen
because they represent the earliest stages of development in mice and are good indicators of
sensorimotor function. Specifically, 1) geotaxis reflex studies for vestibular and proprioceptive
function123; 2) cliff aversion reflex tests the maladaptive impulse behavior123, 3) grip test assesses
force and fatigability123. These neurobehavioral tests for the determination of the functional
recovery of animals have been well-documented in previous studies from my own lab77, 124, 125.
Long term neurobehavioral test, passive avoidance test126, 127 was also used to assess contextual
fear learning, memory deficits, and also long term motor functional improvements. These abilities
are not well-developed until later in life, thus, they will be tested in post-surgery week 3, indicating
long term neurobehavioral recovery.
11.1 Geotaxis reflex
Geotaxis reflex is an automatic, stimulus-bound orientation movement. Pups were placed head
down in the middle of a board inclined with an angle of 45°. The latency for the pup to rotate 180°
was recorded.
11.2 Cliff avoidance test
Pups were placed on the edge of a platform, and the latency for the pup to remove both paws from
the edge, by turning away from the cliff was recorded.
31
11.3 Grip test
Pups were suspended by their forepaws on a wire stretched over a cotton pad in a cage. The latency
for the pup to fall was recorded.
11.4 Passive avoidance test
Passive Avoidance Test is a 3 day, 1 trailed long-term behavioral test for testing both the motor
functional recovery as well as learning and memory recoveries. 3 weeks after the HI injury, mice
in all three groups (sham, HI + vehicle, HI + AG490) underwent the Passive Avoidance Test. The
protocol consists of day 1 habituation, day 2 acquisition/conditioning and day 3 testing. The
apparatus consists of two parts: a large (250 (W) x 250 (D) x 240 (H) mm) illuminated
compartment and a small (195 (W) x 108 (D) x 120 (H) mm) dark compartment with electrified
grid floor (LE872, Panlab, Harvard Apparatus, BCN, Spain). The two compartments are separated
by a guillotine gate. Mice have innate preference toward dark. During the habituation session, the
mouse was placed into the illuminated compartment and allowed to explore for 1 min. After 1 min,
the door for entering the dark compartment was opened, the latency of entering the dark room was
recorded and the door was closed for 30 s before the mouse was returned to its home cage. During
the acquisition/conditioning session, the mouse was allowed to explore the illuminated
compartment for 30 s and had access to the dark compartment. The mouse received a foot shock
(0.4 mA, 2 s) 3 s after entering the dark compartment. During the testing session, which took place
24 hrs after the acquisition/conditioning session, the mouse was placed into the illuminated
compartment, the door to the dark compartment was opened after 5 s and the latency to enter the
dark compartment was recorded (step-through maximal latency: 300 s). The latency to enter the
dark compartment during the retention session was taken as an index of memory performance.
32
12 Immunohistochemistry and Confocal Imaging
Brain tissues were collected 7 days after HI (P14) and fixed in 4 % paraformaldehyde/30 % sucrose
solution at 4°C overnight. Brains samples were sectioned coronally into ~50 μm slices using a
vibratome (Tissue Sectioning System Microtome Vibratome, HuiYou, China) which underwent
immunohistochemical staining. Samples were probed with mouse anti-neuronal nuclei (NeuN)
antibody (MAB377, 1:500; Chemicon, Temecula, USA) and anti-glial fibrillary acidic protein
(GFAP) (ab7260, 1:1000; Abcam, Cambridge, UK) antibodies overnight at 4°C. Next, the sections
were incubated with secondary antibodies Alexa 488 and 568 (#835724, #632115, 1:200; Cell
Signaling Technology) for 1 hr at room temperature and mounted on glass coverslips with ProLong
Gold antifade reagent (P36930; Thermo Fisher Scientific, Burlington, CA). Confocal laser
microscope (LSM700 Zeiss; Oberkochen, Germany) was used to image the immunostained brain
slices. Three brains per treatment group were collected, and 3 to 5 coronal slices per brain were
imaged. All the treatment groups were imaged at the same laser settings with a 40× lens. Cortical
areas directly adjacent to the injury site were imaged. At least 5 randomly chosen fields were
imaged and the number of cells per field was quantified using the Cell Counter plugin for ImageJ
software (National institute of Health, Bethesda, MD, USA).
13 Western Blot
24 hrs after HI, the ipsilateral and contralateral hemispheres of the mice brains were collected and
frozen in dry ice. To study the underlying mechanism of HI, protein was extracted from the cortex.
The brain samples were homogenized in RIPA buffer with a cocktail of proteinase and phosphatase
inhibitors, then incubated at 4 ºC for 1 h and centrifuged for 15 min at 13,000 rpm. The protein
concentrations were measured using the Bio-Rad Protein Assay reagent (Bio-Rad, Hercules, CA).
Samples of the mouse brain (30 μg) were separated on a 10 % SDS-PAGE gel that was transferred
33
to a nitrocellulose membrane (350 mA, 90 min). Blots blocking was carried out using 5 % non-fat
milk in Tris-buffered saline (TBS). Blots were incubated with primary antibodies at 4ºC overnight
and secondary antibodies at room temperature, respectively anti-phospho-Akt (#9271S, Ser473,
1:1000); anti-Akt (#9272S, 1:1000); anti-GAPDH (#2118S, 1: 10,000). Protein signals of interest
were tested using enhanced chemiluminescent reagents (PerkinElmer, Mass, USA) and analyzed
through exposure to film (Helot CL, NJ, USA).
14 Statistics and Data Analysis
Data were presented as means ± SEM. Student’s t-test was performed to assess the statistical
significance of the difference in 2 groups between vehicle group and the AG490 treated group. In
multiple groups, one-way ANOVA following with the Bonferroni test were used. Significance was
defined by the probability level of lower than 0.05 (P<0.05).
34
Chapter 5 Results
1. The level of TRPM2 mRNA expression in the hippocampus and
cortex increases consistently with the developmental age
In order to choose the optimal materials and specific time points for my study, I extracted in situ
hybridization data from the Allen Brain Atlas and searched for the developmental pattern of
TRPM2 in the brain. Due to the lack of availability of developing mouse brain TRPM2 data, I
calculated corresponding mouse developmental age as described in Workman et al., 2013 and used
data from BrainSpan atlas151 of the Developing Human Brain.
From the extracted data, I first confirmed TRPM2 expression in the brain with high expression in
the hippocampus and cortex. The mRNA expression in these two specific regions reach a peak
point at 1 year of age (Figure 8). In my study, I chose the cortex for cell culture in my in vitro
experiments. TRPM2 reaches highest expression level at 37 weeks after conception, which is a
time period equivalent to P7 in a mouse. Therefore, I used P7 mice pups for my in vivo
experiments. There are abundant TRPM2 channel proteins for activation in the event of H2O2
accumulation in the neonatal brain121, and therefore the TRPM2 inhibitor AG490, which acts as a
hydroxyl radical, could be suitable for addressing my scientific questions.
35
Figure 8. TRPM2 expression in the cortex and hippocampus increases with development.
According to data extracted from the BrainSpan Atlas of Developing Human Brain for the TRPM2
gene, TRPM2 is highly expressed in the developing human brain. In the hippocampus and cortex
TRPM2 expression increases with age and peaks at 1 year of age. Each human developmental age
is correlated with the associated mouse age with similar whole brain development. Data are
expressed as mean log2 of reads per kilobase ± SEM. For cortical measurements n = 27, 11, 22,
11, 11. For hippocampal measurements n = 2, 1, 2, 1, 1. RPKM: reads per kilobase per million.
pcw: post conception week.
36
2 AG490 as a pharmacological inhibitor of the TRPM2 channel
AG490 structurally belongs to the tyrphostin family and was initially found to be a JAK2
inhibitor128. It has been recently reported by Mori and colleagues that AG490 significantly reduced
H2O2-induced TRPM2 activation, but not ADPR-induced TRPM2 current120. Therefore, AG490
is a suitable pharmacological tool to test the effect of inhibition of H2O2-activated TRPM2 channel
activity following neonatal HI brain injury.
To verify the efficiency of the inhibitory effect of AG490 on TRPM2 current, I first carried out
whole-cell patch clamp recording on TRPM2 overexpression HEK293 cells. 1 μg/ml doxycycline
was used to induce TRPM2 overexpression in HEK293 cells, with the cells being induced at least
24 hrs before whole cell patch clamping. As shown in Figure 9C, H2O2 (200 μM) elicited a large
and inwardly rectifying current in TRPM2 overexpression HEK293 cells with the current density
of 1497.97 ± 452.03 mA (n=3), whereas pretreatment of HEK293 cells with AG490 reduced the
current to 67.33 ± 31.63 mA (n=3; p=0.0343). Spontaneous TRPM2 current without exposure to
H2O2 was small (Figure 9A).
These results suggest that TRPM2 channel activity is sensitive to inhibition by AG490 and that
AG490 is a valid pharmacological tool for further in vitro and in vivo studies.
37
Figure 9. AG490 efficiently inhibited H2O2-induced TRPM2 current in TRPM2
overexpression HEK293 cells. A. Spontaneous TRPM2 currents in doxycycline-induced TRPM2
overexpression HEK293 cells. B. H2O2-induced TRPM2 currents in doxycycline-induced TRPM2
overexpression HEK293 cells. C. AG490 inhibited H2O2-induced TRPM2 currents in
doxycycline-induced TRPM2 overexpression HEK293 cells. D. Representative I-V trace (black
line is trace of bath solution; red line is trace of perfusion with 200 µΜ H2O2; blue line is trace of
pretreatment with 50 µΜ for 2 hrs and perfusion with 200 µΜ H2O2). E. Summary bar chart
comparing the H2O2-induced TRPM2 currents at +90 mV with and without application of AG490.
*represents p < 0.01 (Student's t-test, n = 3/group).
38
3 AG490 protects neurons from H2O2-induced cell injury in vitro
After verifying that AG490 efficiently blocks TRPM2 current activity in TRPM2 overexpression
HEK293 cells, I investigated the effects of AG490 on the viability and proliferation of cortical
neurons. Cortical neurons are vulnerable to H2O2. To test whether AG490 can protect neurons
from H2O2-induced cell death in vitro, cortical neurons were cultured and treated as described in
the methods section. The MTT assay was carried out to assess cell viability and propidium iodide
(PI) staining was performed to assess cell death. Hydrogen peroxide produced a progressive
apoptotic effect on cortical neurons in a dose-dependent manner from 6-100 μM (Figure 10A, p <
0.05, n=12). Figure 10B shows that AG490 pre-treatment (40 min prior to exposure to H2O2)
improved cell viability of cortical neurons at the optimum concentration of 50 μM. Propidium
iodide (PI) fluorescence intensity in cortical neurons was found significantly greater after exposure
to H2O2 in a dose-dependent manner from 6-50 μM (Figure 10C, p < 0.05, n=9). Figure 10D shows
that AG490 pre-incubation for 40 min significantly reduced PI fluorescence intensity at 50μM
(Figure 10D, p < 0.05, n = 6). This in vitro data indicates that AG490 could protect cultured
neurons from insult caused by H2O2 exposure.
39
Figure 10. TRPM2 inhibitor AG490 pre-treatment reduced neuronal cell death following
H2O2-induced cell injury. A & C. Hydrogen peroxide produced a progressive apoptotic effect on
cortical neurons in a dose-dependent manner from 6-100 μM after 24 hrs of incubation with
AG490. B & D. 50 μM AG490 significantly protected neurons from H2O2-induced injury (CTL,
control; Results are mean ± SEM; *, versus CTL group, #, versus non-treated group, p<0.05; One-
way ANOVA with subsequent Bonferroni test, n as indicated on the bars).
A B
C D
40
4 The Effect of AG490 Pre-treatment on Hypoxic-Ischemic Brain
Injury in vivo.
The neonatal hypoxic-ischemic injury model was implemented on postnatal 7-day old (P7) CD-1
mice. AG490 (30 mg/kg) or vehicle were administered as a single intraperitoneal injection to P7
pups according to the timeline in Figure 11.
Figure 11. Timeline of neonatal hypoxic-ischemic injury and experimental procedures.
Postnatal 7-day old pups (P7) were randomly grouped into 3 groups (sham, vehicle, AG490) and
injected with either vehicle or AG490 (no injection for sham group) 20 min prior to ischemia
induction for pre-treatment and immediately after ischemia induction for post-treatment 1 and
immediately after hypoxia for post-treatment 2. This was followed by 90 min of recovery and 60
min of hypoxia with 7.5 % O2 and 92.5 % N2. TTC staining and western blot were performed 24
hrs after HI (P8). Whole brain imaging, Nissl staining and immunohistochemistry was performed
7 days after the HI (P14). Short term neurobehavioral assessment was performed 1 day, 3 days and
7 days after HI (P8, P10, P14). Long term neurobehavioral assessment was performed 3 weeks
after the HI (P35).
41
4.1 Pre-treatment with TRPM2 inhibitor AG490 reduced the brain
infarct volume of hypoxic-ischemic brain injury in vivo.
After confirming that AG490 delivered neuroprotective effect following H2O2-induced cell death,
I next investigated whether AG490 generated neuroprotective effects in vivo. By using a mouse
neonatal hypoxic-ischemic brain injury model followed by TTC staining, I found that a lower dose
of AG490 (15 mg/kg, i.p., 20 min before HI) did not afford neuroprotection (Figure 13). However,
AG490 pre-treatment with a higher dose (30 mg/kg i.p., 20 min before HI) significantly reduced
brain infarct volume in comparison to the vehicle treated group (Figure 13). TTC is a redox
indicator and it stains for metabolically active tissues. The white area represents the infarct area
and illustrates the damaged tissue which is not metabolically active. TTC staining was carried out
on coronal sections of mouse brains 24 hrs after HI. Representative images of TTC staining are
shown in Figure 13C, where the white un-stained areas indicated the infarct volume. Infarct
volume in the vehicle-treated HI group (Vehicle) was 58.00 ± 5.11 % (n = 13 pups). AG490 pre-
treatment (30 mg/kg) significantly reduced the infarct volume to 22.76 ± 3.11 % (n = 17 pups) in
comparison to the vehicle-treated group (*, p < 0.05). There was no detectable infarction in the
sham group (data not shown). There was no detectable protective effect at 15 mg/kg AG490 pre-
treatment (Figure 12, infarct volume in the Vehicle group was 50.79 ± 6.53 %, n=8; infarct volume
in the 15 mg/kg AG490 treated group was 53.40 ± 9.03 %, n=11).
42
Figure 12. Lower dose of AG490 (15 mg/kg) did not provide neuroprotection. The dose of 15
mg/kg was chosen based on literature review129-132. With three repeated sets of experiments, there
was no observable neuroprotective effect. A. TTC result summary chart from the 1st experiment;
B. TTC result summary chart from 2nd experiment; C. TTC result summary chart from 3rd
experiment; D. Summary chart of all combined TTC results from 3 sets of experiments; E.
Representative brain slices for TTC staining. All data presented as mean ± SEM. Statistical
analysis was done by student’s t-test. There was no significant difference between vehicle group
and the AG490 group.
A B C
D E
43
Figure 13. Pre-treatment with TRPM2 currents inhibitor AG490 (30 mg/kg) reduced the
brain infarct volume of hypoxic-ischemic brain injury in vivo. A.B. Representative triphenyl
tetrazolium chloride (TTC) staining of brains treated with vehicle (5 % DMSO + 5 % Tween 80
in 0.9 % saline) and AG490 (30 mg/kg) 20 min before the onset of injury (pre-treatment). The
brains were harvested and stained 24 hrs following injury. Corrected infarct volume of vehicle-
treated (58.00 ± 5.109, n=13) and AG490-treated (22.76 ± 3.106, n=17) groups respectively 24 hrs
following injury. All data presented as mean ± SEM. Statistical analysis was done by student’s t-
test (*p<0.05).
1st Timeline TTC
Veh
icle
AG49
0
0
20
40
60
80
13 17
*
Infa
rcti
on
Vo
lum
e (
%)
A B
44
4.2 Pre-treatment with TRPM2 inhibitor AG490 reduced brain
damage following hypoxic-ischemic brain injury.
Next, I tested whether the neuroprotective action of AG490 was effective 7 days after HI brain
injury. Whole brains were collected, fixed, imaged, and then sectioned for Nissl staining 7 days
after HI (P14). On the 7th day after HI insult, the brain infarction had already undergone
liquefactive necrosis resulting in loss of brain weight (Figure 14A). Whole brain weight was used
as an indicator of the liquefaction level and measured in all groups. The sham and AG490-treated
groups had greater brain weights in comparison to the vehicle-treated group (Figure 14C, sham
group 0.43 ± 0.01 g, vehicle-treated + HI group 0.33 ± 0.01 g and AG490-treated + HI group 0.37
± 0.01 g). Consistent with this data, the AG490 pre-treatment (30 mg/kg) group demonstrated
significantly less brain damage (both in whole brains and coronal sections, n = 15 pups, Figure
14B) in comparison to vehicle treatment group (n = 21 pups). Whole brains were coronally sliced
into ~100 μm sections for Nissl (cresyl violet) staining to reveal that the AG490 treated group
sustained less brain damage in comparison to the vehicle treated group. There was no detectable
brain damage in sham group. With respect to the neuroprotective effects of AG490, whole brain
imaging at 7 days further verified the TTC staining results at 24 hrs after HI.
45
Figure 14. TRPM2 inhibitor AG490 reduced brain damage following hypoxic-ischemic brain
injury. A. Overall brain morphology was preserved in the pre-treatment paradigm 7 days after HI
injury. Nissl staining showed reduced liquefaction volume in the AG490-treated group in
comparison to the vehicle-treated group during the pre-treatment paradigm. B. Ipsilateral
liquefaction volume was significantly reduced in the AG490-treated group in comparison to the
vehicle group during the pre-treatment paradigm (sham: 0, n=16; vehicle: 63.72 ± 2.938 %, n=21;
AG490: 31.15 ± 4.410 %, n=15). C. Brain weight was significantly higher in the AG490-treated
group in comparison to the vehicle-treated group in the pre-treatment paradigm (vehicle: 0.33 ±
0.006 g, n=21; AG490: 0.38 ± 0.01g, n=15). All data presented as mean ± SEM. Statistical analysis
was done by one-way ANOVA followed by the Bonferroni post-hoc (*p<0.05). * comparison of
vehicle versus sham group; # comparison of AG490 versus vehicle group.
46
4.3 Pre-treatment with TRPM2 inhibitor AG490 promotes recovery
after HI challenge
Body weight is one of the most frequently used indicators of the general health of a mouse pup77,
133. Pups were randomly assigned to different experimental groups with no significant difference
in body weights between groups (sham group 5.05 ± 0.08 g, vehicle-treated + HI group 5.08 ±
0.06 g and AG490-treated + HI group 4.93 ± 0.09 g). The body weights of each group were
measured at 4 timelines: prior to the onset of HI as well as 1, 3, and 7 days after HI (Figure 15).
On day 1 after HI, the mean body weight of the pups that underwent HI surgery was significantly
reduced in comparison to the sham group. 7 days after HI, mice in sham (9.98 ± 0.49 g) and
AG490-treated groups (9.81 ± 0.50 g) gained significantly more weight than vehicle-treated HI
mice (8.32 ± 0.37 g, p < 0.05). These results indicated that AG490 treatment promoted general
health recovery after the HI procedure.
Figure 15. AG490 pre-treatment (20 min before HI injury) improves general health recovery
after HI challenge. Body weight, as an indicator of recovery after HI, was found to be
significantly higher in sham and AG490-treated groups than in vehicle-treated group on the 1st day
and 7 days after HI. All data presented as mean ± SEM. Statistical analysis: one-way ANOVA
followed by Boferroni post-hoc, *p<0.05. * comparison of vehicle versus sham group; #
comparison AG490 versus vehicle group.
47
4.4 Pre-treatment with TRPM2 inhibitor AG490 improves short-
term neurobehavioral performance after HI
To further examine the neuroprotective effect generated by AG490 pre-treatment, I further
assessed the functional outcomes in sham animals (n = 16 pups), vehicle-treated animals (n = 21
pups) and AG490 pre-treated animals (n = 15 pups). Short-term neurobehavioral tests evaluating
the geotaxis reflex, cliff avoidance and grip tests were performed on 1, 3, and 7 days after HI in
the 3 groups respectively. In comparison to pups in the sham group, the neurobehavioral
functioning of pups in the vehicle-treated HI group was significantly impaired 1, 3 and 7 days after
HI (Figure 16, *, p < 0.05). The AG490-treated group performed significantly better in the geotaxis
test 7 days after HI (Figure 16A) compared to the vehicle-treated group (2.33 ± 0.25 s in AG490-
treated HI group versus 8.74 ± 1.63 s in the vehicle-treated HI group; p < 0.05). The cliff avoidance
test performance was also significantly better in the AG490-treated group 3 and 7 days after HI
(Figure 16B) compared to the vehicle-treated group (2.58 ± 0.22 s in the AG490-treated group
versus 4.36 ± 1.33 s in the vehicle-treated group 3 days after HI; 3.86 ± 0.85 s in the AG490-
treated group versus 6.94 ± 1.15 s in the vehicle-treated group 7 days after HI; p < 0.05). Grip test
performance was also significantly better 1, 3 and 7 days after HI in the AG490-treated group
compared to the vehicle-treated group (Figure 16C) (p < 0.05). Therefore, AG490 treatment not
only reduced brain damage but also improved neurobehavioral outcomes after HI brain injury.
48
Figure 16. AG490 pre-treatment (20 min before HI injury) improved neurobehavioral
performance after HI challenge. A.B.C. Neurobehavioral evaluation was performed as described
in the methods section. Geotaxis reflex (A), cliff avoidance test (B) and grip test (C) of sham (n =
16), vehicle (HI + vehicle, n = 21) and AG490 (30 mg/kg) pretreatment (HI + AG490, n = 15)
groups were measured 1 day, 3 days and 7 days after HI (*, p < 0.05 versus sham group; #, p <
0.05 versus vehicle group). All data presented as mean ± SEM. Statistical analysis: one-way
ANOVA followed by the Boferroni post-hoc, *p<0.05.
49
4.5 Pre-treatment with TRPM2 inhibitor AG490 also improves long-
term neurobehavioral performance after HI
To determine whether AG490 pre-treatment would also improve long-term functional recovery, I
subjected mice to the passive avoidance test 3 weeks after HI brain injury and assessed whether
AG490 treatment attenuated memory impairment after HI.
I found that AG490-treated mice groups showed significantly better memory function in
comparison to the vehicle-treated mice, via evidence of significantly longer latency to enter the
dark room 24 hrs after foot shock (Figure 17A). Whole brains extracted 30 days after HI procedure
were used for assessing morphology changes Nissl staining was carried out to indicate the
histology changes. In Figure 17B left panel, representative images of whole brains and Nissl
staining all shown reduced liquefaction volume in AG490-treated group compared to vehicle-
treated group. Summary bar chart in Figure 17B right panel shown that AG490-treated group has
significant less ipsilateral liquefaction comparing to vehicle-treated group (sham: 0, n=11; vehicle:
49.65 ± 10.24 %, n=8; AG490: 24.22 ± 6.039 %, n=10).
By this, my data show that despite short term motor functional recovery improvement, AG490
treatment helps alleviate long term neurobehavioural deficits such as memory deficits following
neonatal HI brain injury in pre-treatment paradigm.
50
Figure 17. Long – term behavioral assessment of functional recovery in the pre-treatment
paradigm following hypoxic-ischemic injury. A. In the Passive Avoidance test, AG490-treated
mice showed better memory function compared to vehicle-treated mice, as was evident from
significantly longer latency to enter the dark room 24 hrs after the conditioning (foot shock). B.
Representative overall brain morphology image and Nissl staining images all shown reduced
liquefaction volume in AG490-treated group compared to vehicle-treated group. Summary bar
chart comparing AG490-treated group and vehicle-treated group (sham: 0, n=11; vehicle: 49.65 ±
10.24 %, n=8; AG490: 24.22 ± 6.039 %, n=10). All data presented as mean ± SEM. Statistical
analysis: one-way ANOVA with Boferroni post-hoc, *p<0.05.
P a s s iv e A v o id a n c e T e s t
0
5 0
1 0 0
1 5 0
2 0 0
2 5 0 *
La
ten
cy
to
En
ter (
s)
S h a m V e h ic le A G 4 9 0
a fte r fo o t s h o c k
b e fo re fo o t s h o c k
*
16 8 1 016 8 1 0
A
B
51
4.6 Pre-treatment with TRPM2 inhibitor AG490 reduces reactive
astrocyte activation
To assess the effects of AG490 on apoptotic signaling and neuronal survival, I performed
immunohistochemical staining and analysis of the penumbra area of the brain slices in sham,
vehicle- and AG490-treated groups 7 days after HI injury. Representative confocal images are
shown in Figure 18A. NeuN is the neuronal nuclear antigen, which is commonly used as a
biomarker for neurons77, 134. GFAP expression in astrocytes represent astroglial activation and
reactive gliosis77, 134, which are hallmarks following neurodegenerative conditions. As seen in
Figure 18B, AG490 pre-treatment significantly reduced the loss of NeuN-positive cells when
compared to the vehicle-treated group. I also found an upregulation of GFAP expression in
astrocytes, which indicates reactive gliosis and astroglial activation during neurodegeneration in
the vehicle-treated group. The expression of GFAP was significantly less in the AG490-treated
group in comparison to the vehicle group.
52
Figure 18. AG490 pretreatment restores neuronal cell numbers and reduces reactive
astrocyte activation. A. Representative confocal images of immunohistochemical staining. NeuN
stains for neurons, GFAP stains for reactive astrocytes. B. Summary bar charts of NeuN- and
GFAP-positive cells per 40x field.
N e u N -p o s it iv e c e lls p e r 4 0 x f ie ld
Sh
am
Veh
icle
AG
490
0
5 0
1 0 0
1 5 0
2 0 0
3 3 3
*
#
Ne
uN
- p
os
itiv
e c
ell
s
G F A P -p o s it iv e c e lls p e r 4 0 x f ie ld
Sh
am
Veh
icle
AG
490
0
5 0
1 0 0
1 5 0
2 0 0
2 5 0*
#
3 3 3
GF
AP
- p
os
itiv
e c
ell
s
A B NeuN GFAP
53
4.7 Pre-treatment with TRPM2 inhibitor AG490 may reduce HI
brain damage through Akt mediated signaling pathways
To determine the potential underlying signalling cascades affected by AG490 treatment, I
examined several signalling cascades that are known to be affected during neonatal HI injury.
During development, the p-Akt/t-Akt level is high at P7 in mice135. My data found that HI injury
significantly reduced Akt phosphorylation levels in the ipsilateral hemisphere. However, AG490
pre-treatment restored the normal levels of these proteins, indicating the protective effect of this
inhibitor.
Figure 19. Biochemical assessment of signalling pathways affected by hypoxic-ischemic
insult on the neonatal brain in a pre-treatment paradigm. A. Akt activation was reduced in
the ipsilateral hemispheres of the vehicle-treated group in comparison to sham group; AG490
pre-treatment restored Akt signalling (vehicle: 0.33 ± 0.09, n=3; AG490: 0.99 ± 0.02, n=3; data
normalized to sham). B. Representative western blots of proteins extracted from the ipsilateral
hemispheres of sham, vehicle-treated and AG490 pre-treated groups 24 hrs after HI. All data
presented as mean ± SEM. Statistical analysis: one-way ANOVA followed by the Boferroni
post-hoc, *p<0.05.
p-A
kt/G
AP
DH
(%
to
sh
am
)
sham
vehic
le
AG
490
0 .0
0 .5
1 .0
1 .5
*
3 3 3
t-A
kt/G
AP
DH
(%
to
sh
am
)
sham
vehic
le
AG
490
0 .0
0 .5
1 .0
1 .5
3 3 3
p-A
kt/t
-A
kt
(%
to
sh
am
)
sham
vehic
le
AG
490
0 .0
0 .5
1 .0
1 .5
3 3 3
*
p-Akt
t-Akt
GAPDH
Sham Vehicle AG490
A
B
54
5 The Effect of AG490 Post-treatment on Hypoxic-Ischemic Brain
Injury in vivo.
5.1 AG490 *post-treatment 1 (30 mg/kg, i.p.) reduced brain infarct
volume of hypoxic-ischemic brain injury in vivo.
*post-treatment 1: immediately after ischemia induction
Next, instead of injecting AG490 20 min prior to the onset of HI brain injury, I administrated
AG490 immediately after ischemia induction in order to test whether AG490 can also provide a
neuroprotective effect in a post-treatment paradigm. By using the same methodology as during
pre-treatment, TTC staining following the post-treatment paradigm showed that AG490
administration right after ischemia induction also reduced brain damage in the neonatal hypoxic-
ischemic brain injury model (Figure 20). AG490 post-treatment (30 mg/kg, i.p., immediately after
ischemia induction) significantly reduced the brain infarct volume to 28.84 ± 3.609 (n=10) in
comparison to the vehicle treated group (50.51 ± 5.100, n=12, *, p < 0.05).
5.2 AG490 *post-treatment 1 (30 mg/kg, i.p.) reduced brain
damage following hypoxic-ischemic brain injury
*post-treatment 1: immediately after ischemia induction
Using the same methodology as during pretreatment, whole brains for post-treatment 1 were
collected, fixed and imaged 7 days after HI with the whole brain weights being measured. The
AG490 post-treatment (30 mg/kg) group demonstrated significantly less brain damage in
comparison to the vehicle treatment group (Figure 21, n = 21 pups). The sham group and AG490-
treated groups had greater brain weights in comparison to the vehicle-treated group (sham: 0, n=5;
vehicle: 57.81 ± 2.434 %, n=19; AG490: 34.24 ± 3.914 %, n=11). Therefore, I conclude that post-
treatment of AG490 also reduced brain damage following HI brain injury.
55
Figure 20. Post-treatment 1 (30 mg/kg, i.p.) reduced brain infarct volume and brain damage
following hypoxic-ischemic brain injury in vivo. Upper panel shows the timeline for TTC
staining and whole brain imaging in AG490 post-treatment. Lower panel A and B show
representative images for TTC staining and a summary chart for corrected brain infarction volume
following AG490 post-treatment. All data presented as mean ± SEM. Statistical analysis was done
by student’s t-test (*p<0.05).
A B
56
Figure 21. TRPM2 inhibitor AG490 reduced morphological and histological damage
following hypoxic-ischemic brain injury. A. Representative images for whole brain and Nissl
staining. B. The ipsilateral liquefaction volume was significantly reduced in the AG490-treated
group compared to vehicle group in post-treatment paradigm (sham: 0, n=16; vehicle: 57.81 ±
2.434 %, n=17; AG490: 34.24 ± 3.914 %, n=11). B. Brain weight was significantly higher in the
AG490-treated group compared to the vehicle-treated group in post-treatment paradigm (sham:
0.15 ± 0.006 g, n=16; vehicle: 0.35 ± 0.014 g, n=17; AG490: 0.40 ± 0.007 g, n=11). All data
presented as mean ± SEM. Statistical analysis was done by one-way ANOVA following by the
Bonferroni post-hoc (*p<0.05). * comparison of vehicle versus sham group; # comparison of
AG490 versus vehicle group.
B
Sh
am
Veh
icle
AG
490
0 .0
0 .1
0 .2
0 .3
0 .4
0 .5
1 6 1 7 1 1
*
#
bra
in w
eig
ht
(g)
A C
he
mis
ph
ere
liq
ue
fac
tio
n v
olu
me
(%
)
Sh
am
Veh
icle
AG
490
0
2 0
4 0
6 0
8 0
1 6 1 7 1 1
*
#
57
5.3 AG490 *post-treatment 1 (30 mg/kg, i.p.) improves
neurobehavioral performance and general recovery after HI
*post-treatment 1: immediately after ischemia induction
Body weight in each group was measured and used as indicators for the general health of mice.
Similar to pre-treatment, pups were randomly assigned to different experimental groups with no
significant difference in body weight between groups (sham group 5.05 ± 0.08 g, vehicle-treated
+ HI group 5.08 ± 0.06 g and AG490-treated + HI group 4.93 ± 0.09 g). The body weights of each
group were measured at 4 timelines: prior to the onset of HI as well as 1, 3, and 7 days after HI
(Figure 22A). On day 3 and day 7 after HI, the mean body weight of pups in the vehicle treated
group was significantly lower than those in the sham and AG490 treated groups. Therefore, AG490
post-treatment 1 also promoted body weight. Consistent with these results, AG490 post-treatment
1 also demonstrated improvement in short-term neuronal behavioral tests (see Figure 22B, C, D).
These results indicated that AG490 can also improve general health recovery as well as neuronal
behavioral outcomes following HI injury using a post-treatment paradigm.
58
Figure 22. AG490 post-treatment 1 (immediately after ischemic injury) improves general
health and neurobehavioral performance after HI challenge. A. Body weights were measured
1, 3 and 7 days after HI. B.C. D. Neurobehavioral evaluation was performed as described in the
methods section. Geotaxis reflex (B), cliff avoidance test (C) and grip test (D) in the sham (n =
16), vehicle (HI + vehicle, n = 19) and AG490 (30 mg/kg) pretreatment (HI + AG490, n = 11)
groups were measured 1, 3 and 7 days after HI (*, p < 0.05 versus sham group; #, p < 0.05 versus
vehicle group). All data presented as mean ± SEM. Statistical analysis: one-way ANOVA followed
by the Boferroni post-hoc, *p<0.05.
C D
B A
59
6 AG490 *post-treatment 2 (30mg/kg, i.p., immediately after HI
induction) demonstrate a trend towards neuroprotection following
HI brain injury
*post-treatment 2: immediately after hypoxia ischemia induction
To further evaluate the therapeutic potential of AG490, I evaluated another time point of
administration. In this case, AG490 was administrated immediately after whole HI induction
(including ischemia and hypoxia processes). The same TTC method as before was used to evaluate
the brain infarct volume between groups.
According to my results, the administration of AG490 immediately after HI brain injury
demonstrated a trend towards neuroprotection, though this effect was not yet significant.
Figure 23. *Post-treatment 2 (30 mg/kg, i.p.) shows a trend towards neuroprotection
following hypoxic-ischemic brain injury. Upper panel shows the timeline for TTC staining for
post-treatment in AG490 post-treatment. Lower panel shows representative images for TTC
staining and a summary chart for corrected brain infarction volume following AG490 post-
treatment. All data presented as mean ± SEM. Statistical analysis was done by student’s t-test.
60
Discussion
1. Connection between clinics and the current study
Despite major advances in understanding fetal and neonatal pathologies, hypoxic-ischemic (HI)
brain injury remains a serious issue that causes significant mortality and long-term morbidity in
neonates. Due to lack of efficient treatment for neonatal HI brain injury, survivors and their
families suffer from the burden of lifetime health care costs. The current licensed and most efficient
treatment for neonatal HI brain injury is hypothermia14. However, clinical studies have shown that
this therapy is not effective for all infants affected by HI. Therefore, in considering the global
prevalence of this ailment and the poor long-term outcomes, novel neuroprotective treatments that
may be used in conjunction with hypothermia are urgently required in the treatment of this
disorder.
Traditional glutamate driven mechanisms were long thought to be the major pathways involved in
the ischemic cascade. Hence, glutamate receptors including NMDA and AMPA receptors were
considered as promising therapeutic targets22. However, all clinical trials using compounds
targeting glutamate receptors failed to generate the expected neuroprotective outcomes22. TRPM2
channels, among the major proteins whose activities were investigated under non-glutamate driven
mechanisms, have been since implicated to be involved in numerous physiological and
pathological processes including HI brain injury. The absence of TRPM2 channel activity by either
using siRNA to silence TRPM2 in vitro 79or knocking out TRPM2 channels in vivo83, 115 was
observed to provide neuroprotective effects. Therefore, TRPM2 channels are important therapeutic
targets for further study. In my study, I used a newly identified TRPM2 channel current inhibitor
61
AG490 to verify the role of this structure in neonatal HI brain injury as well as tested the potential
clinical use of this compound.
2. Summary of major findings
Here, I evaluated a novel inhibitor of the TRPM2 channel (AG490) during treatment in vitro
following H2O2-induced neuronal cell death as well as in vivo in a mouse model of neonatal
hypoxic-ischemic brain injury. AG490 was initially studied as a JAK2 inhibitor. AG490 blocks
TRPM2 activity by acting as a hydroxyl radical scavenger, indicating that AG490 significantly
reduces H2O2-induced TRPM2 activation through scavenging hydroxyl radicals rather than Jak2-
dependent mechanisms. By using a H2O2-induced neuronal cell death model, I found that AG490
protected neurons from apoptosis. I then showed the strong preventative and therapeutic potential
of AG490 in our mouse model of neonatal HI brain injury. I found that pre-treatment with AG490
immediately after hypoxic ischemic exposure significantly reduced brain infarction volumes
compared to vehicle controls 24 hrs after HI. I also found that AG490 administration reduced brain
mass loss and preserved overall brain morphology up to 7 days after HI injury in comparison to
the vehicle group. General health, short-term and long-term functional recovery was significantly
improved with AG490 pretreatment 20 min prior to the onset of HI brain injury as well as
following AG490 posttreatment immediately after ischemic induction. The underlying
mechanisms of this neuroprotective effect requires further evaluation. My data suggests that HI
injury significantly reduced Akt phosphorylation levels in the ipsilateral hemisphere, and that
AG490 pre-treatment restored this deficit. Therefore, the neuroprotective effect of AG490 may be
related to the Akt cascade. Taken together, my study conclusively demonstrates that AG490 has
neuroprotective and therapeutic properties in a mouse model of neonatal HIE.
62
3. Significance of the current study
My study has revealed that the inhibition of TRPM2 channel activity provides neuroprotection
against neonatal hypoxic-ischemic brain injury, and that TRPM2 channel inhibitors may be
potential pharmacological treatments for HI brain injury. This knowledge may help in providing
improved healthcare options for hypoxic-ischemic brain injury in children. It may also divulge
potential preventive measures for HI related neurological complications such as hypoxic-ischemic
encephalopathy and cerebral palsy. The principle findings may also be applied to the study of other
related neurological disorders as well.
4. Differences between neonatal HI brain injury and adult stroke
Neonatal HI brain injury has important differences in comparison to adult ischemic stroke5. For
example, liquifactive disintegration can be the result of severe HI events in the infant brain, but is
not seen following adult ischemic stroke4. Additionally, newly formed blood vessels in neonates
are prone to rupture. The blood brain barrier (BBB) is also compromised following neonatal HI
brain injury4, 5. The autoregulation of the cerebrovasculature is another factor of concern in
infants136. Pre-term neonates demonstrate a “pressure passive” cerebral circulation while sick term
infants show impairment in autoregulation3, 4. Moreover, the concentrations and actions of various
signaling molecules including caspase-3 are different in the developing brain4.
Neonatal HI injury may also evolve over time while adult ischemic stroke does not15. By using
MRI scanning system, it has been found that injuries within the first few hours following HI were
subtle and restrictively diffused only in putamen and thalami, then the injuries progress over within
the next 3-4 days and diffused to other areas in the brain15.
63
To add to this complexity, the NMDA receptor is relatively over-expressed in the developing
brain140, 141. In P6 rats, the NMDA receptor is expressed at 150–200 % of the adult levels142. The
combination of NMDA receptor subunits in the perinatal period are thought to be favored of
prolonged calcium influx for a given excitation143. Increased level of glutamate has been found in
the cerebrospinal fluid (CSF) of infants who have suffered from severe HI injury137, 144. The
neonatal brain is more sensitive to seizure activity compared to the mature brain, suggesting a
prominent role for neuronal hyperexcitability and excitotoxicity145, 146. Neonatal brains are also
more vulnerable to hydrogen peroxide elevation, which is one of the characteristics following HI
brain injury121. The accumulation of hydrogen peroxide coupled with low antioxidant activity in
neonates results in sensitivity to oxidative stress induced by HI.
5. Proposed mechanism of neonatal HI brain injury
GSK-3 was first identified as a regulatory protein for glycogen metabolism. It has two isoforms
named GSK-3α and GSK-3β respectively147. GSK-3 is involved in numerous cellular processes in
the brain, including the Wnt-1/β–catenin and phosphoinositide 3-kinase/protein kinase
B(PI3K/Akt) signaling pathways147, 148. The kinase activity of GSK-3 may be inhibited through
phosphorylation of GSK-3α (pGSK-3α) at the Ser21 site and GSK-3β (p-GSK-3β) at the Ser9
site147, 149.
Studies have shown that phosphorylation of Akt could inhibit GSK-3β kinase activity and
consequently result in the downregulation of caspase-3 mediated apoptotic signaling135. This
suggests that the regulation of GSK-3β kinase activity may be linked to cell death as observed in
our model. Our previous study has shown that using TDZD-8135, which is a specific blocker of
64
Figure 24. Propose mechanism for inhibitory effect of AG490. The mechanism of inhibitory
effect of AG490 needs further evaluation. It may inhibit TRPM2 activities through i) direct block
of TRPM2 channels; ii) inhibit TRPM2 activity through ADPR-independent pathways and reduce
the level of TRPM2 activator H2O2; iii) inhibit TRPM2 activity through ADPR-dependent
pathways and reduce the level of H2O2 first then block the ADPR binding site. The inhibitory
effect may get involved in p-Akt – GSK-3β – caspase 3 signaling pathways.
65
GSK-3β, can also generate a neuroprotective effect. Therefore, AG490’s neuroprotective and
therapeutic effects in our mouse model may act through the Akt/GSK-3β/Caspase-3 pathway.
Further experimentation needs to be carried out in order to verify the link between AG490 to GSK-
3β and Caspase-3.
6. Pitfalls in the current study and proposed future directions
My study tested the inhibitory effect of AG490 on TRPM2 channel activity and found that this
drug demonstrated a promising neuroprotective effect both in vitro and in vivo. The in vivo
neuroprotective effect of AG490 could be observed at different time points. In order to limits gaps
between the mouse model and a future clinical study, the following improvements may be made
to the current study:
i) Instead of injecting AG490 only once, the drug could be administrated multiple times.
Multiple administrations of the compound may result in stronger and even more
prolonged neuroprotective effects. As shown in my TTC data, the neuroprotective
effect of AG490 was strong within the 1st and 2nd timelines. However, the data showed
a trend of decreasing neuroprotective effect according to the time period of AG490
administration.
ii) To better evaluate the effects of AG490, different doses of the compound could be
tested on our HI mouse model. The lower dose of AG490 (15 mg/kg) initially utilized
in the study was chosen based on literature review. As we did not see a difference
between the vehicle treated and AG490 treated groups, we increased the dose to 30
66
mg/kg to observe a neuroprotective effect. To further test the effects of AG490, 2 more
doses (1 dose lower than 30 mg/kg but higher than 15mg/kg; the other dose higher than
30 mg/kg) could be chosen for additional experiments in our HI mouse model. By this,
we could verify whether AG490 generates a neuroprotective effect in a dose-dependent
manner. Additionally, higher doses of AG490 may lead to prolonged neuroprotective
effects.
iii) AG490 was initially studied as a JAK2 inhibitor, and is therefore not a specific inhibitor
for TRPM2 channels. An in vitro study indicated that AG490 may inhibit H2O2-induced
TRPM2 activation in a JAK2-independent manner. In this case, Mori and colleagues
used the whole cell patch clamp technique to test the inhibitory effect of AG490 on
TRPM2 HEK293 cells. Interestingly, AG490 efficiently blocked H2O2-induced
TRPM2 channel currents while Jak inhibitor 1 and staurosporine, which are commonly
used as JAK2 inhibitors (Lawrie et al., 1997; Nakagawa et al., 2011), did not affect
TRPM2 channel activity at all. These observations suggest that inhibition of H2O2-
induced TRPM2 channel activation by AG490 is independent of JAK2 mechanisms.
As a future direction for my study, the effects of JAK2 specific inhibitors like Jak2
inhibitor 1 and staurosporine could be tested in vivo. If this in vivo data is not consistent
with or even opposite to the effects observed under AG490, we could then verify that
the inhibitory effect of AG490 on TRPM2 channel activity in vivo is also independent
from JAK2 mechanisms. If the in vivo data generated using other JAK2 specific
inhibitors shows a similar effect as AG490, then more experiments need to be carried
out to further evaluate the action of AG490 in either JAK2 dependent or independent
67
mechanisms. For example, the combined administration of AG490 and a JAK2 specific
activator/inhibitor into the HI mouse model could be one possible experiment.
iv) Since AG490 acts as a hydroxyl radical scavenger, it is important to test whether the
neuroprotective effect generated by this compound is specifically mediated via
inhibition of TRPM2 channel activity. Due to the availability of TRPM2 knockout
mice, a proposed next step could be the administration of AG490 into this model to
view changes in neuroprotective effects in comparison to the controls.
v) Another future direction could include the observance of additional long-term
behavioral tests to fully assess the functional recovery outcomes of TRPM2 inhibition
by AG490. For instance, a novel objective test could be conducted150.
68
REFERENCES
1. Perlman, J.M. Intrapartum hypoxic-ischemic cerebral injury and subsequent cerebral
palsy: medicolegal issues. Pediatrics 99, 851-859 (1997).
2. Perlman, J.M. & Risser, R. Severe fetal acidemia: neonatal neurologic features and short-
term outcome. Pediatric neurology 9, 277-282 (1993).
3. Shah, P.S., Beyene, J., To, T., Ohlsson, A. & Perlman, M. Postasphyxial hypoxic-
ischemic encephalopathy in neonates: outcome prediction rule within 4 hours of birth. Archives
of pediatrics & adolescent medicine 160, 729-736 (2006).
4. Millar, L.J., Shi, L., Hoerder-Suabedissen, A. & Molnar, Z. Neonatal Hypoxia Ischaemia:
Mechanisms, Models, and Therapeutic Challenges. Frontiers in cellular neuroscience 11, 78
(2017).
5. Ferriero, D.M. Neonatal brain injury. The New England journal of medicine 351, 1985-
1995 (2004).
6. Lawn, J.E., Cousens, S., Zupan, J. & Lancet Neonatal Survival Steering, T. 4 million
neonatal deaths: when? Where? Why? Lancet 365, 891-900 (2005).
7. Finer, N.N., Robertson, C.M., Richards, R.T., Pinnell, L.E. & Peters, K.L. Hypoxic-
ischemic encephalopathy in term neonates: perinatal factors and outcome. The Journal of
pediatrics 98, 112-117 (1981).
8. Richer, L.P., Shevell, M.I. & Miller, S.P. Diagnostic profile of neonatal hypotonia: an 11-
year study. Pediatric neurology 25, 32-37 (2001).
9. Levene, M.I., Sands, C., Grindulis, H. & Moore, J.R. Comparison of two methods of
predicting outcome in perinatal asphyxia. Lancet 1, 67-69 (1986).
10. Ruth, V.J. & Raivio, K.O. Perinatal brain damage: predictive value of metabolic acidosis
and the Apgar score. Bmj 297, 24-27 (1988).
11. Laptook, A.R., et al. Outcome of term infants using apgar scores at 10 minutes following
hypoxic-ischemic encephalopathy. Pediatrics 124, 1619-1626 (2009).
12. Robertson, N.J., Cowan, F.M., Cox, I.J. & Edwards, A.D. Brain alkaline intracellular pH
after neonatal encephalopathy. Annals of neurology 52, 732-742 (2002).
13. Weeke, L.C., et al. Role of EEG background activity, seizure burden and MRI in
predicting neurodevelopmental outcome in full-term infants with hypoxic-ischaemic
encephalopathy in the era of therapeutic hypothermia. European journal of paediatric neurology
: EJPN : official journal of the European Paediatric Neurology Society 20, 855-864 (2016).
14. Choi, H.A., Badjatia, N. & Mayer, S.A. Hypothermia for acute brain injury--mechanisms
and practical aspects. Nature reviews. Neurology 8, 214-222 (2012).
15. Fatemi, A., Wilson, M.A. & Johnston, M.V. Hypoxic-ischemic encephalopathy in the
term infant. Clinics in perinatology 36, 835-858, vii (2009).
16. Gunn, A.J., Battin, M., Gluckman, P.D., Gunn, T.R. & Bennet, L. Therapeutic
hypothermia: from lab to NICU. Journal of perinatal medicine 33, 340-346 (2005).
17. Gunn, A.J. & Gunn, T.R. The 'pharmacology' of neuronal rescue with cerebral
hypothermia. Early human development 53, 19-35 (1998).
18. Kracer, B., Hintz, S.R., Van Meurs, K.P. & Lee, H.C. Hypothermia therapy for neonatal
hypoxic ischemic encephalopathy in the state of California. The Journal of pediatrics 165, 267-
273 (2014).
69
19. Pauliah, S.S., Shankaran, S., Wade, A., Cady, E.B. & Thayyil, S. Therapeutic
hypothermia for neonatal encephalopathy in low- and middle-income countries: a systematic
review and meta-analysis. PloS one 8, e58834 (2013).
20. Savman, K. & Brown, K.L. Treating neonatal brain injury - promise and inherent
research challenges. Recent patents on inflammation & allergy drug discovery 4, 16-24 (2010).
21. Arundine, M. & Tymianski, M. Molecular mechanisms of glutamate-dependent
neurodegeneration in ischemia and traumatic brain injury. Cellular and molecular life sciences :
CMLS 61, 657-668 (2004).
22. Besancon, E., Guo, S., Lok, J., Tymianski, M. & Lo, E.H. Beyond NMDA and AMPA
glutamate receptors: emerging mechanisms for ionic imbalance and cell death in stroke. Trends
in pharmacological sciences 29, 268-275 (2008).
23. Anderson, C.M. & Swanson, R.A. Astrocyte glutamate transport: review of properties,
regulation, and physiological functions. Glia 32, 1-14 (2000).
24. Chinopoulos, C., Gerencser, A.A., Doczi, J., Fiskum, G. & Adam-Vizi, V. Inhibition of
glutamate-induced delayed calcium deregulation by 2-APB and La3+ in cultured cortical
neurones. Journal of neurochemistry 91, 471-483 (2004).
25. Hetman, M. & Kharebava, G. Survival signaling pathways activated by NMDA
receptors. Current topics in medicinal chemistry 6, 787-799 (2006).
26. Liu, Y., et al. NMDA receptor subunits have differential roles in mediating excitotoxic
neuronal death both in vitro and in vivo. The Journal of neuroscience : the official journal of the
Society for Neuroscience 27, 2846-2857 (2007).
27. Gladstone, D.J., Black, S.E., Hakim, A.M., Heart & Stroke Foundation of Ontario Centre
of Excellence in Stroke, R. Toward wisdom from failure: lessons from neuroprotective stroke
trials and new therapeutic directions. Stroke; a journal of cerebral circulation 33, 2123-2136
(2002).
28. Ikonomidou, C. & Turski, L. Why did NMDA receptor antagonists fail clinical trials for
stroke and traumatic brain injury? The Lancet. Neurology 1, 383-386 (2002).
29. Krebs, C., Fernandes, H.B., Sheldon, C., Raymond, L.A. & Baimbridge, K.G. Functional
NMDA receptor subtype 2B is expressed in astrocytes after ischemia in vivo and anoxia in vitro.
The Journal of neuroscience : the official journal of the Society for Neuroscience 23, 3364-3372
(2003).
30. Bonfoco, E., Krainc, D., Ankarcrona, M., Nicotera, P. & Lipton, S.A. Apoptosis and
necrosis: two distinct events induced, respectively, by mild and intense insults with N-methyl-D-
aspartate or nitric oxide/superoxide in cortical cell cultures. Proceedings of the National
Academy of Sciences of the United States of America 92, 7162-7166 (1995).
31. Matute, C., Alberdi, E., Ibarretxe, G. & Sanchez-Gomez, M.V. Excitotoxicity in glial
cells. European journal of pharmacology 447, 239-246 (2002).
32. Wong, R. NMDA receptors expressed in oligodendrocytes. BioEssays : news and reviews
in molecular, cellular and developmental biology 28, 460-464 (2006).
33. Karadottir, R., Cavelier, P., Bergersen, L.H. & Attwell, D. NMDA receptors are
expressed in oligodendrocytes and activated in ischaemia. Nature 438, 1162-1166 (2005).
34. Newcomer, J.W., Farber, N.B. & Olney, J.W. NMDA receptor function, memory, and
brain aging. Dialogues in clinical neuroscience 2, 219-232 (2000).
35. Coyle, J.T., Basu, A., Benneyworth, M., Balu, D. & Konopaske, G. Glutamatergic
synaptic dysregulation in schizophrenia: therapeutic implications. Handbook of experimental
pharmacology, 267-295 (2012).
36. Bargiotas, P., Monyer, H. & Schwaninger, M. Hemichannels in cerebral ischemia.
Current molecular medicine 9, 186-194 (2009).
70
37. Alibrahim, A., et al. Neuroprotective effects of volume-regulated anion channel blocker
DCPIB on neonatal hypoxic-ischemic injury. Acta pharmacologica Sinica 34, 113-118 (2013).
38. Blaustein, M.P. & Lederer, W.J. Sodium/calcium exchange: its physiological
implications. Physiological reviews 79, 763-854 (1999).
39. Bojarski, C., Meloni, B.P., Moore, S.R., Majda, B.T. & Knuckey, N.W. Na+/Ca2+
exchanger subtype (NCX1, NCX2, NCX3) protein expression in the rat hippocampus following
3 min and 8 min durations of global cerebral ischemia. Brain research 1189, 198-202 (2008).
40. Khananshvili, D. Sodium-calcium exchangers (NCX): molecular hallmarks underlying
the tissue-specific and systemic functions. Pflugers Archiv : European journal of physiology 466,
43-60 (2014).
41. Gomez-Villafuertes, R., Mellstrom, B. & Naranjo, J.R. Searching for a role of
NCX/NCKX exchangers in neurodegeneration. Molecular neurobiology 35, 195-202 (2007).
42. Meng, F., To, W.K. & Gu, Y. Role of TRP channels and NCX in mediating hypoxia-
induced [Ca(2+)](i) elevation in PC12 cells. Respiratory physiology & neurobiology 164, 386-
393 (2008).
43. Matsuda, T., et al. SEA0400, a novel and selective inhibitor of the Na+-Ca2+ exchanger,
attenuates reperfusion injury in the in vitro and in vivo cerebral ischemic models. The Journal of
pharmacology and experimental therapeutics 298, 249-256 (2001).
44. Kormos, A., et al. Efficacy of selective NCX inhibition by ORM-10103 during simulated
ischemia/reperfusion. European journal of pharmacology 740, 539-551 (2014).
45. Pignataro, G., et al. Evidence for a protective role played by the Na+/Ca2+ exchanger in
cerebral ischemia induced by middle cerebral artery occlusion in male rats. Neuropharmacology
46, 439-448 (2004).
46. Boscia, F., et al. Permanent focal brain ischemia induces isoform-dependent changes in
the pattern of Na+/Ca2+ exchanger gene expression in the ischemic core, periinfarct area, and
intact brain regions. Journal of cerebral blood flow and metabolism : official journal of the
International Society of Cerebral Blood Flow and Metabolism 26, 502-517 (2006).
47. Belousov, A.B., Fontes, J.D., Freitas-Andrade, M. & Naus, C.C. Gap junctions and
hemichannels: communicating cell death in neurodevelopment and disease. BMC cell biology 18,
4 (2017).
48. Thompson, R.J., Zhou, N. & MacVicar, B.A. Ischemia opens neuronal gap junction
hemichannels. Science 312, 924-927 (2006).
49. Johansen, D., Cruciani, V., Sundset, R., Ytrehus, K. & Mikalsen, S.O. Ischemia induces
closure of gap junctional channels and opening of hemichannels in heart-derived cells and tissue.
Cellular physiology and biochemistry : international journal of experimental cellular
physiology, biochemistry, and pharmacology 28, 103-114 (2011).
50. Contreras, J.E., et al. Role of connexin-based gap junction channels and hemichannels in
ischemia-induced cell death in nervous tissue. Brain research. Brain research reviews 47, 290-
303 (2004).
51. Davidson, J.O., et al. Connexin hemichannel blockade is neuroprotective after asphyxia
in preterm fetal sheep. PloS one 9, e96558 (2014).
52. Davidson, J.O., et al. Connexin hemichannel blockade improves outcomes in a model of
fetal ischemia. Annals of neurology 71, 121-132 (2012).
53. Kim, Y., et al. Tonabersat Prevents Inflammatory Damage in the Central Nervous System
by Blocking Connexin43 Hemichannels. Neurotherapeutics : the journal of the American Society
for Experimental NeuroTherapeutics (2017).
54. Xu, J., Chen, L. & Li, L. Pannexin hemichannels: A novel promising therapy target for
oxidative stress related diseases. Journal of cellular physiology (2017).
71
55. Nielsen, B.S., Hansen, D.B., Ransom, B.R., Nielsen, M.S. & MacAulay, N. Connexin
Hemichannels in Astrocytes: An Assessment of Controversies Regarding Their Functional
Characteristics. Neurochemical research (2017).
56. Best, L., Brown, P.D., Sener, A. & Malaisse, W.J. Electrical activity in pancreatic islet
cells: The VRAC hypothesis. Islets 2, 59-64 (2010).
57. Voss, F.K., et al. Identification of LRRC8 heteromers as an essential component of the
volume-regulated anion channel VRAC. Science 344, 634-638 (2014).
58. Pedersen, S.F., Okada, Y. & Nilius, B. Biophysics and Physiology of the Volume-
Regulated Anion Channel (VRAC)/Volume-Sensitive Outwardly Rectifying Anion Channel
(VSOR). Pflugers Archiv : European journal of physiology 468, 371-383 (2016).
59. Jentsch, T.J., Lutter, D., Planells-Cases, R., Ullrich, F. & Voss, F.K. VRAC: molecular
identification as LRRC8 heteromers with differential functions. Pflugers Archiv : European
journal of physiology 468, 385-393 (2016).
60. Bowens, N.H., Dohare, P., Kuo, Y.H. & Mongin, A.A. DCPIB, the proposed selective
blocker of volume-regulated anion channels, inhibits several glutamate transport pathways in
glial cells. Molecular pharmacology 83, 22-32 (2013).
61. Fujii, T., et al. Inhibition of gastric H+,K+-ATPase by 4-(2-butyl-6,7-dichloro-2-
cyclopentylindan-1-on-5-yl)oxybutyric acid (DCPIB), an inhibitor of volume-regulated anion
channel. European journal of pharmacology 765, 34-41 (2015).
62. Han, Q., et al. DCPIB, a potent volume-regulated anion channel antagonist, attenuates
microglia-mediated inflammatory response and neuronal injury following focal cerebral
ischemia. Brain research 1542, 176-185 (2014).
63. Minieri, L., et al. The inhibitor of volume-regulated anion channels DCPIB activates
TREK potassium channels in cultured astrocytes. British journal of pharmacology 168, 1240-
1254 (2013).
64. Trouet, D., et al. Inhibition of VRAC by c-Src tyrosine kinase targeted to caveolae is
mediated by the Src homology domains. American journal of physiology. Cell physiology 281,
C248-256 (2001).
65. Wang, J.J. & Xu, T.L. [Acid-sensing ion channels as a target for neuroprotection:
acidotoxicity revisited]. Sheng li xue bao : [Acta physiologica Sinica] 68, 403-413 (2016).
66. Yermolaieva, O., Leonard, A.S., Schnizler, M.K., Abboud, F.M. & Welsh, M.J.
Extracellular acidosis increases neuronal cell calcium by activating acid-sensing ion channel 1a.
Proceedings of the National Academy of Sciences of the United States of America 101, 6752-
6757 (2004).
67. Voilley, N. Acid-sensing ion channels (ASICs): new targets for the analgesic effects of
non-steroid anti-inflammatory drugs (NSAIDs). Current drug targets. Inflammation and allergy
3, 71-79 (2004).
68. Xiong, Z.G., et al. Neuroprotection in ischemia: blocking calcium-permeable acid-
sensing ion channels. Cell 118, 687-698 (2004).
69. Allen, N.J. & Attwell, D. Modulation of ASIC channels in rat cerebellar Purkinje neurons
by ischaemia-related signals. The Journal of physiology 543, 521-529 (2002).
70. Immke, D.C. & McCleskey, E.W. Lactate enhances the acid-sensing Na+ channel on
ischemia-sensing neurons. Nature neuroscience 4, 869-870 (2001).
71. Baron, A. & Lingueglia, E. Pharmacology of acid-sensing ion channels - Physiological
and therapeutical perspectives. Neuropharmacology 94, 19-35 (2015).
72. Li, H. TRP Channel Classification. Advances in experimental medicine and biology 976,
1-8 (2017).
72
73. Gees, M., Colsoul, B. & Nilius, B. The role of transient receptor potential cation channels
in Ca2+ signaling. Cold Spring Harbor perspectives in biology 2, a003962 (2010).
74. Morelli, M.B., Amantini, C., Liberati, S., Santoni, M. & Nabissi, M. TRP channels: new
potential therapeutic approaches in CNS neuropathies. CNS & neurological disorders drug
targets 12, 274-293 (2013).
75. Nilius, B., Owsianik, G., Voets, T. & Peters, J.A. Transient receptor potential cation
channels in disease. Physiological reviews 87, 165-217 (2007).
76. Aarts, M., et al. A key role for TRPM7 channels in anoxic neuronal death. Cell 115, 863-
877 (2003).
77. Chen, W., et al. TRPM7 inhibitor carvacrol protects brain from neonatal hypoxic-
ischemic injury. Molecular brain 8, 11 (2015).
78. Aarts, M.M. & Tymianski, M. TRPMs and neuronal cell death. Pflugers Archiv :
European journal of physiology 451, 243-249 (2005).
79. Kaneko, S., et al. A critical role of TRPM2 in neuronal cell death by hydrogen peroxide.
Journal of pharmacological sciences 101, 66-76 (2006).
80. Sun, H.S., et al. Suppression of hippocampal TRPM7 protein prevents delayed neuronal
death in brain ischemia. Nature neuroscience 12, 1300-1307 (2009).
81. Miller, B.A. & Cheung, J.Y. TRPM2 protects against tissue damage following oxidative
stress and ischemia-reperfusion. The Journal of physiology (2015).
82. Shimizu, T., et al. Extended therapeutic window of a novel peptide inhibitor of TRPM2
channels following focal cerebral ischemia. Experimental neurology 283, 151-156 (2016).
83. Alim, I., Teves, L., Li, R., Mori, Y. & Tymianski, M. Modulation of NMDAR subunit
expression by TRPM2 channels regulates neuronal vulnerability to ischemic cell death. The
Journal of neuroscience : the official journal of the Society for Neuroscience 33, 17264-17277
(2013).
84. Olah, M.E., et al. Ca2+-dependent induction of TRPM2 currents in hippocampal neurons.
The Journal of physiology 587, 965-979 (2009).
85. Ye, M., et al. TRPM2 channel deficiency prevents delayed cytosolic Zn2+ accumulation
and CA1 pyramidal neuronal death after transient global ischemia. Cell death & disease 5, e1541
(2014).
86. Venkatachalam, K. & Montell, C. TRP channels. Annual review of biochemistry 76, 387-
417 (2007).
87. Ramsey, I.S., Delling, M. & Clapham, D.E. An introduction to TRP channels. Annual
review of physiology 68, 619-647 (2006).
88. Hellmich, U.A. & Gaudet, R. Structural biology of TRP channels. Handbook of
experimental pharmacology 223, 963-990 (2014).
89. Eisfeld, J. & Luckhoff, A. Trpm2. Handbook of experimental pharmacology, 237-252
(2007).
90. Jiang, L.H., Yang, W., Zou, J. & Beech, D.J. TRPM2 channel properties, functions and
therapeutic potentials. Expert opinion on therapeutic targets 14, 973-988 (2010).
91. Kraft, R. & Harteneck, C. The mammalian melastatin-related transient receptor potential
cation channels: an overview. Pflugers Archiv : European journal of physiology 451, 204-211
(2005).
92. Clapham, D.E., Runnels, L.W. & Strubing, C. The TRP ion channel family. Nature
reviews. Neuroscience 2, 387-396 (2001).
93. Shimizu, S., et al. Sensitization of H2O2-induced TRPM2 activation and subsequent
interleukin-8 (CXCL8) production by intracellular Fe(2+) in human monocytic U937 cells. The
international journal of biochemistry & cell biology 68, 119-127 (2015).
73
94. Jang, Y., et al. TRPM2, a Susceptibility Gene for Bipolar Disorder, Regulates Glycogen
Synthase Kinase-3 Activity in the Brain. The Journal of neuroscience : the official journal of the
Society for Neuroscience 35, 11811-11823 (2015).
95. Sumoza-Toledo, A. & Penner, R. TRPM2: a multifunctional ion channel for calcium
signalling. The Journal of physiology 589, 1515-1525 (2011).
96. Jiang, L., et al. Mitochondria dependent pathway is involved in the protective effect of
bestrophin-3 on hydrogen peroxide-induced apoptosis in basilar artery smooth muscle cells.
Apoptosis : an international journal on programmed cell death 18, 556-565 (2013).
97. Ogawa, N., Kurokawa, T. & Mori, Y. Sensing of redox status by TRP channels. Cell
calcium (2016).
98. Zeng, B., Chen, G.L. & Xu, S.Z. Divalent copper is a potent extracellular blocker for
TRPM2 channel. Biochemical and biophysical research communications 424, 279-284 (2012).
99. Simon, F., Varela, D. & Cabello-Verrugio, C. Oxidative stress-modulated TRPM ion
channels in cell dysfunction and pathological conditions in humans. Cellular signalling 25,
1614-1624 (2013).
100. Kolisek, M., Beck, A., Fleig, A. & Penner, R. Cyclic ADP-ribose and hydrogen peroxide
synergize with ADP-ribose in the activation of TRPM2 channels. Molecular cell 18, 61-69
(2005).
101. Naziroglu, M. & Luckhoff, A. A calcium influx pathway regulated separately by
oxidative stress and ADP-Ribose in TRPM2 channels: single channel events. Neurochemical
research 33, 1256-1262 (2008).
102. Starkus, J., Beck, A., Fleig, A. & Penner, R. Regulation of TRPM2 by extra- and
intracellular calcium. The Journal of general physiology 130, 427-440 (2007).
103. Gelderblom, M., et al. Transient receptor potential melastatin subfamily member 2 cation
channel regulates detrimental immune cell invasion in ischemic stroke. Stroke; a journal of
cerebral circulation 45, 3395-3402 (2014).
104. Kahya, M.C., Naziroglu, M. & Ovey, I.S. Modulation of Diabetes-Induced Oxidative
Stress, Apoptosis, and Ca2+ Entry Through TRPM2 and TRPV1 Channels in Dorsal Root
Ganglion and Hippocampus of Diabetic Rats by Melatonin and Selenium. Molecular
neurobiology 54, 2345-2360 (2017).
105. Takahashi, N., Kozai, D., Kobayashi, R., Ebert, M. & Mori, Y. Roles of TRPM2 in
oxidative stress. Cell calcium 50, 279-287 (2011).
106. Akpinar, H., Naziroglu, M., Ovey, I.S., Cig, B. & Akpinar, O. The neuroprotective action
of dexmedetomidine on apoptosis, calcium entry and oxidative stress in cerebral ischemia-
induced rats: Contribution of TRPM2 and TRPV1 channels. Scientific reports 6, 37196 (2016).
107. Chong, Z.Z., Li, F. & Maiese, K. Oxidative stress in the brain: novel cellular targets that
govern survival during neurodegenerative disease. Progress in neurobiology 75, 207-246 (2005).
108. Fonfria, E., et al. Amyloid beta-peptide(1-42) and hydrogen peroxide-induced toxicity
are mediated by TRPM2 in rat primary striatal cultures. Journal of neurochemistry 95, 715-723
(2005).
109. Mattson, M.P., et al. beta-Amyloid peptides destabilize calcium homeostasis and render
human cortical neurons vulnerable to excitotoxicity. The Journal of neuroscience : the official
journal of the Society for Neuroscience 12, 376-389 (1992).
110. Ostapchenko, V.G., et al. The Transient Receptor Potential Melastatin 2 (TRPM2)
Channel Contributes to beta-Amyloid Oligomer-Related Neurotoxicity and Memory Impairment.
The Journal of neuroscience : the official journal of the Society for Neuroscience 35, 15157-
15169 (2015).
74
111. Haraguchi, K., et al. TRPM2 contributes to inflammatory and neuropathic pain through
the aggravation of pronociceptive inflammatory responses in mice. The Journal of neuroscience
: the official journal of the Society for Neuroscience 32, 3931-3941 (2012).
112. Xie, Y.F., et al. Dependence of NMDA/GSK-3beta mediated metaplasticity on TRPM2
channels at hippocampal CA3-CA1 synapses. Molecular brain 4, 44 (2011).
113. Kraft, R., et al. Hydrogen peroxide and ADP-ribose induce TRPM2-mediated calcium
influx and cation currents in microglia. American journal of physiology. Cell physiology 286,
C129-137 (2004).
114. Uchida, K., et al. Lack of TRPM2 impaired insulin secretion and glucose metabolisms in
mice. Diabetes 60, 119-126 (2011).
115. Huang, S., et al. Transient receptor potential melastatin 2 channels (TRPM2) mediate
neonatal hypoxic-ischemic brain injury in mice. Experimental neurology 296, 32-40 (2017).
116. Zhong, Z., et al. TRPM2 links oxidative stress to NLRP3 inflammasome activation.
Nature communications 4, 1611 (2013).
117. Chen, G.L., et al. Pharmacological comparison of novel synthetic fenamate analogues
with econazole and 2-APB on the inhibition of TRPM2 channels. British journal of
pharmacology 167, 1232-1243 (2012).
118. Hill, K., McNulty, S. & Randall, A.D. Inhibition of TRPM2 channels by the antifungal
agents clotrimazole and econazole. Naunyn-Schmiedeberg's archives of pharmacology 370, 227-
237 (2004).
119. Togashi, K., Inada, H. & Tominaga, M. Inhibition of the transient receptor potential
cation channel TRPM2 by 2-aminoethoxydiphenyl borate (2-APB). British journal of
pharmacology 153, 1324-1330 (2008).
120. Shimizu, S., et al. Inhibitory effects of AG490 on H2O2-induced TRPM2-mediated
Ca(2+) entry. European journal of pharmacology 742, 22-30 (2014).
121. Lafemina, M.J., Sheldon, R.A. & Ferriero, D.M. Acute hypoxia-ischemia results in
hydrogen peroxide accumulation in neonatal but not adult mouse brain. Pediatric research 59,
680-683 (2006).
122. Rice, J.E., 3rd, Vannucci, R.C. & Brierley, J.B. The influence of immaturity on hypoxic-
ischemic brain damage in the rat. Annals of neurology 9, 131-141 (1981).
123. Lubics, A., et al. Neurological reflexes and early motor behavior in rats subjected to
neonatal hypoxic-ischemic injury. Behavioural brain research 157, 157-165 (2005).
124. Xiao, A.J., et al. Marine compound xyloketal B reduces neonatal hypoxic-ischemic brain
injury. Marine drugs 13, 29-47 (2015).
125. Xu, B., et al. Neuroprotective Effects of a PSD-95 Inhibitor in Neonatal Hypoxic-
Ischemic Brain Injury. Molecular neurobiology (2015).
126. Jarvik, M.E. & Kopp, R. An improved one-trial passive avoidance learning situation.
Psychological reports 21, 221-224 (1967).
127. Castellano, C. & Pavone, F. Effects of ethanol on passive avoidance behavior in the
mouse: involvement of GABAergic mechanisms. Pharmacology, biochemistry, and behavior 29,
321-324 (1988).
128. Seo, I.A., et al. Janus Kinase 2 Inhibitor AG490 Inhibits the STAT3 Signaling Pathway
by Suppressing Protein Translation of gp130. The Korean journal of physiology &
pharmacology : official journal of the Korean Physiological Society and the Korean Society of
Pharmacology 13, 131-138 (2009).
129. Chai, H.T., Yip, H.K., Sun, C.K., Hsu, S.Y. & Leu, S. AG490 suppresses EPO-mediated
activation of JAK2-STAT but enhances blood flow recovery in rats with critical limb ischemia.
Journal of inflammation 13, 18 (2016).
75
130. Davoodi-Semiromi, A., Hassanzadeh, A., Wasserfall, C.H., Droney, A. & Atkinson, M.
Tyrphostin AG490 agent modestly but significantly prevents onset of type 1 in NOD mouse;
implication of immunologic and metabolic effects of a Jak-Stat pathway inhibitor. Journal of
clinical immunology 32, 1038-1047 (2012).
131. Higuchi, T., et al. Prevention of acute lung allograft rejection in rat by the janus kinase 3
inhibitor, tyrphostin AG490. The Journal of heart and lung transplantation : the official
publication of the International Society for Heart Transplantation 24, 1557-1564 (2005).
132. Toda, T., Yamamoto, S., Yonezawa, R., Mori, Y. & Shimizu, S. Inhibitory effects of
Tyrphostin AG-related compounds on oxidative stress-sensitive transient receptor potential
channel activation. European journal of pharmacology 786, 19-28 (2016).
133. Chen, W.L., et al. Xyloketal B suppresses glioblastoma cell proliferation and migration
in vitro through inhibiting TRPM7-regulated PI3K/Akt and MEK/ERK signaling pathways.
Marine drugs 13, 2505-2525 (2015).
134. Turlova, E., et al. TRPM7 Regulates Axonal Outgrowth and Maturation of Primary
Hippocampal Neurons. Molecular neurobiology 53, 595-610 (2016).
135. Wang, H., et al. Tideglusib, a chemical inhibitor of GSK3beta, attenuates hypoxic-
ischemic brain injury in neonatal mice. Biochimica et biophysica acta 1860, 2076-2085 (2016).
136. Volpe, J.J. Neonatal encephalopathy: an inadequate term for hypoxic-ischemic
encephalopathy. Annals of neurology 72, 156-166 (2012).
137. Hagberg, H., Andersson, P., Kjellmer, I., Thiringer, K. & Thordstein, M. Extracellular
overflow of glutamate, aspartate, GABA and taurine in the cortex and basal ganglia of fetal
lambs during hypoxia-ischemia. Neuroscience letters 78, 311-317 (1987).
138. Choi, D.W. Calcium-mediated neurotoxicity: relationship to specific channel types and
role in ischemic damage. Trends in neurosciences 11, 465-469 (1988).
139. Choi, D.W. Excitotoxic cell death. Journal of neurobiology 23, 1261-1276 (1992).
140. McDonald, J.W., Roeser, N.F., Silverstein, F.S. & Johnston, M.V. Quantitative
assessment of neuroprotection against NMDA-induced brain injury. Experimental neurology
106, 289-296 (1989).
141. Represa, A., Tremblay, E. & Ben-Ari, Y. Transient increase of NMDA-binding sites in
human hippocampus during development. Neuroscience letters 99, 61-66 (1989).
142. Tremblay, E., Roisin, M.P., Represa, A., Charriaut-Marlangue, C. & Ben-Ari, Y.
Transient increased density of NMDA binding sites in the developing rat hippocampus. Brain
research 461, 393-396 (1988).
143. Danysz, W. & Parsons, C.G. Glycine and N-methyl-D-aspartate receptors: physiological
significance and possible therapeutic applications. Pharmacological reviews 50, 597-664 (1998).
144. Riikonen, R.S., Kero, P.O. & Simell, O.G. Excitatory amino acids in cerebrospinal fluid
in neonatal asphyxia. Pediatric neurology 8, 37-40 (1992).
145. Holmes, G.L. The long-term effects of seizures on the developing brain: clinical and
laboratory issues. Brain & development 13, 393-409 (1991).
146. Holmes, G.L. & Ben-Ari, Y. The neurobiology and consequences of epilepsy in the
developing brain. Pediatric research 49, 320-325 (2001).
147. Beurel, E., Grieco, S.F. & Jope, R.S. Glycogen synthase kinase-3 (GSK3): regulation,
actions, and diseases. Pharmacology & therapeutics 148, 114-131 (2015).
148. Jope, R.S., Yuskaitis, C.J. & Beurel, E. Glycogen synthase kinase-3 (GSK3):
inflammation, diseases, and therapeutics. Neurochemical research 32, 577-595 (2007).
149. Shim, S.S. & Stutzmann, G.E. Inhibition of Glycogen Synthase Kinase-3: An Emerging
Target in the Treatment of Traumatic Brain Injury. Journal of neurotrauma (2016).
76
150. Nadler, J.J., et al. Automated apparatus for quantitation of social approach behaviors in
mice. Genes, brain, and behavior 3, 303-314 (2004).
151. Website: © 2015 Allen Institute for Brain Science. Allen Developing Mouse Brain Atlas
[Internet]. Available from: http://developingmouse.brain-map.org. 2017.