Stroke Study: Novel Animal Models and Innovative Treatment Strategy
A dissertation presented to
the faculty of
the College of Arts and Sciences of Ohio University
In partial fulfillment
of the requirements for the degree
Doctor of Philosophy
Xinge Yu
April 2016
© 2016 Xinge Yu. All Rights Reserved. 2
This dissertation titled
Stroke Study: Novel Animal Models and Innovative Treatment Strategy
by
XINGE YU
has been approved for
the Department of Biological Sciences
and the College of Arts and Sciences by
Yang V. Li
Professor of Neuroscience
Robert Frank
Dean, College of Arts and Sciences 3
ABSTRACT
YU, XINGE, Ph.D., April 2016, Biological Sciences
Stroke Study: Novel Animal Models and Innovative Treatment Strategy
Director of Dissertation: Yang V. Li
As one of the leading causes of death and long-term disability, stroke brings
detrimental burden to its patients and the society. The most common subtype of stroke is
ischemic stroke. Current treatment for ischemic stroke is limited. The only FDA
approved drug is tissue plasminogen activator (tPA). The application of tPA is restricted
within 4.5 hours of stroke onset, due to its severe side effects and broad
contraindications. The lethal side effect of tPA is hemorrhage. The usage of tPA and its
therapeutic effect has to be compromised to avoid hemorrhage. Studies have been
proposing that Zn2+ accumulation has deleterious effect in ischemic neuronal damage, and reducing intracellular Zn2+ overload by Zn2+ chelation improve neuronal survival from cerebral ischemia.
In this study, the therapeutic effect of Zn2+ chelation is investigated not only in
mice, but also in a newly developed zebrafish model. Severe hypoxia was performed to
induce a hypoxic-ischemic brain injury on zebrafish, to mimic the global cerebral stroke
in a cardiac arrest. A self-designed chamber was used to perform stable and effective
hypoxia. The brain injury was quantified by histological staining of the brain, and the
overall survival and behavioral changes of zebrafish after hypoxia. Photothrombotic
method was adopted and modified to develop adult zebrafish as a model for focal
thrombotic stroke. Rose Bengal was intraperitoneally injected to the zebrafish and a light 4
probe was placed on the optic tectum of the brain. Brain damage was quantified by 2,3,5-
Triphenyltetrazolium chloride (TTC) staining and overall recovery. Treatment of tPA
was used to confirm thrombosis-induced brain injuries and the feasibility of using
zebrafish model to screen thrombolytic candidates. In vitro experiment,
spectrophotometry was used to quantify blood clot-lysis (thrombolysis). Thrombosis
model in vivo was achieved by photothrombotic method on the mouse femoral artery.
The artery reperfusion induced by tPA was monitored under microscope and quantified
by ImagePro.
Results of this study show that adult zebrafish are sensitive to hypoxia. Detectable
brain injury can be achieved by 8 minutes of severe hypoxia. Longer time of hypoxic
treatment leads to moving disability and mortality of zebrafish. Zn2+ chelation improves neuronal viability under oxygen deprivation, and therefore increases the behavioral recovery and overall survival rate. During thrombolysis, the blood clot releases high concentration of Zn2+. Data suggest that increase of Zn2+ around the thrombosis reduces tPA-induced thrombolysis. Zn2+ chelation, when combined with tPA, increase the overall
thrombolysis in vitro and the rate of successful reperfusion in vivo. Moreover, the
application of Zn2+ chelation improves efficacy and potency of tPA-induced
thrombolysis.
In conclusion, Zn2+ chelation has shown promoting effect in neuronal rescue, and also in assisting thrombolysis during thrombotic stroke. It can be a treatment combining with tPA to improve successful reperfusion and reduces tPA-induced cytotoxicity. Taken 5
together, Zn2+ chelation is promising to be used to improve outcomes of stroke and cardiovascular occlusion.
6
ACKNOWLEDGMENTS
I would like to express my sincere thanks to my advisor Dr. Yang Li for guiding me through my graduate study, for being wise, patient and encouraging, and for showing me the beautiful art of science. I have learned countless valuable lessons from Dr. Li, which not only guides my scientific study, but also benefits my life.
I would like to thank my lab mates Dr. Christian Stork, Dr. Chinthasagar Bastian,
Kira Slepchenko, Zihui Wang, Qiping Lu, Hariprakash Haragopal, and Dr. Zhijun Shen for their assistance, their instructive discussions, and friendship through the ups and downs of research.
I would also like to thank members of my advisory committee, Dr. Robert Colvin,
Dr. Gary Cordingley and Dr. Daewoo Lee for their guidance and advices throughout the completion of this project.
7
TABLE OF CONTENTS
Page
Abstract ...... 3 Acknowledgments ...... 6 List of Figures ...... 11 List of Abbreviations ...... 14 1. Introduction ...... 16 1.1 Cerebral ischemia ...... 17 1.1.1 Classification ...... 18 1.1.2 Mechanisms of the cerebral ischemia ...... 19 1.1.3 Treatment of cerebral ischemia ...... 21 1.2 Models for cerebral ischemia ...... 24 1.2.1 Models for global and focal cerebral ischemia ...... 25 1.2.2 Limitations of current models ...... 26 1.2.3 Zebrafish as an alternative model for cerebral ischemia...... 29 1.2.3.1 Zebrafish biology ...... 29 1.2.3.2 Zebrafish as a research model ...... 29 1.2.3.3 Zebrafish as a cerebral ischemia model ...... 31 1.3 Dyshomeostasis of Zn2+ in cerebral ischemia ...... 33 1.3.1 Zn2+ in cellular biology ...... 33 1.3.2 Zn2+ induced toxicity ...... 34 1.3.3 Zn2+ in cerebral ischemia ...... 36 1.3.4.1 Blood clot formation and clot-lysis...... 37 1.3.4.2 Formation of thrombosis ...... 38 1.3.4.3 Involvement of Zn2+ in thrombosis and thrombolysis ...... 39 1.4 Conclusions of the introduction ...... 42 2. Specific Aims ...... 44 3. Materials and Methods ...... 48 3.1 Animals ...... 48 3.2 Specimen ...... 48 8
3.3 Hypoxia chamber and global hypoxic treatment ...... 49 3.4 Photothrombotic treatment ...... 50 3.5 Brain slices preparations ...... 51 3.6 Cresyl violet staining ...... 52 3.7 2,3,5-triphenyltetrazolium chloride (TTC) staining and quantification ...... 52 3.8 Quantification of TTC staining ...... 53 3.9 Intracellular Zn2+ measurements ...... 53 3.10 In vitro study of blood clot-lysis ...... 54 3.11 Custom-modified cuvettes for absorbance measurement ...... 54 3.12 Preparation of fibrin clots ...... 56 3.13 Western blot assay for the determination of the level of tPA-induced fibrinolysis ...... 57 3.14 Femoral artery occlusion and reperfusion ...... 58 3.15 Images acquisition and analysis of thrombolysis ...... 59 3.16 Chemicals delivery ...... 59 3.17 Chemicals and reagents ...... 59 4. Results ...... 61 4.1 Developing zebrafish as a novel model of cerebral ischemia and thrombotic stroke...... 61 4.1.1 Zebrafish as a model for global hypoxic ischemia ...... 61 4.1.1.1 Treatment of hypoxia and the set-up of the end point of hypoxic treatment ...... 61 4.1.1.2 Abnormal behavior during the recovery from hypoxic treatment ...... 65 4.1.1.3 Histological staining and quantification of brain damage after hypoxic treatment...... 66 4.1.2 Zebrafish as a model of thrombotic stroke ...... 70 4.1.2.1 Zebrafish exhibited abnormal behavior after photothrombotic treatment. 70 4.1.2.2 Photothrombotic treatment causes quantitative brain damage...... 72 4.1.2.3 Photothrombosis induced cerebral damage is responsive to tPA treatment...... 75 4.2 The neuroprotective role of Zn2+ chelation in oxygen-deprived brain injuries in vivo...... 79 4.2.1 DEDTC reduces mortality rate of zebrafish after hypoxia...... 79 9
4.2.2 DEDTC enhanced the hypoxia tolerance in zebrafish...... 82 4.2.3 DEDTC preserves brain viability from hypoxic attack...... 83 4.3 The promoting effect of Zn2+ chelation in thrombolysis in vitro...... 86 4.3.1 Measurement of blood clot-lysis in vitro...... 86 4.3.2 Thrombolysis in vitro is measured by spectrophotometry...... 89 4.3.3 Inhibiting effect of zinc and iron on tPA-induced thrombolysis in vitro...... 98 4.3.4 Effect of ion chelation on tPA-induced thrombolysis in vitro...... 112 4.3.5 Effect of Zn2+ chelation on tPA-induced fibrinolysis in vitro...... 114 4.4 Effect of ion chelation on tPA-induced thrombolysis in vivo...... 115 5. Discussion ...... 124 5.1 The zebrafish is developed as a model of global cerebral ischemia...... 124 5.2 Advantages of using zebrafish as an ischemia model ...... 127 5.3 Advantages of adult zebrafish being a thrombotic stroke model...... 129 5.4 The in vivo neuroprotective effect of Zn2+ chelation in oxygen deprivation...... 131 5.5 Spectrophotometric measurement of thrombolysis...... 133 5.6 Zn2+ in blood dynamic and its inhibiting effect in tPA-induced thrombolysis. .... 135 5.7 Iron in blood dynamic and thrombolysis...... 138 5.8 Metal ion chelation with EDTA and CaEDTA...... 139 5.9 Summary of the study ...... 142 References ...... 144 10
LIST OF TABLES Page
Table 1: Compounds that have failed recently in clinical evaluation for the treatment of acute ischemic stroke…………………………………………………………………….23
Table 2: The effect of hypoxia on zebrafish……………………………………………..64
Table 3: Behavioral changes of zebrafish after light exposure, or Rose Bengal treatment…………………………………………………………………………………70
Table 4: The behavioral changes of zebrafish in recovery after photothrombotic treatment…………………………………………………………………………………72
Table 5: The recovery rate between the tPA treated group and the non-tPA treated group after photothrombosis……………………………………………………………………78
Table 6: The recovery rate from oxygen deprivation……………………………………82 11
LIST OF FIGURES
Page
Figure 1: Hypoxia Chamber……………………………………………………………..50
Figure 2: Schematic diagram of the custom-designed hypoxia chamber………………..51
Figure 3: Schematic diagram of the custom-modified cuvette to describe the set-up of blood clot-lysis (thrombolysis) for spectrophotometric measurement…………………..55
Figure 4: Fibrin clot forms after centrifuge……………………………………………...57
Figure 5: Percentage of zebrafish death after hypoxic treatment………………………..62
Figure 6: The analysis of zebrafish hypoxia time distribution…………………………..64
Figure 7: Swimming capability of zebrafish during recovery…………………………...66
Figure 8: Cresyl violet staining of optic lobe……………………………………………67
Figure 9: Quantification of brain viability……………………………………………….69
Figure 10: Brain damage of zebrafish after photothrombotic treatment…………………74
Figure 11: The evaluation of the brain damage after photothrombotic treatment……….77
Figure 12: The outcomes of photothrombotic treatment between the tPA treated group and the non-tPA treated group…………………………………………………………...78
Figure 13: The images are slices from the optic lobe of the zebrafish brain stained with a fluorescent Zn2+ indicator, Newport Green……………………………………………...80
Figure 14: DEDTC increased the survival rates…………………………………………81
Figure 15: DEDTC enhanced hypoxia tolerance in zebrafish…………………………...83
Figure 16: Zebrafish brain injury detected by TTC staining………………………….....85
Figure 17: The effect of serum in tPA-induced clot-lysis in vitro…………………….....87 12
Figure 18: In vitro blood clot-lysis (in percentage) mediated by different concentrations
of tPA treatment……………………………………………………………………...... 88
Figure 19: The effect of TPEN in tPA-induced blood clot-lysis………………………...89
Figure 20: The set-up of blood clot-lysis (thrombolysis) for spectrophotometric
measurement……………………………………………………………………………..91
Figure 21: The measurement of tPA-induced in vitro thrombolysis over time……….....93
Figure 22: In vitro thrombolysis at different doses of tPA………………………………95
Figure 23: The reaction rate of tPA-induced thrombolysis at different doses of tPA in vitro………………………………………………………………………………………97
Figure 24: Zinc inhibited tPA-induced thrombolysis……………………………………99
Figure 25: Fe3+ inhibited tPA-induced thrombolysis…………………………………...101
Figure 26: The inhibiting effect of Fe2+ on tPA-induced thrombolysis………………...103
Figure 27: The maximal inhibiting effect of different ions on the overall amount of
thrombolysis…………………………………………………………………………….104
Figure 28: The rate of thrombolytic activity in different treatments of Zn2+…………...106
Figure 29: The rate of thrombolytic activity in different treatments of Fe3+…………...108
Figure 30: The rate of thrombolytic activity in different treatments of Fe2+…………...110
Figure 31: Time taken to reach the maximum rate of tPA-induced thrombolysis……...111
Figure 32: EDTA, a chelating agent, facilitated tPA-induced thrombolysis…………...113
Figure 33: Western blots of tPA-induced fibrinolysis………………………………….115
Figure 34: Photothrombosis and thrombolysis in vivo…………………………………117
Figure 35: Chelating agent CaEDTA facilitated tPA-induced thrombolysis in vivo…...119 13
Figure 36: Real-time thrombolysis in vivo..………………………………………………...121
Figure 37: Change of light transmission of thrombosis in tPA group and (1/2 tPA +
CaEDTA) group………………………………………………………………………...123
Figure 38: Zn2+ as an inhibitor of blood clot-lysis……………………………………...138
14
LIST OF ABBREVIATIONS
Abs: absorbance
ACSF: artificial cerebrospinal fluid
CaEDTA: ethylenediaminetetraacetic acid calcium disodium salt
DCF: dichlorofluorescein dMCAo: distal middle cerebral artery occlusion
DO: dissolved oxygen
EDTA: ethylenediaminetetraacetic acid
IP: intraperitoneal injection
MCAo: middle cerebral artery occlusion
MPT: mitochondria permeability transition mPTP: mitochondria permeability transition pore
MS-222: Ethyl 3-aminobenzoate methanesulfonate
NG: Newport Green
NMDA receptor: N-methyl-D-aspartate receptor pMCAo: proximal middle cerebral artery occlusion
ROS: reactive oxygen species
SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis
TBST: Tris-buffered saline with Tween
TIA: transient ischemic attack tPA: tissue plasminogen activator
TPEN: N, N, N', N', tetrakis (2-pyridylmethyl) etylenediaminepentaethylene 15
TTC: 2,3,5-triphenyltetrazolium chloride
uPA: urokinase plasminogen activator
2VO: two-vessel occlusion method
4VO: four-vessel occlusion method
16
1. INTRODUCTION
Ischemic stroke is rapid loss of brain function because of the interruption of blood flow and subsequently brain damage from oxygen-glucose deprivation due to thrombosis or embolism.1-3 It can cause permanent neurological damage including moving and
speaking disability, seizures, and brain death. Half of Americans have at least one of
these risk factors of stroke. They include old age, obesity, high blood pressure, previous
stroke or transient ischemic attack (TIA), high cholesterol, smoking, diabetes, atrial
fibrillation and heart diseases2. CDC reports that stroke affects more than 795,000 every year, 87% of which is ischemic stroke. The only drug approved by FDA for treatment of acute thrombotic stroke so far is tissue plasminogen activator (tPA).4, 5 The therapeutic
effect of tPA is to degrade the blood clot and reperfuse the occluded artery, which is
critical for saving neurons under stroke. Unfortunately, only 1-3% of stroke patients in
the US receive this therapy because of the narrow timing window, severe side effects.6
For these reasons, stroke is still one of the leading causes of death and serious long-term
disability in United States7. It has been a big burden to individuals, the society, and national health care.8
Safer and efficient therapies are highly needed to increase the survival rate and
quality of surviving, which means, the mechanisms of the ischemic brain damage has to
be revealed. A large amount of evidence has shown that the ischemia-induced cell death
are involved in both necrosis and apoptosis. Major contributors include Ca2+ overload, increase of reactive oxygenate species (ROS), and mitochondria depolarization. There is also growing body of evidence showing a detrimental role of Zn2+ overload during 17
ischemia. Intracellular Ca2+ overload is found to be proceeded by Zn2+ accumulation
during ischemia. Reducing Zn2+ overload has been reported to be inhibiting Ca2+
increase, mitochondria depolarization, and production of ROS. In blood dynamics, Zn2+ triggers platelet activation and aggregation, and enhances blood coagulation. Zn2+ is also found to interact with plasmin(ogen), and some thrombolytic agents such as tPA and urokinase plasminogen activator (uPA)9. The effect of Zn2+ on thrombolytic agents is not
conclusive. However, some recent studies report that Zn2+ attenuates tPA activities.
Therefore, Zn2+ may play a detrimental role in thrombolysis therapy.
For all the reasons above, reducing Zn2+ is proposed be a good strategy in stroke treatment. Zn2+ chelation is hypothesized to be able to protect neurons from ischemic
stress, as well as promote blood clot degradation during thrombolysis.
In the following parts of the introduction, three major topics will be looked
through: A.) the classification, mechanisms and current therapies of cerebral ischemia,
B.) a brief introduction of current cerebral ischemia models, and the advantages of using
zebrafish as a model system, and C.) roles of Zn2+ in physiology and stroke pathology, as well as Zn2+ chelation being a therapeutic candidate for stroke.
1.1 Cerebral ischemia
Cerebral ischemia is the fourth leading cause of death in the United States and one of the most common diseases leading to long-term disability7. The brain is about 2% of
the total body mass, yet 15-20% of blood flow travels from the heart to the brain and the
brain accounts for 20% of total oxygen consumption. Due to its high metabolic demand,
brain cells are extremely sensitive to oxygen deprivation. The neuronal death initiates 18
within five minutes after oxygen supply has been cut off. Symptoms of cerebral ischemia
include dysfunction in vision, weakness of the body, problems with movements and
coordination, impairment of speaking, and unconsciousness. Cerebral ischemia can be
caused by thrombosis, embolism, or systemic hypofusion7.
1.1.1 Classification
Several different classification methods of the cerebral ischemia are widely used
in clinics and research based on different aspects, such as causes, locations, territories and
clinical symptoms7. According to the damage regions, cerebral ischemia is
subcategorized into focal cerebral ischemia and global cerebral ischemia.
Focal cerebral ischemia is characterized by a reduction of cerebral blood flow in a
distinct region of the brain due to an acute occlusion of cerebral vessels10. Both thrombosis and embolism can cause focal brain ischemia. Thrombotic ischemia is damage of brain cells due to starvation when the blood clot forms locally and blocks the blood perfusion. Embolismic ischemia is caused by the occlusion of a clot detaching from the heart or major arteries and getting caught in a narrow artery. The signs and symptoms of a stroke depend on which region of the brain is affected and how severe of the ischemia.
Global cerebral ischemia is the transient or permanent oxygen-glucose deprivation caused by the interruption of the blood flow being followed by brain hypofusion, which commonly results from cardiac arrest. If therapeutic intervention cannot be performed in time, such as cardiopulmonary resuscitation in the cases of cardiac arrest, global cerebral ischemia can quickly lead to coma and brain death. Even 19
when the cardiopulmonary resuscitation is successfully performed, prolonged ischemia of
the brain may yield instant and/or delayed brain damage11, 12. Among the patients of the cardiac arrest who restored circulation by successful cardiopulmonary resuscitation, 60% of them subsequently die of severe brain damage. Less than 10% of the resuscitated patients recovered back to their normal condition13, 14. Under those circumstances,
revealing the molecular mechanisms of the cell damage, as well as developing effective
neuroprotective candidates are critical for improving clinical treatment.
1.1.2 Mechanisms of the cerebral ischemia
According to the literatures, necrosis and apoptosis have been proved to be both
involved in ischemic injury in neurons. Although both leading to cell death, necrosis and
apoptosis are morphologically and mechanically different from each other. Necrosis is a
unregulated cell death, which is morphologically characterized by (1) swelling of the cell
and organelles, (2) the breaking down of the organelles and plasma membrane, (3)
disintegration of the nucleus, and (4) the discharge of cell contents to extracellular
space15-19. Apoptosis, on the other hand is a programed cell death, which can be
intervened. It is characterized by (1) compaction and margination of the nuclear
chromatin, (2) cytoplasmic shrinkage and nucleus condensation, and (3) formation of the
membrane-bound cell fragments called apoptotic bodies20-24. Apoptotic bodies, in normal
condition are phagocytized by macrophages, and therefore, cell contents from the
apoptosis do not enter extracellular space19.
On molecular level, apoptosis is also characterized by “programmed cell death”,
which is associated with depolarization of the plasma membrane, activation of apoptosis- 20
specific proteases such as caspases, and DNA degradation. Despite the morphologic and
molecular distinctions, necrosis and apoptosis may share some molecular events, such as
the overload of intracellular Ca2+, increase of reactive oxygen species, mitochondria
permeability transition (MPT) and the depolarization of mitochondria19, 25-27. As necrosis
occurs prominently in core areas, and apoptosis occurs in adjacent areas, potential
interventions are targeting apoptotic molecular pathways in hoping to save or reduce
brain damage28.
The disruption of Ca2+ homeostasis leading to the overload of intracellular Ca2+ is
well accepted to play a causal role in neuronal cell death during cerebral ischemia and
reperfusion29. Intracellular high concentration of Ca2+ triggers lipolysis, proteolysis, and
DNA degradation30, 31. The overload of cytosolic Ca2+ also triggers the production of
nitric oxide and the kinase/phosphatase activation32-36. Nitric oxide introduces chromatin condensation and caspase-like activity37-39. Kinase/phosphatase activation signals for function of apoptosis-associated proteins and the gene expression19. Ca2+ influxes into
mitochondria and causes increased free radical production and the latter one triggers
MPT40-42.
Free radicals are chemicals species that contain unpaired electrons that are highly
reactive. Free radicals can damage DNA, proteins, and lipids43, 44. Oxygen free radicals, also known as reactive oxygen species (ROS), are produced by mitochondria under oxidative stress. Toxicity from free radicals is normally monitored and prevented by endogenous protective mechanisms such as antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase) and free radical scavengers45. However, in the 21
process of ischemic cell damage, ROS increase rapidly and overcome the degradation
ability of the cells and result in severe damage of the cell by breaking down DNA and
proteins, as well as triggering MPT19, 46-51.
MPT, the mitochondria permeability transition is an increase of permeability in
mitochondria caused by the opening of the permeability transition pore (PT pore). The PT
pore is an ion channel located in the inner membrane of mitochondria52. Growing literatures support that mitochondria play a key role in both necrotic and apoptotic cell death53-57. The major event on mitochondria, which is leaded by several cell damage pathway during ischemia, is MPT58, 59. The opening of the PT pore channel can be triggered by ATP depletion, intracellular Ca2+ overload and free radicals, and MPT causes the swelling of mitochondria, the oxidative phosphorylation, increased production of superoxidase, and release of intermembrane space proteins (cytochrome c and apoptosis inducing factor (AIF)) that activate apoptotic effector caspases19, 60, 61.
1.1.3 Treatment of cerebral ischemia
Early and adequate medical intervention is critical for improving stroke outcomes.
Potential treatments for improving neuronal survival have been under intensive studies
for decades. So far, the only FDA approved drug for the treatment of acute thrombotic
stroke is tissue plasminogen activator (tPA)5. The use of tPA in thrombolysis have been heavily studied since 1980s62-66. By binding with tPA, plasminogen is cleaved and
converted to plasmin. Plasmin degrades the fibrin bundles into soluble fibrin pieces
(degradation fibrin products), and fulfills thrombolysis67. 22
Currently, according to National Institutes of Health (NIH), only 1-3% of stroke
patients in the US receive this therapy because of the narrow timing window, severe side
effects and broad contraindication6, 68. The most lethal side effect of tPA is cerebral
hemorrhage, which is tPA dose-dependent69, 55. Furthermore, the application of tPA
during stroke increases permeability of blood-brain barrier, thereby causes brain edema.57
Additionally cytotoxicity of tPA contributes to neuronal death.60,61 For these reasons, the dose of tPA has to be tightly controlled, while the effect of thrombolysis may be compromised. Therefore, reducing side effects and toxicity of tPA is an effective route to improve the therapeutic outcomes of stroke. Studies have been focused on improving efficacy of tPA for higher rate of reperfusion,70 and increasing potency (reduce the necessary dose) of tPA for enhancement of safety.
Another promising treatment under intense investigation is neuroprotectants, which is a group of chemicals that has anti-necrosis or anti-apoptosis features. Even though necrosis and apoptosis are morphologically and molecularly different, it is well accepted that they are both involved in ischemic cell death and they share common
(upstream) molecular pathways, such as the disruption of Ca2+ homeostasis, excess free
radicals and the dysfunction of mitochondria results from permeability transition are
major contributors to ischemic cell injury. As necrosis occurs prominently in core areas
and apoptosis in adjacent areas, potential interventions targeting apoptotic molecular
pathways in hoping to save or reduce brain damage71. Potential treatment includes the
calcium channel blocker, glutamate antagonists, NMDA channel blockers, free radical
scavengers as well as hypothermia. Treatments have been proposed to be neuroprotective 23
by targeting proteins such as calpains and caspases in the cell death cascade. However
several chemicals have recently been proved efficacy in animal models but have failed in
clinical trials. The following table is adopted from the previous study, showing several
agents, which are targeting the NMDA receptor, Ca2+ channel blocker and cell membrane
stabilizer, are less efficient in clinical trials72.
Table 1: Compounds that have failed recently in clinical evaluation for the treatment of acute ischemic stroke
Compound Mechanism of action Outcome (clinical
phase)
Selfotel NMDA receptor antagonist Adverse events
Gavestinel Antagonist at the glycine site of the Lack of efficacy
NMDA receptor
Citicoline Cell-membrane stabilizer Lack of efficacy
Ca2+ antagonists Ca2+ channel antagonists Lack of efficacy
Aptiganel NMDA receptor antagonist Lack of efficacy
One of possible reasons is that we are still lack of understanding the mechanism
of brain damages caused in stroke. Also, It seems reasonable to assume that drugs that
work on a specific biochemical mechanism must be given at the time that the mechanism
is active, which makes the window of opportunity too narrow for clinical administration.
Additionally, earlier studies in animal models relied on histological evaluation and placed 24 little emphasis on functional outcome measures. However, clinical outcome is almost invariably determined functionally, primarily by measures of motor ability8. Another possible reason is that animal models failed to predict clinical efficacy, optimal doses and toxicity, because some compounds that clinically important may have adverse events that limit the possibility of achieving dose levels that have maximal neuroprotective effects in animals73. Therefore, it is important to use reliable animal models in high throughput studies.
1.2 Models for cerebral ischemia
Since the late 1970s, animal models of cerebral ischemia has been developed with the aim of studying mechanisms that cause tissue damage and to provide the basis for the development of new therapies for stroke. Nowadays, reliable animal models for stroke are available in a variety of species including primates and lower animals such as
Mongolian gerbils, rabbits, rats and mice. Although the using and developing primates for stroke models is valuable, small animals actually have a privilege at preclinical studies.
Using small animals in biomedical research is well accepted. For example, rates and mice are both well-suited for ischemic strokes. Physiological variables can be monitored, and sufficient numbers for statistical analysis can be reached without excessive costs74. In stroke studies the rat is the most commonly used animal because that the size of rats allows easy monitoring of the physiologic variables and handling of vascular structures. The mouse is the best-characterized animal in genetics and molecular 25
biology. Many transgenic animals are available in this species. An increasing number of
stroke studies are carried out in mice from the 1990s onwards10.
1.2.1 Models for global and focal cerebral ischemia
The idea to make global ischemia on rodents and mammals is to occlude the major supply arteries of the brain. Global ischemia can be induced by means of different approaches. The four-vessel occlusion method (4VO) is to occlude the two vertebral arteries accompanied with brief bilateral common carotid artery occlusion75-79. Another
method is the two-vessel occlusion method (2VO) is to occlude the two common carotid
arteries together with systematic hypotension10, 80-83. Gerbils are widely used as a global ischemia model due to the incomplete circle of Willis. However, blood supply still manages to get the brain in those methods mentioned above.
Stroke caused by an acute cerebral vessel occlusion can be reproduced by different techniques. They are named by mechanical occlusion of either the proximal middle cerebral artery (pMCAo) (large vessel occlusion) or distal MCA (dMCAo) (small vessel occlusion), or by thrombotic occlusion either via injection of blood clots or thrombin into the MCA, or by photothrombosis after intravenous injection of Rose
Bengal10. Models of pMCAo belong to the most frequently used procedures in stroke
research, which is usually induced by direct mechanical occlusion84-88. The mechanical occlusion is most often achieved by the insertion of a silicon-coated nylon suture into the internal carotid artery and subsequently towards the circle of Willis to occlude the
MCA89-94. The severity of ischemic injury can be modeled by leaving the suture filament in place either transiently for a variable duration of time. The suture is then removed to 26
allow tissue reperfusion. In cases of permanent pMCAo, the suture is left in place and no
reperfusion is allowed95.
As the more commonly used models (suture model and distal middle cerebral
artery occlusion) are often technically challenging, using simpler models to induce a
focal ischemic lesion have been developed as an alternative. One of these methods is
photothrombosis which is induced by the trans-cranial illumination of the brain after the
systemic delivery of a photosensitive dye (Rose Bengal)96. Rose Bengal releases singlet oxygen to break the endothelial cells of the blood vessel under light exposure, and triggers the coagulation pathway in the location of the irradiated tissue. This model has the advantage that the region of ischemia can be predefined and highly circumscribed, allowing the possibility to coagulate distinct cortical areas with stereotactical precision97.
1.2.2 Limitations of current models
Gerbils are widely used as a global ischemia model due to the incomplete circle of
Willis. However they might present various problems when used in models for global
cerebral ischemia, as many gerbils do not have a totally incomplete circle of Willis, i.e.
small communicating arteries give collateral supply to the forebrain97. Lately, growing
number of studies use mild brain insults to study brain cell death and test neuroprotective
agents. One disadvantage of this idea is that the findings may not be translated to cardiac
arrest-introduced damage because the latter one is much more extensive98.
The most widely used model for focal brain ischemia is the middle cerebral artery
occlusion (MCAo) on rats and mice. It has several limitations. 27
(1) The surgery of MCAo requires tying off the external carotid artery temporarily or
permanently depending on the experimental design74. This process does not
happen in physiological condition of strokes. Thus, it cannot be a model for study
of thrombolysis, which is still the only effective therapy for brain stroke.
(2) Infarct volume varies largely between different laboratories even though with the
same reported duration of occlusion and survival time. The range is from 5% to
55% of the ipsilateral hemisphere. Under those circumstances, evaluation of
therapeutic methods or neuroprotective agents becomes even harder74.
(3) It produces the stroke volume larger than the real cases receiving medical care in
the hospital. In most of the clinical cases, the stroke size is fairly small, ranging
from 3.25% to 13% of the ipsilateral hemisphere. According to a study by
Carmicheal in 2005, most of the rodent MCAo models have infarct size larger
than15% of the ipsilateral hemisphere, which has rarely happened in human
stroke except for the malignant infarction74.
(4) Evaluating the behavior changes and the recovery after different therapeutic
treatments in the MCAo models is difficult because the occlusion causes
widespread damage of motor, sensory and cognitive functions74.
(5) The surgery requires invasive procedures and skillful techniques, either of which
may lead to variable results among different laboratories.
The Photothrombosis model, which is lack of those limitations of the MCAo method, is to induce localized stroke by injection a photosensitive dye (Rose Bengal)10.
Rose Bengal is a photo-excited dye. When irradiated, it forms singlet oxygen which 28
breaks down the cell connection and damage vessels endothelium by which triggers the
clotting cascade and vessel obstruction96, 99. Studies have shown that, photothrombotic
treatment induces irradiated neuronal damage from the blood vessel occlusion, as well as
subsequently ischemic injuries surrounding the occluded location100, 101. This method
produces detectable progressive neuronal damage such as inflammation filtration and
increase of casepase3102-105. Several advantages make the photothrombosis model
superior than the MCAo model for particular study purposes.
(1) The stroke location is predictable. Stroke on cortex can be induced by exposure of
cold light outside the skull106. Stroke of inner brain is manipulated by implanting
optic fiber with source of cold light into the location107. Through this method,
functionally distinctive region of the brain can be accurately chosen to study the
neuronal damage, as well as the functional loss108, 109.
(2) The damage is more localized and correlated to time and intensity of the light
exposure.
(3) The infarct volume is smaller than MCAo models, more closed to cases of the
non-malignant stroke74.
However, using rats or mice as photothrombosis models still have the problem of invasive procedures. Due to the thickness of the skull, surgeries are required for either removing or exposing part of the skull106, or injecting the optic fiber into the brain
tissue107.
In either focal or global cerebral ischemia models, attacks from invasive procedures and demanding techniques as well as the unstable effects of treatments are the 29
major limitations110. Although these limitations did not make those models less important and valuable, studies performing on alternative models are necessary to provide more objective results. The zebrafish can be one of the choices. It has been extensively explored as a research model these days using both embryos and adults. In the following part adult zebrafish will be proposed as a model for cerebral ischemia.
1.2.3 Zebrafish as an alternative model for cerebral ischemia
1.2.3.1 Zebrafish biology
The zebrafish (danio rerio) is a teleost fish living in freshwater. They develop fast from embryos to sexually mature individuals. Larval zebrafish have gained hunt and escape ability by five days post fertilization. Their reproductive system is completely mature by three months111, 112. The adult zebrafish reaches an average length of 4.5 cm,
has a slim compressed shape. Indistinct secondary sex characteristics occasionally make
it difficult to distinguish between immature males and females; however, in the adult, the
body contour of the gravid female is sufficiently different from the male to allow for easy
identification113. Males generally have larger anal fins while females have distinct genital
papilla114.
1.2.3.2 Zebrafish as a research model
The zebrafish has served as a useful study model for detecting environmental
hazards115. Lately, growing numbers of studies has been published using zebrafish as the research model113, 116-122. Because zebrafish absorb small molecules dissolved in the
surrounding water through their skin and gills, chemical treatment is easily manipulated.
In addition, comparing to testing in other animal models, a statistically significant 30
number of zebrafish can be used for each treatment condition and small amount of
chemicals is required for tested groups121. Therefore, zebrafish permit highly efficient phenotype analysis and rapid in vivo drug screening.
As an emerging research model, the zebrafish distinguishes itself from others by high throughput, which is extremely useful for not only drug-screening but also therapeutic study before clinical trials123. Zebrafish have well studied database, from
gene, organisms to behavior phenotypes124, 125. Many behavioral assays have been developed to quantify zebrafish behaviors. Studies have found that zebrafish exhibit circling behavior after optic nerve transection113, 126. Later, a more strong correlation between damage of the optic lobe and abnormal behaviors has been revealed, suggesting a premotor function besides the sensory function127. Some studies have developed a
behavior test to quantify the visually mediated escape response on adult zebrafish112, 128.
Also, zebrafish share a lot of similarity with mammalians genetically and functionally. In studies of neuroscience, zebrafish have been used as a model of
Alzheimer’s disease129 and anxiety130, 131. All of these make the zebrafish a simple model, yet with complexity, suitable to study human diseases and drug testing132.
Zebrafish as an experimental organism within biomedical research is already widely used
in several areas, including neuroscience120, 130, 133-136. The basic structure of the CNS in
fish has all the major domains found in mammalian brain, and the same neurotransmitters
such as GABA, glutamate, dopamine, noradrenaline, serotonin, histamine, and
acetylcholine are found in both interneuron systems and in long pathways137. Many of the 31
genes being involved in human neurodegenerative diseases have been identified in
zebrafish, and their expression patterns are known or easily studied.
For the past 20 years, the blood physiology of zebrafish has been intensively
studied. Zebrafish have erythrocytes, granulocytes and lymphocytes, being analogous to
those of human138, 139. The thrombocytes of zebrafish are analogous to platelets in human,
with nuclei. Those thrombocytes are activated during blood coagulation and going
through morphological changes during activation, similar to platelets139, 140. Zebrafish also share same coagulation cascades with human. Therefore, zebrafish has been used as a model to study hemostasis and thrombosis, especially in gene modification for targeting particular proteins139-148.
1.2.3.3 Zebrafish as a cerebral ischemia model
In this study, as a model of global ischemia, zebrafish have low tolerance to severe hypoxia (0.5-0.6 mg/L)149. According to preliminary data, zebrafish can only sustain severe hypoxia for 10-15 minutes. Therefore, severe hypoxia performs extensive insult to the brain, similar to which is caused by a cardiac arrest. Transient global hypoxia can be turned on and off by controlling the dissolved oxygen of the water. Previous studies on rodents and mammalians showed variable results due to the incomplete occlusion of the major arteries of the brain. In the study using zebrafish, transient global hypoxia can be simply achieved by oxygen depletion and reoxygenation in water. No invasive procedures are needed.
32
In this study, zebrafish will also be used as a photothrombosis stroke model. Several reasons make the zebrafish a superior model for focal ischemia than rats or mice.
(1) As a model of photothrombosis, zebrafish need no surgery, because of the thin
skull and half-transparent skin. This advantage contributes to a more stable data
set.
(2) The optic tectum (also known as optic lobe) of zebrafish communicates the
brain with the body150, and also is required by optomotor response while
swimming127. As a result, consistent abnormal swimming such as rotating and
circling can be observed after local ischemia of the optic tectum, which makes
zebrafish a reliable model for behavioral evaluation after brain damage. Delayed
behavioral changes are observed in zebrafish 24h to 48h after photothrombosis,
which makes the evaluation and measurements practical for delayed brain
damages and recovery.
(3) Changes of motions and balance are easier to be observed and evaluated on
fish than rats and mice. This is because, firstly, fish have three-dimensional
movements in water, which makes unbalanced movements such as rotating and
circling clear. On the other hand, rats and mice mostly have two-dimensional
movements, which makes measuring the abnormal behaviors after stroke harder
and less reproducible. Recently, neurobehaviors of zebrafish are well described
and even quantified151-153.
(4) Zebrafish have a large group of similarity to mammalians. For example,
zebrafish have been proved to have the same clotting factors and the clotting 33
pathways as in mammals154-157, which makes zebrafish a promising model for
thrombolysis.
(5) Applying chemicals on zebrafish is much easier than on mammalians.
Zebrafish can take water-dissolved chemicals through gills without injection
procedures. This advantage is especially attractive in drug testing when studying
different combinations of chemicals.
(6) Zebrafish models are good for high throughput studies. The genetic
modification of zebrafish took less time, with significantly large sample
number141. The low cost of husbandry is also a huge advantage for high
throughput studies123.
1.3 Dyshomeostasis of Zn2+ in cerebral ischemia
1.3.1 Zn2+ in cellular biology
Zn2+ is one of the most important cations for cells to maintain their normal
biological activity158-160. It is associated with a large number of essential proteins
including enzymes involving in signaling transduction pathways and in transcription.
Among the Zn2+-associated proteins, some of them are mainly extracellular
(metalloproteins and growth factors), whereas others are intracellular (a large variety of cytoplasmatic enzymes such as dehydrogenases, aldolases, peptidases and phosphatases) and even intranuclear (nuclear replication and transcriptional enzymes and transcriptional regulators)161, 162.
About 30 to 40% of the cellular Zn2+ is localized in the nucleus, 50% in the cell
plasma and cytosolic organelles and the rest is attached to membranes163. In the 34
intracellular environment, Zn2+ interacts with many regulatory enzymes and, similar to calcium, may act as a secondary messenger triggering/inhibiting important cellular processes, e.g., cell death and apoptosis164, 165. Zn2+ has been proved to play a key role on cell growth and differentiation by those observations of Zn2+-deficiency induced growth
retardation163, 166, 167.
Cell growth and differentiation are introduced by intracellular signaling transductions, including the binding of hormones to receptors, second messengers being triggered, cascades of protein kinases, protein phosphatases and finally leading to transcription factors binding to promoters of the genes164. Zn2+ is involved in all of those activities as a regulator or a structural component168. Zn2+ can also interfere with Ca2+
regulation169. Zn2+ can flux into the cell through the voltage dependent Ca2+ channel, as
well as the NMDA channel that modulates Ca2+. An increase of intracellular Zn2+ also
elevates the intracellular Ca2+ release by triggering the hormone sensitive intracellular
Ca2+ stores170, 171. Zn2+ homeostasis is tightly controlled since intracellular and
extracellular Zn2+ concentrations are considerably low comparing to other divalent.
Therefore, an excess of Zn2+ accumulation is a detrimental factor that is critical to the
neuron viability.
1.3.2 Zn2+ induced toxicity
Intracellular overload with Zn2+ is cytotoxic and can introduce neuronal damage
in apoptosis and necrosis by generating reactive oxygen species (ROS) and inhibiting
cellular energy production in mitochondria3, 165, 172. The overload of intracellular Zn2+ in brain ischemia promotes the increase of Ca2+ and they together contribute to the 35
depolarization of mitochondria membrane. A number of targets have been proposed,
including complexes of the mitochondrial electron transport chain, components of the
tricarboxylic acid cycle, and enzymes of glycolysis173-175. Consequences of cellular Zn2+ overload may include increased cellular ROS productions, loss of mitochondrial membrane potential, and further reduced cellular ATP levels176-180. Additionally, Zn2+ toxicity might be related to Zn2+ uptake by mitochondria and induction of mitochondrial permeability transition181, 182. By removing the excess Zn2+, damaged mitochondria recover faster after transient oxygen-glucose-deprivation169, 183.
Zn2+ overload appears to cause the dysfunction of mitochondria. Under normal
conditions, Zn2+ is taken by mitochondria through the activated cation permeable channel, the mitochondrial Ca2+ uniporter, and other unidentified routes184-186.
Mitochondrial Zn2+ uptake functions as organelle storage and may provide clearance of
cytosolic accumulation, especially in neurons undergoing excitotoxicity187. Excessive and
prolonged intramitochondrial Zn2+ overload can increase ROS production by inhibiting
the activity of complex III of the electron transport chain or by interfering with complex I
and α-ketoglutarate dehydrogenase 188. Zn2+ overload also contributes to the increase of
multiconductance cation channels activity in the inner mitochondrial membrane that is
consistent with the activation of the mitochondrial permeability transition pore
(mPTP)189. Consequences of mPTP opening are involved in further uptake of Zn2+, and
the release of pro-apoptotic mitochondrial proteins such as cytochrome C and apoptosis-
inducing factor (AIF). Therefore, mitochondria may be one of the early victims of
cytosolic Zn2+ accumulation186. After being disturbed by Zn2+ uptake, injured 36
mitochondria release Zn2+ that worsens Zn2+ dyshomeostasis, and ROS that triggers the pro-apoptotic signaling pathways, and results in neuronal death.
Zn2+ overload also triggers mitochondrial independent apoptotic signaling pathways, by activating NADPH oxidase through protein kinase C (PKC)190, and by increasing the activity of neuronal nitric oxide synthase (nNOS)181. The latter one is a key
enzyme for the production of NO and peroxynitrite (ONOO−). NO or NO-derived
molecules increasing intracellular Zn2+ from oxidation and nitrosylation of
metalloproteins has been directly demonstrated by studies in vitro and in vivo191, 192. Zn2+
also interferes with the glycolytic metabolism of neurons and inhibits glyceraldehyde-3-
phosphate dehydrogenase (GAPDH) through a reduction of cytosolic NAD+, leading to
ATP depletion and neuronal death172. Zn2+-induced cell death seems to occur through
poly-ADP-ribose polymerase (PARP) activation, which has been shown in other cases of
predominantly necrotic cell death193.
1.3.3 Zn2+ in cerebral ischemia
The mechanisms of cytotoxic of Zn2+ overload are also applied in cerebral
ischemia. Zn2+-induced ischemic brain damage started to draw attention in 1990s194-196.
Increasing studies have shown that during hypoxic-ischemic stress, the plasma membrane of the neurons depolarizes due to the ATP depletion197-203. Depolarization triggers the
opening of the voltage-gated Ca2+ channel. The depolarization of the plasma membrane also causes the release of the neurotransmitters that opens ligand-gated Ca2+ channels
such as NMDA receptors and AMPA receptors. Extracellular Zn2+ enters the neurons
through Zn2+ transporters (ZIP, ZnT) on the plasma membrane204-206. Zn2+ can also flux 37
into the cell through various ion channels including voltage-gated Ca2+ channels, Ca2+ permeable AMPA receptors, and NMDA receptors207-211. Na+–Ca2+ exchanger is another
route for Zn2+ exchange212. Neuronal Zn2+ uptake is also contributed by the Zinc/proton antiport213. Endogenous Zn2+ increase may come from endoplasmic reticulum (ER) and
other intracellular storage214, 215.
Zn2+ accumulation in mitochondria deteriorates the function of the mitochondria
by inhibiting the production of ATP and by increasing the activation of the mitochondria
permeability transition pores (mPTP)216. The activation of mPTP worsens the membrane
depolarization and the loss of the membrane integrity of the mitochondria. Zn2+ overload
also increases the production of ROS, pro-apoptotic factors such as cytochrome C and
caspase-3, which are the key factors for cell necrosis and apoptosis217-220.
1.3.4 Zn2+ in blood coagulation and thrombolysis
1.3.4.1 Blood clot formation and clot-lysis
The injury of endothelial cells in blood vessels triggers vasoconstriction and
platelet aggregation. The activated platelets are bridged to each other and form the
platelet plug on the site of the injury221. The platelet plug itself is not stable. For
reinforcement, fibrin bundles are formed and attach to the platelet plug, which traps more
blood cells and becomes a blood clot222. The formation of fibrin is activated through a series of cascade reactions. The cascade reaction requires FXa, which is the enzyme that induces the conversion of prothrombin to thrombin223. Thrombin attaches to the platelet
surface and cleaves fibrinogen to form fibrin. Fibrin monomers cross-link with Factor
XIII and form fibrin bundles, and together with the platelet plug forms a fibrin clot224. 38
The activation of Factor X (FXa) is achieved through two pathways, which are known as
the intrinsic pathway and extrinsic pathway. The intrinsic pathway is triggered by the
exposure of subendothelial matrix such as collagen. The intrinsic pathway initiates as
Kininogen and Kallikrein contacting the injury site of endothelium and inducing
activation of Factor XI225, 226. The extrinsic pathway starts as tissue factors releasing from subendothelial matrix and triggering the activation of Factor VII. Both intrinsic and extrinsic pathways eventually lead to the activation of Factor X, and initiate the fibrin formation cascade226.
On the other hand, the injured endothelial cells and subendothelial connective
tissue releases tissue plasminogen activator, which cleaves plasminogen in blood plasma
and converts it to plasmin. Plasmin degrades the fibrin clot into fibrin degradation
products, and removes the blood clot. In pathological condition, the continuous formation
of the blood clot outcompetes the degradation of fibrin by plasmin, and thus forms
thrombosis.
1.3.4.2 Formation of thrombosis
Main causes of thrombosis have been known as Virchow’s triad since 1856, which are
endothelial cell injury, hypercoagulability (change of blood constitution), and alternations
of blood flow227-229. Injuries of endothelial cells lead to the release of tissue factor from subendothelial matrix, which triggers activation of zymogens (coagulation factors) through intrinsic and extrinsic pathways. The activation of coagulation factors changes the blood constitution and makes the blood hypercoagulability. The alternation of 39
rheology followed by changes of shear force also makes the blood vessel more
susceptible for thrombosis230.
The arterial thrombosis, which is most commonly seen in thrombotic stroke, is majorly caused by rapture of atheroma. Atherosclerosis is initiated by the accumulation of low-density lipoproteins (LDLs), which eventually leads to dysfunction of endothelial cells of arteries67.
1.3.4.3 Involvement of Zn2+ in thrombosis and thrombolysis
The concentration of free Zn2+ in blood plasma is 12.2- 21.4 µM231-233. The majority of zinc is bound with albumin in a low affinity, with 0.5 µM free Zn2+ in blood
plasma234. There is more zinc in cell plasma and cytosolic organelles (mitochondria and endoplasmic reticulum)162, 165, 186, 235, 236. The most zinc-enriched site in bloodstream is the
α-granule of platelets, where the zinc concentration reaches 30-60 times more than that of in blood plasma233, and may reach to 500 µM231, 232.
The function of Zn2+ in blood coagulation was neglected in the studies in the past.
One reason is that Ca2+ has been long identified as a critical divalent ion in blood coagulation and shadows the importance of Zn2+, due to the higher concentration of Ca2+
in blood plasma. Additionally, blood samples are commonly treated by sodium citrate as
an anticoagulant for preparation of studies on platelet functions and blood coagulation.
Later, Ca2+ is routinely re-introduced to start blood coagulation or platelets aggregation.
However, sodium citrate chelates Zn2+ with higher affinity than Ca2+. Without reconstitution of Zn2+, the important role of Zn2+ tends to be overlooked9, 237.
40
Zn2+ plays critical roles in blood coagulation and platelets aggregation. For
example, Zn2+ binds and modulates FXII activation, which is the required for proper
function of Kininogen/Kallikrein contacting endothelium in the initiation of intrinsic
pathway238. Zn2+ amplifies FXII activation by at least 10-fold9, 239-242. Zn2+ has been
proved to promote collagen and ADP-induced platelets aggregation243, 244. Zn2+ also
strengthens fibrin bundles by promoting fibrinogen binding to platelet surface and
enhancing the binding of adhesive proteins to platelets 9, 245. Zn2+ binds to fibrinogen and
fibrin, which leads to the diameter of fibrin bundles being 10-time larger than that in
absence of Zn2+246, 247. By promoting fibrinogen binding to platelets, Zn2+ also amplifies platelets aggregation248. By enhancing Ca2+ influx towards platelets to modulate intracellular Ca2+ signaling, Zn2+ promotes platelets activation249-251.
During thrombolysis, a large amount of zinc releases from platelets in the blood clot and leads to locally high concentration of Zn2+. Zn2+ enhances fibrin bundles, making it more resistant to thrombolysis245. Zinc has been suggested to have inhibiting effect on
tPA activity252. Some studies have shown that zinc binds to tPA and prevents
plasminogen activation253. However, the effect of Zn2+ on thrombolysis is not conclusive, because very few studies performed involving fibrin, and the physiological complexity of the blood clot has not been investigated. In this study, the effect of zinc on thrombolysis will be tested on the whole blood clot, which is a more physiologically relevant model.
The in vivo investigation will also be performed to verify the results.
41
1.3.5 Application of Zn2+ chelators
Applying Zn2+ chelators to intervene Zn2+ accumulation has been investigated as a treatment for cerebral ischemia. Moderate Zn2+ chelators have been shown promising results254. The advantage for using these relative weak chelators is to maintain optimal range of Zn2+ to fulfill normal physiology function and avoid Zn2+ deficiency, while preventing the damage introduced by excess Zn2+ 187. Chelation of Zn2+ has been studied as an alternative or supportive therapy to prevent cell death3, 255-257. Membrane-permeable
Zn2+ chelators are developed and used to inhibit death of cultured neurons. High affinity
Zn2+ chelator, N, N, N', N', tetrakis (2-pyridylmethyl) etylenediaminepentaethylene
(TPEN), or the low affinity Zn2+ chelator, 1- hydroxypyridine-2-thione (pyrithione) have
been proved to be neuroprotective in vitro studies172.
At present, despite clear demonstration of numerous agents that can prevent the
cascade of events leading to ischemic neuronal death in animal models, it is not known
whether any particular neuroprotectant is more effective than others to conclusively
improve stroke outcome in humans14, 258. As discussed above, Zn2+ induced cell death has various serial and parallel events. Therefore, the effective strategy may be a combined treatment that targets multiple events including Zn2+ dyshomeostasis186. In this study,
Zn2+ chelation is applied not only for protecting neurons during ischemia, but also for
improving the efficacy of thrombolysis. Extracellular Zn2+ chelator CaEDTA can reduce
Zn2+ from extracellular environment, such as blood stream, which will be investigated in
thrombolysis. 42
In conclusion, Zn2+ is a key component for normal activities of a lot of functional proteins. It is also a very active second messenger that regulates the intracellular signaling pathways. Zn2+ induced cytotoxicity may cause the damage of plasma
membrane, increase permeability of mitochondria membrane, and increase the production
of free radicals. As well as Ca2+, Zn2+ is a key ion playing an important role in ischemic
brain damage. Zn2+ is also a key component in blood coagulation and the formation of
thrombosis. The degradation of blood clot release excessive Zn2+ and may further inhibiting blood clot degradation. For all the events that are Zn2+-related in thrombotic
stroke, the neuroprotective effect and thrombolytic effect of Zn2+ chelators together with other drug candidates should be vigorously tested in vitro and in vivo.
1.4 Conclusions of the introduction
Ischemic stroke affects a large population, and brings the heavy burden to health care and the society. Investigations are targeting different events during ischemic, in order to improve the outcome of stroke. Two major directions are involved in studies of ischemic stroke. One is to minimize neuronal damage during ischemia and reoxygenation. The other direction is to improve the effect of thrombolysis and artery reperfusion. Zn2+ is one of the factors that play important roles in both events. First, Zn2+
has been suggested to play a key role cytotoxic cell injuries. Under ischemia stress,
plasma membrane depolarization causes opening of the voltage-gated ion channels and
ligand-gated ion channels and leads to the influx of extracellular Zn2+. Zn2+ may also be
released from intracellular sources such as Endoplasmic Reticulum (ER), which may
contribute to the intracellular Zn2+ overload165, 235. Cytosol excessive Zn2+ is sequestered 43
by mitochondria and results in the depolarization and damage of the mitochondria. Zn2+ has also been shown to increase the production of free radicals that causes cell injuries.
Additionally, Zn2+ is critical in functional blood coagulation, platelets aggregation and
blood clots formation. Zn2+ also participates in pathological thrombosis formation and blood clot degradation. Zn2+ is packaged in high concentration in platelets granules and is released during platelets activation. The local high concentration of Zn2+ further promotes platelet aggregation and fibrin bundle formation, and therefore prevents clot degradation.
Zn2+ is also suggested to inhibit enzyme activities of thrombolytic agents such as tPA.
For these reason, I hypothesize that reducing excessive Zn2+ may not only benefit the neuronal survival under ischemia stress, but also promote thrombolysis and eventually improve recovery from thrombotic stroke. Although mechanisms of the neuronal death in cerebral ischemia have been intensively studied and relative neuroprotective chemicals are suggested to be effective on different cerebral ischemia models, clinical trials are currently not efficient. Strong and stable supports have to be provided by different research models before any chemical being moved to preclinical studies. Alternative models with good balance of complicity and simplicity, as well as similarity with human are required to answer detailed questions about mechanisms of cerebral ischemia, as well as testing candidate agents. For all these reasons, the zebrafish will be investigated as a new model of cerebral ischemia because of genetic advantages, high throughput, the similarities it shares with human on cerebral structures and constructions, and well established studies on the zebrafish brain and behaviors. In this study, the effect of Zn2+ chelation in stroke will be investigated in multi-fields in both mice and zebrafish models. 44
2. SPECIFIC AIMS
In spite of the growing understanding of mechanisms of brain damage in
thrombotic stroke, the clinical treatment has remained limited to thrombolysis, and its
administration is often compromised by narrow timing window and broad
contraindications. Current studies of stroke focus on preventing neuronal damage and
increasing efficacy and safety of thrombolytic treatment. One of examples is the
increasing awareness of detrimental role of Zn2+ overload during ischemia, and that the neuroprotective effect of reducing Zn2+ has been heavily investigated. However, it is still
difficult to translate bench research to clinical application. One of the reasons that
promising agents failed in clinical trials is due to inadequate basic research on optimal
doses and toxicity of the candidate. In this study, I develop new animal models and
investigate strategies of in vivo treatment for thrombotic stroke. The goal of this study is
to elucidate the relations between ion accumulation and stroke, including ischemic
neuronal damage and blood vessels reperfusion. The long-term goal of my research is to
propose new therapy to attenuate brain injury and promote thrombolysis in stroke and
heart attack.
The overall hypothesis behind this study is that reducing accumulated Zn2+ by
Zn2+ chelation targets multiple events in thrombotic stroke, including preventing
ischemic cell damage and improving thrombolysis. This hypothesis is based on the
following key observations: First, rapid intracellular Zn2+ accumulation in brain slices has been observed during ischemia in rodents. Secondly, the application of Zn2+ chelators
effectively reduces Zn2+ and attenuates ischemic neuronal injuries in vitro. Third, 45
platelets, one of the major components in thrombosis, contain concentrated Zn2+ packaging in α-granules. Those Zn2+ is functionally important to platelets activation and aggregation. During thrombolysis, α-granules are released and create locally Zn2+
accumulation around the blood clot. I hypothesize that the local high Zn2+ prevent the blood clot from being lysed. The experimental focus (objective) of this study is to investigate how reducing Zn2+ improves the outcome of thrombotic stroke.
Specific aim 1: To develop adult zebrafish as a novel stroke model for in vivo drug investigation. Zebrafish is widely used in biomedical research, and has shown its superiority in various high throughput studies. However, adult zebrafish has not been used in studies of cerebral ischemia. I hypothesize that the adult zebrafish can be developed as a stable and reproducible model for cerebral ischemia and thrombotic stroke. Specifically, I hypothesize that the adult zebrafish is sensitive to oxygen deprivation, and exhibits detectable brain damage and behavioral changes. The objective of this aim is to quantify brain injuries induced by cerebral hypoxic-ischemia and thrombotic stroke on adult zebrafish. The significant of this aim is to build a novel model of cerebral ischemia for high throughput studies.
Specific aim 2: To investigate the neuroprotective role of Zn2+ chelation in oxygen-deprived brain injuries in vivo. The objective of this aim is to evaluate the protective effect of Zn2+ chelation in the brain injury from oxygen deprivation in vivo.
This aim bases on the hypothesis that Zn2+ chelation attenuates neuronal death during oxygen deprivation, which may lead to improvement of overall recovery. By reducing cytosol Zn2+ contributed by organelles during oxygen deprivation, Zn2+ chelation reduces 46
brain damage. The neuroprotective effect will be shown in vivo as increased survival rate
from oxygen deprivation, and better behavioral recovery.
Specific aim 3: To determine the effect of Zn2+ in thrombolysis in vitro. During thrombolysis, the platelet, a major component of the blood clot release α-granules containing high concentrated Zn2+. How this Zn2+ release affects hemostasis is not well
studied. However, studies have shown that Zn2+ facilitates blood coagulation through
both intrinsic and extrinsic pathways. I hypothesize that Zn2+ accumulation around the
blood clot plays an inhibiting role in thrombolysis. The objective of this aim is to
quantify the change of thrombolysis when the ionic components around the blood clot are
altered.
Specific aim 4: To investigate the promoting effect of Zn2+ chelation in
thrombolysis in vivo. According to the results from aim 3, the dose of Zn2+ chelator that
can effectively improve thrombolysis is determined. I hypothesize that Zn2+ chelation can be used as an adjunct treatment for thrombolysis. The experimental focus of this aim
(objective) is to quantify the in vivo promoting effect of Zn2+ chelation on thrombolysis and determine a better strategy of treatment for thrombolysis. The significant of this aim is to propose a method that improves the therapeutic effect of current used thrombolytic treatment (tPA) and reduces toxicity.
Results from this study support that the adult zebrafish is a reliable stroke model.
Evidence includes: First, both oxygen deprivation and thrombotic attack induce brain damage on adult zebrafish, which can be labeled by histological staining. Second, the induced brain injury is severe enough to cause swimming disability and death, which 47
mimics the massive stroke. Third, the photothrombosis treated zebrafish responds well to
tPA, which makes zebrafish a reliable model for future investigations of thrombolytic
agents. By using zebrafish as in vivo model, data have shown that reducing Zn2+ during
and after oxygen deprivation increases tolerance for hypoxia, as well as the overall
recovery and survival rate.
In thrombotic stroke, Zn2+ chelation not only protects neurons from ischemic
attack, but also enhances blood clot-lysis. Zn2+ is found inhibiting tPA-mediated thrombolysis in a dose-dependent fashion. Low concentration of Zn2+ is able to postpone the maximum reaction rate of tPA, suggesting competitive binding sites between tPA and
Zn2+. Although Zn2+ chelation itself does not trigger thrombolysis, Zn2+ chelation
increases efficacy and potency of tPA-induced thrombolysis. The enhancement of
thrombolysis by Zn2+ chelation accelerates the reperfusion process, and leads to a significant higher rate of successful artery reperfusion. Taken together, results suggest that Zn2+ chelation is a promising treatment for stroke. It has neuroprotective effect in vivo. Additionally, applying Zn2+ chelation together with tPA is able to reduce the
necessary dose of tPA without compromising thrombolytic effect, and therefore minimize
tPA-induced toxicity.
My future study of stroke includes using both zebrafish and mice for
investigations of hemostasis and thrombolysis. Genetically modified zebrafish model
with protein mutations will be introduced to investigation the functions and roles of
particular proteins in susceptibility of thrombotic stroke, and thrombolysis. Mouse model
will be used to combine with zebrafish model for in vivo examination. 48
3. MATERIALS AND METHODS
3.1 Animals
All work in this study was conducted in accordance with the Ohio University
Institutional Animal Care and Use Committee (IACUC) guidelines.
Zebrafish (Danio rerio) were purchased from a local aquarium supplier and kept
in aquaria under a 12-h light/12-h dark photoperiod with half deionized water plus half
dechlorinated water (made with fresh tap water with sodium thiosulfate, a dechlorinating
agent, 10-15mg/L) at room temperature, which was adopted from the work of Faulk and
Holt in 2006. Zebrafish were fed daily with commercially available dry food. The size of
the zebrafish (mixed sex) used in the present study ranged from 30 mm to 49 mm total
length (mean: 41.56 mm; median: 42.5 mm). Thus, all individuals were well into
adulthood.
Adult CD1 mice (male) were purchased from Harlan Laboratories (Indianapolis,
US). Animals were kept under 12-h light/12-h dark cycle and were acclimatized for at
least 24 hours before surgery.
3.2 Specimen
Citrated blood of male CD1 mice was purchased from Innovative Research
(46430 Peary Court Novi, MI 48377) and BioChemed Services (172 Linden Drive,
Suite101, Winchester, VA 22601). Blood was arrived on the next day of harvest and
stored in 4 °C. Citrated blood was used in 3 days due to its decreased clotting ability. The
aliquot of 200 µl blood was placed in 0.5 ml tubes and recalcified with 3 µl of CaCl2
(1M). The blood samples were incubated in 0.5 ml centrifuge tubes at 37 °C for 3 hours. 49
Blood clots were gently rinsed by saline for 3 times and then transferred to custom-
modified cuvettes for measurement of absorbance.
3.3 Hypoxia chamber and global hypoxic treatment
Hypoxia chamber was made by 1000 ml clear glass bottle with 800 ml water inside (Figure 1). The chamber had two ports, one of which connected to a nitrogen (N2) tank and the other connected the air space inside to the open air outside. Once those two ports were sealed, this was a closed airtight system. Hypoxia in the chamber is created by bubbling pure N2 into the chamber and then hermetically sealed immediately after
stopping N2 perfusion. The dissolved oxygen (DO) reaches 0.6-0.8 mg/L after less than 6
minutes and remains constant during the test course. When the chamber is hypoxia-ready,
one zebrafish was transferred into the chamber. The chamber is continuously perfused
with a steady stream of N2 bubbling for one more minute in the presence of the zebrafish to ensure it is approximate to an anoxia environment.
The endpoint of the time in the hypoxia chamber was determined as when the zebrafish lay on one side at the bottom of the chamber for one minute, motionless (except for occasionally opercular movement). The endpoint is not accumulative. Any time of motionless that was less than one minute will be ignored. Once the endpoint was fulfilled, the hypoxic treatment was terminated and the zebrafish was transferred to the normoxia water for recovery and behavioral observation. A camera (Nikon D300s) was positioned in front of the hypoxia chamber and recovery beaker. All experiments including hypoxic treatment and recovery were recorded onto the video and later replayed for conformation and analysis. 50
A B
N2 O2
c
c
Figure 1: Hypoxia chamber. A. Schematic diagram of the custom-designed hypoxia chamber. B. The average of dissolved oxygen (DO) in the water of the hypoxia chamber decreased as the time of nitrogen perfusion increased. It started decreasing at the beginning of nitrogen perfusion and reached the minimum (0.6-0.8 mg/L) in 6 minutes.
DO stay stable during the rest of measurement (n=3).
3.4 Photothrombotic treatment
The zebrafish was anesthetized by Ethyl 3-aminobenzoate methanesulfonate (MS-
222) (140 mg/ml) and intraperitoneally injected with Rose Bengal solution according to the body weight (50 µg/g, 100 µg/g, and 200 µg/g). The total amount of injection was less than 0.1 ml. Then the zebrafish was placed into a petri-dish in the upright posture.
The media in the petri-dish is made of MS-222 (75 mg/l) to maintain anesthesia. A white light probe was placed right above the optic tectum region of the zebrafish brain. The optic tectum was exposed in different light intensities (4 units, 6 units, 8 units and 10 51
units) for a period of time (5 minutes, 10 minutes, 20 minutes and 30 minutes). The
illuminator and the light probe were purchased from Dolan Jenner (Boxborough, MA
01719). The mortality rate of each group was measured, and the recovery as well as
behavioral changes was recorded daily for 3 days.
Figure 2: Schematic diagram of the custom-designed chamber for photothrombotic
treatment. Rose Bengal was intraperitoneally injected to the zebrafish. The zebrafish was
submerged in the petri-dish with MS-222 solution (75mg/L) to keep zebrafish in
anesthesia. A rubber band (not shown here) was used to keep the zebrafish in upright
position yet was loose enough to allow gill movement. Gentle perfusion of O2 was
provided during the process to ensure adequate O2 supply.
3.5 Brain slices preparations
Adult zebrafish were anesthetized with MS-222 and decapitated. The brain was removed and placed in ice-cold artificial cerebral spinal fluid (ACSF). For cresyl violet staining, the brain was frozen and fixed in matrix of Gum Tragacanth/OCT compound
(Tissue-tek®). The temperature in microtome chamber was set up at -10 °C. The 52
thickness of the brain sections was set up to be 50 µm. For 2,3,5-triphenyltetrazolium
chloride (TTC) staining, coronal sections were cut into 200 µm thick by a vibratome at
300 µm away from the beginning of the optic lobe. ACSF was made by the following (in
mM): 121 NaCl, 1.75 KCl, 5 MgCl2, 1.25 KH2PO4, 26 NaHCO3, and 10 glucose, gassed
256 with 95%O2/5% CO2 .
3.6 Cresyl violet staining
The method was adopted from a previous study259. Brain sections of zebrafish were delipidized by passing them through ascending concentrations of ethanol from 70% to 95%, for 15 second in each, and two baths of xylene for 5 minutes in each. Brain slices were then rehydrated by descending concentrations of ethanol (form 95% to 70%) and then distilled water (for 15 second each). Cresyl violet was made into 5 µg/ml solution in distilled water, and was filtered. Brain sections were stained for 5 minutes, following with 30 seconds of rinse in distilled water, dehydration in ascending concentrations of ethanol from 70% to 95% for 1 minute in each, and differentiation in 95% of ethanol for at least 10 minutes. Two baths in 100% ethanol was followed for complete dehydration
(1 minutes for each). Before observing by microscopy, the sections were cleared in xylene for 5 minutes twice.
3.7 2,3,5-triphenyltetrazolium chloride (TTC) staining and quantification
Slices for TTC staining were taken as 1 mm in thickness, from the middle region
of the optic lobe, which was 300 µm away from the beginning of the optic lobe, and
incubated with TTC solution (2% by weight) in dark (unless it was stated otherwise). For 53
staining only, the incubation time was 40 minutes. Slices were then placed in 10%
formalin overnight and images taken on the next day.
3.8 Quantification of TTC staining
The method of TTC staining quantification adopted and modified from the
previous study260. Slices were incubated in 2% TTC solution for 90 minutes. After staining, the TTC solution was discarded and slices were gently rinsed by 2 drops of
DMSO/ethanol (1:1) solution and then placed individually in a cuvette with 1 ml
DMSO/ethanol (1:1) solution in dark for extraction overnight260. A spectrophotometer
was used to measure absorbance of each cuvet. Slices were taken out from cuvettes
before absorbance values were tested. The area of the brain slice was measured by
software Motic plus 3.0. The hypoxic treatment in experiments of the TTC staining was 8
minutes. According to preliminary data, all the zebrafish survived from 8 minutes of
hypoxia. When the hypoxia was increased to 9 minutes, some of the zebrafish did not
recover. Because the TTC staining was performed one day after the treatment, 8 minutes
of hypoxia was used to treat zebrafish in order to include all the samples.
3.9 Intracellular Zn2+ measurements
Newport Green (NG) staining: Coronal slices (200 µm thick) were loaded with 10
µM Newport Green (NG) DCF diacetate (excitation λ, 505 nm; emission λ, 535 nm)
Slices were maintained in the incubation chamber for 15 minutes in the room temperature. Brain slices were examined under microscope and acquired images by
ImagePro through a microscope-connected camera.
54
3.10 In vitro study of blood clot-lysis
The method was adopted and modified from a previous study261. Centrifuge tubes
(0.5 ml capacity) were weighted in analytical balance, and were labeled with their weight
(W0). Mouse blood was collected as aliquots of 200 µl in each centrifuge tube, and incubated in 37°C for 3 hour. After the blood clot was formed, serum was completely pipetted out and the blood clot was weighed together with the tube (W1). The weight of the blood clot was calculated as the gross weight subtracted by the weight of the tube
(Wclot = W1-W0). Clot-lytic agents were then added in each tube according to the
experimental design. Blood samples were placed in 37°C incubation for 1 hour for blood
clot-lysis. After incubation, lysate in each tube was completely pipetted out and
discarded. Each tube was weighted again (W2). The weight difference of each clot before
and after clot-lysis was calculated and normalized by original weight of the blood clot
(Wclot), to be presented as percentage of lysis.
3.11 Custom-modified cuvettes for absorbance measurement
Schematic diagram of the modified cuvettes was shown in Figure 3. A nylon web was placed at the narrowed part of the cuvette to separate the cuvette into an upper chamber and a lower chamber. The lower chamber was where the light went through during spectrophotometric measurement. The optical intensity change of the light was detected by a spectrophotometer and was output as absorbance value. The upper chamber was to hold the blood clot from entering the lower chamber or interfering the measurement. Cuvettes were filled with tPA dissolving in 1.5 ml saline solution.
Treatments were tPA, tPA plus EDTA, and tPA plus metal ions (Zn2+, Ca2+, Fe3+ or Fe2+) 55 according to the design of experiments. At the beginning of blood clot-lysis, the color of lysate was clear and the absorbance (optical density) value was low. As the blood clots being lysed, more hemoglobin was released into the vehicle, which gave samples red color and increased absorbance (Figure 3, 20). The overall amount of blood clot-lysis was quantified by the absorbance value.
Figure 3: Schematic diagram of the custom-modified cuvette to describe the set-up of blood clot-lysis (thrombolysis) for spectrophotometric measurement. A nylon web was placed to separate the cuvette into an upper chamber (for holding the blood clot) and the lower chamber (for light going through measuring absorbance value).
56
Measurement of absorbance by spectrophotometry: 1) Choosing the optimal
wavelength. The spectrophotometer (Biomate3) from Thermo Spectronic (Waltham, MA,
USA) was used to test the most sensitive wavelength for hemoglobin at 500 nm to 600
nm. The peaks of absorbance were between 540 nm and 580 nm, which was consistent
with previous studies253, 262. The wavelength of 580 nm was chosen to measure the absorbance. 2) Measurement of absorbance. Samples (cuvettes with blood clots and treatments dissolved in saline) were placed in the spectrophotometer. Saline was used to set up the absorbance baseline. The absorbance of each sample is measured in every 5 minutes, and up to 1 hour. 3) The calculation of rate of blood clot-lysis. The absorbance change between two time points divided by the time duration was considered the rate of clot-lysis.
3.12 Preparation of fibrin clots
To make the fibrin clot, citrated mouse blood (5 ml) was recalcified with CaCl2
(1M, 60 µl) and was immediately centrifuged in 2000 rpm for 15 minutes. The blood clot
was formed with minimization of red blood cells (Figure 4). Continuous incubation was
performed at 37°C for 3 hours. Different clot-lysis treatments were assigned for each
fibrin clot sample according to experimental plan, such as tPA, tPA plus EDTA, and
saline. Samples were then incubated in 37°C for 45 minutes for clot-lysis. Liquid (lysate)
in each sample was collected and denatured for protein analysis (SDS-PAGE and western
blot assay).
57
Figure 4: Fibrin clot forms after centrifuge. The fibrin clot is labeled by white dash lines.
Red blood clot formed at the bottom. Scale bar: 5 mm.
3.13 Western blot assay for the determination of the level of tPA-induced fibrinolysis
For determination of D-dimer protein level as a indicator of fibrinolysis, 20 µl of lysate sample was collected from each treatment. Samples were mixed with 20 µl 2X sample buffer individually in eppendorf tubes and immediately boiled in water bath for 5 minutes. Samples were further diluted by 1X sample buffer and the final dilution of the samples was 1: 8. For separation of proteins on 10% Tris-HCl SDS-PAGE, 10 µl of each sample was loaded. Proteins in the gels were electrophoretically transferred onto nitrocellulose membrane at 50 mA for 1.5 hours. The nitrocellulose membrane was then 58
blocked with 5% milk in TBST (50 mM Tris-HCl and 150 mM NaCl in PH 7.4, with 1%
Tween20) for 1.5 hours. The membrane was then incubated with D-dimer antibody (1
µg/ml) in TBST with 1% milk in cold room for overnight. The secondary antibody
conjugated with horseradish peroxidase was used for detection in room temperature for 2
hours. The membrane was developed by ECL reagent and was analyzed under BioRad
ChemiDoc and ImageJ.
3.14 Femoral artery occlusion and reperfusion
The CD1 mouse was anesthetized by intraperitoneal (IP) injection with cocktail of
ketamine, xylazine and aceptromazine. The anesthesia cocktail was made by Ketamine
(100 mg/ml) 1 ml, Xylazine (20 mg/ml) 1 ml, Aceptromazine (10 mg/ml) 0.3 ml and
sterile water or saline 7.7 ml. According to the body weight, 6.5 µl/g anesthesia cocktail was given through IP injection. The mouse (in surgical plane anesthesia) was placed on dorsal recumbency on a heat plate connected with ATC 1000 animal temperature controller (World Precision Instruments, 175 Sarasota Center Boulevard, Sarasota, FL
34240-9258 USA). The femoral artery of the mouse was exposed, followed by the intravenous injection of Rose Bengal (30 mg/kg). Cold white light that was provided by
Fiber-Lite Illuminator through a probe in 2.16 mm diameter (Dolan-Janner Industries)
was placed on the femoral artery. The light intensity was 1300 µW/cm2. The duration of
light exposure was 20 minutes. Occlusion of femoral artery was observed at the location
of light exposure, as a dark blood clot and emptiness observed downstream (Figure 18).
Alteplase (tPA) 2 mg/kg was given to the mouse with 0.01 mg as initial bolus, then 30-
minute perfusion of the rest via Syringe Infusion Pump 22 (Harvard Apparatus, Holliston, 59
MA). Half dose of tPA was 1mg/kg, with 0.01 mg as initial bolus and 30-minute perfusion of the rest.
3.15 Images acquisition and analysis of thrombolysis
Images of the occlusion of femoral arteries were taken under dissection microscope connected with Moticam 2500 camera and Motic Images Plus 2.0 software.
Images from each trial were made into sequenced images and analyzed by ImagePro software to quantify the thrombolysis process.
3.16 Chemicals delivery
In zebrafish experiments, water dissolvable agents such as Zn2+ chelators and
Zn2+ indicators were systematically delivered in the water. Rose Bengal and tPA were delivered intraperitoneally. The doses of water dissolved chemicals are DEDTC (250
µM), CaEDTA (100 µM), TPEN (50 µM), Nimodipine (10 µM), and dimethylthiurea
(1mM) unless it has been told otherwise.
3.17 Chemicals and reagents
Sodium thiosulfate, 2,3,5-triphenyltetrazolium chloride (TTC), sodium diethyldithiocarbamate trihydrate(DEDTC), and Rose Bengal were purchased from
Sigma Aldrich (St Louis, MO). Newport Green DCF was purchased from Invitrogen
(Life Technologies Corporation, Grand Island, NY). Ethyl 3-aminobenzoate methanesulfonate (MS-222) was purchased from Sigma. Activase-tPA was purchased from Genentech (South San Francisco, CA). Ethylenedinitrilo-tetraacetic acid (EDTA) was purchased from Eastman (Kingsport, Tennessee), Rose Bengal and
Ethylenediaminetetraacetic acid Disoudium-Calcium salt (CaEDTA) were purchased 60 from Sigma Aldrich (St Louis, MO). D-dimer antibody (polyclonal, rabbit-anti-mouse) was purchased from Bioss Antibodies (Woburn, Massachusetts). Secondary antibody
(goat-anti-rabbit-HRP) was purchased from Santa Cruz (Dallas, Texas). Clarity™
Western ECL Blotting Substrate was purchased from Bio-Rad (Hercules, California). All chemicals were dissolved in saline unless stated otherwise.
3.18 Data analysis
Values of mean and standard deviation are calculated by Microsoft Excel.
Statistic analysis of percentage of death and survival under different DEDTC concentrations was performed using fisher’s exact test under R program. Statistic analysis of hypoxia tolerance was performed using one-tailed Student t test under Microsoft
Excel.
Statistical analysis of TTC measurements was performed using two-tailed
Student’s t-test with Microsoft Excel. Statistical analysis of percentage of reperfusion under different treatment of lysis was performed using Fisher’s exact test under R program. Statistical analysis of absorbance increase of clot-lysis and the rate change were performed using two-tailed Student t test under Microsoft Excel. P<0.05 is considered significant.
61
4. RESULTS
4.1 Developing zebrafish as a novel model of cerebral ischemia and thrombotic stroke
4.1.1 Zebrafish as a model for global hypoxic ischemia
4.1.1.1 Treatment of hypoxia and the set-up of the end point of hypoxic treatment
To develop zebrafish as a global ischemia model, a prerequisite experiment was to test the sensitivity of zebrafish to oxygen deprivation. To investigate this, a hypoxia chamber was made to perform effective and stable treatment of oxygen deprivation. The
hypoxia chamber was made by a caped bottle with two ports. One was connected to N2
and the other was left open. By clamping both of the ports, the bottle was considered to
be an airtight chamber. Oxygen depletion was achieved by N2 perfusion. Once the severe
hypoxia (dissolved oxygen < 1 mg/L) was achieved, both ports were clamped and the N2
perfusion was stopped (Figure 1A). The oxygen meter (EXTECH, DO-600K) was used to
test the dissolved oxygen level when N2 was bubbling. Dissolved oxygen level dropped rapidly as the N2 perfusion started, and remained below 1 mg/L as long as the N2
perfusion continued (Figure 1B). This observation verified that N2 perfusion produced hypoxic water for zebrafish. More specifically, 6 minutes of N2 perfusion was efficient to create severe hypoxic environment for future experiments.
To test the tolerance of zebrafish to oxygen deprivation, zebrafish were individually put into hypoxia chamber. The zebrafish was treated individually with hypoxia until it was motionless for 1 minute, this point was labeled “endpoint 1”. This measurement was not accumulative, thus, any motionless status (or staying still) that was 62 less than 1 minute was not included and was ignored. Once the endpoint 1 was reached, the zebrafish was transferred to normal water to recover. Some of the zebrafish, treated with hypoxia, recovered as they regain swimming ability. The rest of them did not regain swimming and subsequently died. The mortality rate was 55%. In a separated experiment, the endpoint 2 was set up as zebrafish staying motionless for 2 minutes.
Zebrafish were treated individually with hypoxia until the endpoint 2 criterion was reached, and then transferred to normal water to recover. The mortality rate corresponding to the endpoint 2 was 100% (Figure 5).
Figure 5: Percentage of zebrafish death after hypoxic treatment. Endpoint 1, hypoxic treatment ended when the zebrafish is lying motionless for one minute; Endpoint 2, hypoxic treatment ends when the zebrafish is lying motionless for two minutes. 63
According to the experiment above, the effect of the hypoxic treatment of endpoint 1 was similar to median lethal dose (LD50), because it caused about 50% mortality rate. So the endpoint 1 was used as the endpoint in the following experiments for severe oxygen deprivation treatment. Each zebrafish was placed individually into the hypoxia chamber and remained there until it was motionless (except for occasionally opercular movement) on its side at the bottom of the chamber for 1 minute. Once this point was reached, the subject was transferred to normoxia (normal level of oxygen) water to recover. The period of time that zebrafish was under hypoxic treatment (from beginning to the endpoint) was defined as hypoxia time.
The mean hypoxia time or the average time to the endpoint for tested zebrafish in hypoxia chamber was 679.52 ± 90 seconds (mean ± SD, n =23; Table 2). The shortest hypoxia time was 540 seconds (9 minutes), which was from a subject that didn’t survive.
The longest time was 940 seconds (≈ 16 minutes), also from a subject that didn’t survive.
The distribution analysis of individual zebrafish revealed that hypoxia times for the majority of zebrafish (>70%) were between 10-12 minutes (Figure 6).
Immediately after hypoxic treatment, the zebrafish was transferred to normoxia water for recovery and behavioral observation. We generally observed the zebrafish recovery for two hours. Depending on recovery from hypoxic treatment, tested zebrafish were sorted subsequently into dead or survived groups. Table 2 summarized the results of zebrafish under hypoxic treatment. Although there was no difference in average hypoxia times between these two groups, 14 (60.87%) zebrafish did not recover from hypoxia Zebrafish as a hypoxic-ischemic model
Table 1. Effect of hypoxic treatment on Zebrafish 64 Hypoxia Time Zebrafish Number % Dead with an average hypoxia time of 695.64 ±111 seconds (mean ± SD) while 9 zebrafish (mean ± SD, Sec) Total 23 679.52 ± 90 survivedDead with after hypoxia Hypoxia time of 647.78 ± 56 seconds14 (mean ± SD). 60 695.64 ± 111 Survived after Hypoxia 9 40 647.78 ± 56
ally observed the zebrafish recovery for two hours. De- pending on recovery from hypoxic treatment, tested zebrafish were sorted subse- quently into dead or survived groups. Table 1 summarizes the results of zebrafish under hypoxic treatment. Although there was no difference in average hypoxia times be- tween these two groups, 14 (60.87%) did not recover from hypoxia with an average hypoxia time of 695.64 ± 111 seconds (mean ± SD) while 9 survived with a hy- FigureFigure 6: The 2. The analysis analysis of zebrafish of zebrafish hypoxia hypoxi timea distribution. time distribution. Three-dimensional Three dimen- poxia time of 647.78 ± 56 sional diagram shows the distributions of hypoxia or tolerant time of individ- seconds (mean ± SD). ual zebrafish in the hypoxia chamber (All fish = 23, Survived=9, Dead =14). diagram shows the distributions of hypoxia or tolerant time of individual zebrafish in the The size and distribution of hypoxia chamber (All fish = 23, Survived=9, Dead =14). brain injury were determined by TTC staining. The brains of opercular movement) on its side at the bottom healthy zebrafish were bilaterally stained deep of the chamber for 1 minute. Once this point red (Figure 3A and B). Following hypoxic treat- Table 2: The effect of hypoxia on zebrafish was reached, the subject was transferred to a ment TTC staining reliably delineated the infarct recovery beaker. This measurement was not (pale or unstained areas versus deep red col- accumulative, thus, any motionless status (or ored brain tissue). Bilateral, moderate to com- staying still) that was less than 1 minute was plete TTC decoloration or demarcation of the ignored. The period of time that the zebrafish infarct after 10 minutes of hypoxic treatment was under hypoxic treatment was defined as was clearly visible in the tectum of optic lobes hypoxia time. The mean hypoxia time or the av- (Figure 3C and D). Bilateral TTC decoloration erage time to the endpoint for tested zebrafish expanded to the deep structure of the optic lobe in hypoxia chamber was 679.52 ± 90 seconds with 20 to 35 minutes of hypoxia (Figure 3E-H). (mean ± SD, n =23; Table 1). The shortest hy- We measured the infarct size as percentage of poxia time was 540 seconds (9 minutes), which the total area. In 10 minutes of hypoxia, the was from a subject that didn’t survive. The long- percentage was around 11.6 ± 5.97 % (Mean ± est time was 940 seconds ( 16 minutes), also SD, n = 3), and this number was increased to from a subject that didn’t survive. The distribu- 24.79 ± 3.82 % (n = 3) with 20 minutes of hy- tion analysis of individual zebrafish revealed poxia and up to 60 ± 27.26 % (n = 5) after 30- that hypoxia times for the majority of zebrafish 35 minutes of hypoxia. (>70%) were between 10-12 minutes (Figure 2). Of tested zebrafish, 9 (39.13%) survived hypoxic Immediately after hypoxic treatment, the zebraf- treatment and recovered in the recovery beaker, ish was transferred into the recovery beaker for although the mean hypoxia time (647.78 ± 56 recovery and behavioral observation. We gener- seconds, mean ± SD, n = 9; Table 1) was not
91 Int J Physiol Pathophysiol Pharmacol 2011;3(2):88-96 65
4.1.1.2 Abnormal behavior during the recovery from hypoxic treatment
To observe the behavioral changes during the recovery, the zebrafish was treated individually with hypoxia and transferred to normal water as soon as the endpoint was reached. The recovery was recorded by a camera for 2 hours or until the zebrafish regained the normal behavior. The camera was placed on one side of the recovery beaker.
After the severe hypoxic treatment, the zebrafish stayed motionless in the recovery beaker at first, followed by a twitching, which was considered as first twitching. The next behavior that was observed during recovery was unbalanced swimming, and then the balanced swimming. Unbalanced movement was defined as swimming associated with erratic behaviors such as circling and rotating, or swimming without maintaining the upright position. The time needed for regaining each movement was recorded and used to evaluate recovery (Figure 7). I did not observe any erratic movement from control zebrafish that underwent the transfer from one environment to another without the hypoxic treatment.
Zebrafish as a hypoxic-ischemic model
66
FigureFigure 7: Swimming 4. Swimming capability of zebr capabilityafish during recovery. of zebrafish Bars are times during when zebrafishrecovery. regains each Bars pattern were of swimming times (first when motion, zebrafish unbalanced swimming, regained and each pattern of swimming (first motion, unbalanced balancedswim, swimming) and after balanced hypoxic treatment swim) (Mean after ± SD, hypoxic n = 9). treatment (Mean ± SD, n = 9).
4.1.1.3 Histological staining and quantification of brain damage after hypoxic
treatment
first,To identifyproceed brain damage, by a cresylcouple violet ofstaining seconds was performed of rotating, to label dead and then they were circling again. The surviving neurons. Cresyl violet staining labels the distribution of cells and neurons by staining fish began to show balanced movement after chromatin.about When 15 cells minutes go through cellof derecovery,ath, one of the whichirreversible was phases when is chromatin condensationthe zebrafish of the nucleus. regained Nuclear condensation normal was shownand ascoordinated heavily staining of
cresylswimming, violet and lost ofindicating shape of cell plasma sufficient (Figure 8D), recovery which verified in immediate their cell Figure 3. Brain injury of zebrafish detected by TTC swimming ability. staining. A. TTC stained healthy brain sections. B-H. TTC stained brain sections following various hypoxic treatments (10, 20, 30, 35 min) in the hypoxia cham- Disscusion ber. In this study we demonstrated that hypoxic treatment effectively induced zebrafish brain significantly different from the dead zebrafish damage. Under near complete hypoxia condi- group. We recorded the movements and behav- tions (DO = 0.6-0.8 mg/L), zebrafish quickly lost iors of the tested zebrafish once it was trans- motion and reduced opercular movement with ferred to the recovery chamber. The zebrafish the average hypoxia time of 679.52 seconds or that survived hypoxic treatment showed rapid about 11 mintues. The distribution analysis of opercular movement in a few minutes but didn’t individual zebrafish revealed that hypoxia times move until about 10 minutes after being placed for the majority of zebrafish (>70%) were be- in the recovery beaker. Through recovery follow- tween 10-12 minutes. Essentially, 60.87% of ing hypoxic treatment, all surviving zebrafish tested zebrafish did not recover or died from experienced initial twitching followed by unbal- hypoxia. These data indicate that zebrafish are anced or erratic movements before balanced sensitive to hypoxic attack. Furthermore, the movement was regained. The times when sur- fact that TTC-defined brain damage was ob- viving fish started each movement are listed in served and became worse with increasing Figure 4. We did not observe any erratic move- length of hypoxic treatment indicates the clear ment from control zebrafish that underwent the correlation between hypoxia time and brain transfer from one environment to another with- damage and further suggests that the hypoxia out the hypoxic treatment. Unbalanced move- time or hypoxic treatment reliably predicts sub- ment was defined as swimming associated with sequent severe hypoxic brain damage in zebraf- erratic behaviors such as circling, rotating, and ish. About 40% of tested zebrafish survived hy- upright swimming. Circling and rotating were poxic treatment and recovered by regaining co- both observed on the same zebrafish, but did ordinated swimming. Through recovery following not necessarily have to happen at the same hypoxic treatment, all surviving zebrafish experi- time (Figure 5). Sometimes, zebrafish circled enced erratic movements, including circling and
92 Int J Physiol Pathophysiol Pharmacol 2011;3(2):88-96 67 damage of oxygen deprivation. The zebrafish was sacrificed immediately after hypoxic treatment, and the brain was dissected and prepared as 20 µm slices. The optic lobe region with cresyl violet staining is shown in Figure 8.
Figure 8: Cresyl violet staining of optic lobe. A. Staining of healthy brain slices. B.
Staining after hypoxic treatment. The squared area of each brain slice is magnified as C-
D accordingly. Arrows show nuclear condensation.
68
Histological staining is a reliable way to visualize morphological change.
However, for high throughput measurement and comprehensive quantification of brain
injuries, this method may not be ideal, because it includes complicated preparation of
brain slices and subsequent staining procedure. For fast evaluation of the brain injury
caused by oxygen deprivation, TTC staining was performed on the optic lobe of zebrafish
brain to distinguish between healthy and damaged regions. TTC staining is comparably
fast and accurate method for cell viability.
The white compound tetrazolium chloride (TTC) is reduced by dehydrogenases in
healthy cells to formazan, which yields a red color (Figure 9A). TTC staining had been
used to evaluate cell viability accurately at 6 hours or longer after cell death. In this
study, TTC staining was performed 24 hours after hypoxic treatment to ensure an
accurate measurement. The hypoxic treatment was reduced from median lethal dose to a
sub-lethal dose, which was 8 minutes of hypoxia. The purpose of shortening the time of
hypoxia was to avoid mortality before TTC staining. The 1 mm coronal slices of optic lobe were incubated with 2% TTC solution for 90 minutes. Results showed that oxygen deprivation for 8 minutes generated obvious brain damage, which is shown as a stainless tissue, compared to a bright red in healthy tissue (Figure 9B).
To quantify TTC staining, spectrophotometry was performed to measure the absorbance value of the TTC staining, after it was extracted from the brain slices. After staining the TTC solution was discarded and slices were placed individually in a cuvette with 1ml of DMSO/ethanol (1:1) solution in the dark overnight, to ensure a complete extraction of the staining. The absorbance of the solution which resulted from the 69 extraction was measured by a spectrophotometer at 485 nm wavelength. The volume of the slices was measured by software (Motic plus 3.0). The results showed that TTC staining of the hypoxic treated group was significantly reduced compared to that of the healthy brains (Figure 9C). These data suggest that TTC staining is a practical and reliable method to quantify brain damage in zebrafish.
Figure 9: Quantification of brain viability. A. A healthy brain in TTC staining. B. A hypoxia-treated brain in TTC staining. C. Spectrophotometric measurement shows that hypoxic treatment significantly reduced TTC absorbance comparing to that of the control
(healthy) group (** p < 0.01, n = 6). 70
4.1.2 Zebrafish as a model of thrombotic stroke
4.1.2.1 Zebrafish exhibited abnormal behavior after photothrombotic treatment
Rose Bengal was used to induce thrombotic injuries, however the safety of Rose
Bengal, as well as light exposure had to be determined first. Results suggest that light exposure without Rose Bengal did not induce brain injuries, because zebrafish treated with light only remained healthy after 3 days. High doses of Rose Bengal (200 µg/g or higher) without light exposure induced behavioral disability in zebrafish and eventually caused death. These results suggest the toxicity of Rose Bengal, and strict control of Rose
Bengal concentration had to be implemented. Low dose of Rose Bengal (< 100 µg/g) was found to be safe for zebrafish. No short or long-term (three-day) brain damage was observed in zebrafish treated with this dose of Rose Bengal (Table 3).
Table 3: Behavioral changes of zebrafish after light exposure, or Rose Bengal treatment.
Injected White Exposure Day 0 Day 1 Day 2 Rose light time Bengal intensity (min) (µg/g) (unit)* -- 10 30 Normal Normal Normal 100 (n=3) -- -- Normal Normal Normal 200 -- -- Normal Unbalanced Died 500 -- -- Normal Hardly swim, died * White light intensity: 1 unit = 100 µW/cm2
To establish an effective method to induce photothrombosis, combinations of
different concentrations of Rose Bengal and different durations of light exposure were
applied to zebrafish. The zebrafish was anesthetized by MS-222 (140 mg/l) and 71 intraperitoneally injected with different doses of Rose Bengal (50 µg/g and 100 µg/g) according to the body weight. Then the zebrafish was placed into a petri-dish in the upright position. The solution in the petri-dish had MS-222 (75 mg/l) to maintain anesthesia. The source of white light was placed right above the optic tectum for different periods of time (5 minutes, 10 minutes, 20 minutes and 30 minutes) and different light intensity (4 units, 6 units, 8 units and 10 units, with 1 unit equals to 100 µW/cm2). The mortality rate of each group was recorded and the behavioral changes during the recovery were recorded daily for 3 days.
Rose Bengal in concentration range of 50 – 100 µg/g was adequate to induce thrombotic brain damage, because zebrafish treated with that concentration, displayed behavior changes. The extent of damage depended on the amount of light exposure
(Table 4). Light exposure for 30 minutes with 800 µW/cm2 light intensity, and with Rose
Bengal in 50 – 100 µg/g was an effective combination to induce reproducible thrombotic brain injuries. This was defined as an effective photothrombosis, because the combination of light intensity and duration with particular concentration of Rose Bengal induced reproducible brain damage, which was observed as behavioral changes or death of the subject.
72
Table 4: The behavioral changes of zebrafish in recovery after photothrombotic treatment.
Injected White Exposure Day 0 Day 1 Day 2 Rose light time Bengal intensity (min) (µg/g) (unit)* 100 10 5 Normal Normal Normal 100 10 10 Normal Normal Normal 100 10 20 Normal Circling and Died rotating 100 10 20 Normal Normal Normal 100 (n=2) 8 30 Died 100 8 30 Rotating Died and circling 100 (n=2) 8 30 Normal Circling and Died rotating 100 (n=2) 8 30 Normal Circling and Circling rotating and rotating 50 (n=2) 10 30 Died 50 10 30 Rotating Rotating and Died circling 50 (n=2) 8 30 Normal Circling and Circling rotating and rotating 50 8 30 Normal Normal Circling and rotating 50 (n=2) 6 30 Normal Circling and Circling rotating and rotating 50 (n=2) 6 30 Normal Normal Circling and rotating * White light intensity: 1 unit = 100 µW/cm2
4.1.2.2 Photothrombotic treatment causes quantitative brain damage
We considered zebrafish as completely recovered from anesthesia, if they retained the ability of normal and balanced swimming behavior. Zebrafish were kept in the dark during recovery. To prevent further excitation of Rose Bengal, only dim red light was 73
used as necessary for observation. There were two types of abnormal swimming behavior
observed by 24 hours after photothrombotic treatment: rotating and circling (Figure 10A and B). Rotating behavior was observed when zebrafish was turning around its longitudinal axis. Circling behavior was observed when the zebrafish was turning around an external axis. These abnormal behaviors were not observed in controls treated with
Rose Bengal or light only. By 24 hours after photothrombotic treatment, 96.8% of zebrafish died or exhibited circling and rotating behavior (Table 4). These results indicate that photothrombosis generated delayed brain damage in zebrafish that could be observed as behavioral changes.
74
Figure 10: Brain damage of zebrafish after photothrombotic treatment. A. Circling after photothrombotic treatment. The zebrafish turns around its longitudinal axis. Scale bar,
5cm. B. Rotating after photothrombotic treatment. The zebrafish turns around an external axis. Scale bar, 5cm. Sequential images of zebrafish circling and rotating movements were separately from one-second of videos. 75
To confirm the brain damage, TTC staining was performed at 24 hours after photothrombotic treatment. Zebrafish in the first control group were treated with Rose
Bengal only, without the light exposure. In the second control group zebrafish received light exposure for 30 minutes, without Rose Bengal treatment. TTC staining and behavior observation indicated no brain damage from light exposure alone or Rose Bengal alone
(Figure 11A). TTC staining of photothrombotic treated brain slices showed lack of staining in the area of light exposure, indicating the brain injury. Quantified measurement of TTC staining showed a significant reduction of brain cell viability with 20 minutes of light exposure, comparing with the control group, and even greater reduction with 30 minutes of light exposure. Results showed photothrombotic brain damage in a dose- dependent manner (Figure 11B). Results also supported a strong correlation between abnormal swimming pattern and the injuries of the brain (Figure 10, 11).
4.1.2.3 Photothrombosis induced cerebral damage is responsive to tPA treatment
In a separate group (tPA group), the tPA was injected immediately after photothrombosis. TTC staining after 24 hours showed that the application of tPA increased viability of brain tissue if compared with photothrombosis without tPA. 24- hour-recovery of zebrafish was compared between tPA-treated and non-treated groups. In non-tPA treated group, 41.9% of the zebrafish exhibited abnormal swimming, and 54.8% of zebrafish died by 24 hours after photothrombosis. Only 3% of the subjects fully recovered without exhibiting behavioral changes. Treatment with tPA increased the recovery rate from 3% to 55% at 24 hours. The rate of abnormal swimming was decreased to 15% and the mortality rate was reduced to 30%. Detailed data are shown in 76
Table 5 and Figure 12. Treatment with tPA also improved brain viability after photothrombosis, quantified by TTC staining (Figure 11). Results suggest that the zebrafish photothrombotic model is responsive to tPA treatment, which makes zebrafish a promising model for stroke studies and drug screening.
77
Figure 11: The evaluation of the brain damage after photothrombotic treatment. A. TTC staining of the zebrafish brain at 24 hours of photothrombotic treatment. Scale bar, 1mm.
B. Spectrophotometric measurement shows that photothrombotic damage was dose- dependent with light exposure, and treatment of tPA significantly improved TTC absorbance comparing to non-tPA treated group. (* p < 0.05, *** p= 4.36587E-05, n =
7). 78
Table 5: The recovery rate between the tPA treated group and the non-tPA treated group after photothrombosis.
Figure 12: The outcomes of photothrombotic treatment between the tPA treated group and the non-tPA treated group. n = 20 in tPA treated group, 31 in non-tPA treated group.
***, p = 3.359e-5.
79
4.2 The neuroprotective role of Zn2+ chelation in oxygen-deprived brain injuries in vivo
4.2.1 DEDTC reduces mortality rate of zebrafish after hypoxia
Oxygen and glucose deprivation can induce a significant increase in intracellular
Zn2+ and neuronal injury/death3, 256, 257. The staining of affected brain regions with Zn2+
indicators and cell viability probes revealed a striking correlation or co-localization
between Zn2+ accumulation in neurons cell death3, 256, 263. In this experiment, fluorescent
Zn2+ indicator Newport Green was used to label Zn2+ accumulation in hypoxic-damaged cells. In the hypoxia-treated zebrafish, the increases in intracellular Zn2+ were detected throughout the brain regions (Fig. 13A). When DEDTC was applied during the zebrafish recovery following hypoxic treatment, the fluorescence intensity of Zn2+ staining was significantly reduced in the zebrafish brain (Figure 13B). Results suggest that adding
DEDTC in the water effectively delivered this Zn2+ chelator into the brain.
80
Figure 13: DEDTC decreases Zn2+ accumulation. The images are slices from the optic
lobe of the zebrafish brain stained with a fluorescent Zn2+ indicator, Newport Green. A.
The image taken after hypoxic treatment showing bright Zn2+ staining. B. The image
taken after hypoxic but was treated with 250 µM DEDTC. Scale bar, 10 µm.
In control experiments, Zn2+ chelator DEDTC was added in the water and it did not alter zebrafish swimming behavior. The application of DEDTC increased survival rate of hypoxic-treated zebrafish in a concentration-dependent manner (Figure 14). In this set of experiments, DEDTC was included in the recovery chamber (containing normoxia water) in different concentrations for 30 minutes. The maximum concentration of
DEDTC applied was 250 µM, which yielded 85% survival rate of hypoxic-treated zebrafish. This improvement of the survival rate was significantly higher than the controls (45%)(Figure 14). Hypoxic treatment induced a significant increase in intracellular Zn2+ and neuronal injury/death, which were attenuated by zinc chelation
(Figure 14 and table 6).
81
Figure 14: DEDTC increases the survival rate. The concentration-dependent improvement of survival rate happens in DEDTC-treated zebrafish. DEDTC concentration range of 10–250 µM was tested. DEDTC was presented in recovery chamber. The numbers of animals tested: 31 zebrafish in control (0 µM DEDTC), 14 in 5
µM, 26 in 50 µM, 14 in 100 µM, and 24 in 250 µM DEDTC treatment. *p < 0.05, indicating the significant difference between the control and the zebrafish treated in 250
µM DEDTC.
82
Table 6: The recovery rate from oxygen deprivation
4.2.2 DEDTC enhanced the hypoxia tolerance in zebrafish
The effect of DEDTC on hypoxia tolerance in zebrafish was tested by applying
DEDTC to the hypoxia chamber (not recovery chamber as in the experiments described above). The hypoxia tolerance or tolerance time was defined as the period from the initiation of hypoxia to the endpoint (or the time to endpoint, TTE) during the hypoxic treatment of each zebrafish. The presence of DEDTC during the hypoxic treatment significantly increased TTE of zebrafish (TTEcontrol = 584 ± 75 sec, TTEDEDTC =670 ±
83 sec, mean ± SD; p<0.05, n=9). The extended period of hypoxia treatment in the present of DEDTC suggests that removal of Zn enhances hypoxia tolerance in zebrafish
(Figure 15).
83
Figure 15: DEDTC enhances hypoxia tolerance in zebrafish. The bar chart shows TTE
(time to endpoint) of zebrafish in hypoxic treatment. DEDTC treatments (50 µM) significantly increased TTE when comparing to control (no DEDTC application). Mean ±
SD, n = 9; * p < 0.05.
4.2.3 DEDTC preserves brain viability from hypoxic attack
The brain damage by hypoxic treatment was evaluated post-mortem by TTC
(2,3,5-triphenyltetrazolium chloride) staining, which is a widely used method to measure hypoxic-ischemic brain damages. The brains of healthy zebrafish were stained deep red
(Figure 16A). In the zebrafish that were treated with hypoxia, moderate to complete TTC decoloration or demarcation of the infarct (pale or unstained areas versus deep red colored brain tissue) was clearly visible in the tectum of optic lobes (Figure 16B). In 84 some zebrafish, TTC decoloration expanded to the deep structure of the optic lobe.
DEDTC, when applied in hypoxia chamber, significantly improved TTC staining in zebrafish, even cancelled the TTC decoloration in some cases (Figure 16C).
To quantify TTC staining and the brain damage, the TTC staining in the zebrafish brain was extracted and the absorbance of the extraction solution was measured using the spectrophotometric assay following standard procedures (detailed in methods). There were very significant increases in TTC absorbance with DEDTC treatment during hypoxia, indicated by more TTC stained tissue, which was 1.373 ± 0.163mm3 (mean ±
SD) in comparison to 0.829 ± 0.211 mm3 (mean ± SD) in the hypoxia group without
DEDTC treatment. The overall TTC staining of DEDTC treated hypoxic group was of similar intensity to TTC staining of healthy brain, which suggests that early DEDTC treatment prevents hypoxia-induced brain damage. Treatment with DEDTC after hypoxia did not yield results as desirable as DEDTC treatment during hypoxia, however this treatment rescued the brain from hypoxic injuries. DEDTC alone treated zebrafish without hypoxia (shown as “DEDTC control” in Figure 16D) did not yield a higher TTC staining compared to that of healthy brains (shown as ‘‘control’’ in Figure 16D). These data suggest that DEDTC had no chemical reaction with TTC and did not interfere with the staining and the intensity of the staining was comparable to the healthy group. Taking together these results further support that application of DEDTC reduced the hypoxic brain damage.
85
Figure 16: Zebrafish brain injury detected by TTC staining. A-C. TTC stained brain sections: healthy (A), following hypoxic treatments (B), and following hypoxic treatment with DEDTC application in the hypoxic chamber (C). Scale bar, 1 mm. D.
Spectrophotometric measurement shows that DEDTC significantly improved TTC absorbance comparing to hypoxia only group (*p < 0.05, **p < 0.01, n = 6). 86
4.3 The promoting effect of Zn2+ chelation in thrombolysis in vitro
4.3.1 Measurement of blood clot-lysis in vitro
Mouse blood (200 µl) was coagulated in the centrifuge tube at 37 °C for 3 hours.
Once the blood clot was formed, serum was extracted and the blood clot was weighed.
Treatments with clot-lysing agents were added according to the experimental design.
After 45 minutes of clot-lysis, the blood clot was weighed again and compared to the weight before clot-lysis. The reduction in weight was considered as the amount of blood clot-lysis.
First experiments were done to determine whether serum was necessary to trigger tPA-induced blood clot-lysis. The experiments compared clot-lysis between two groups.
One group was treated with tPA without serum. The other group was treated with tPA together with the serum which was previously extracted from the centrifuge tube during blood clot preparation. The results showed that including serum during clot-lysis did not increase the overall clot-lysis. These data suggest that enough plasminogen was attached to the blood clot for tPA to activate conversion of plasminogen to plasmin, and in turn to initiate clot-lysis (Figure 17). Therefore, subsequent experiments did not contain serum during clot-lysis.
87
Figure 17: The effect of serum in tPA-induced clot-lysis in vitro. The application of serum is not necessary to initiate tPA-induced blood clot-lysis in vitro. Values are mean ±
SEM. n=4 in (no serum) group. n=10 in serum group.
Different doses of tPA were examined to determine whether this method of in vitro measurement was able to reflect different amount of blood clot-lysis. Data showed that tPA induced blood clot-lysis in a dose-dependent fashion. The treatment with tPA at
1 mg/ml produced 61% clot-lysis. The blood clot was lysed 50% in 0.5 mg/ml tPA. The lower tPA concentration generated less clot-lysis. The treatment with tPA of 0.05 mg/ml concentration had 37% clot-lysis, and 31% clot-lysis was yielded by 0.01 mg/ml tPA 88
(Figure 18). The following experiments were performed to investigate the modulation of
Zn2+/Zn2+ chelation in clot-lysis. Therefore a relatively mild dose of tPA (0.05 mg/ml) with moderate amount of clot-lysis was chosen.
Figure 18: The clot-lysis mediated by tPA is dose-dependent. In vitro blood clot-lysis (in percentage) mediated by different concentrations of tPA treatment. Values are mean ±
SEM. n= 3-8 in each group.
TPEN, a cell permeable Zn2+ chelator, which can bind to free zinc intracellularly and extracellularly, was added together with tPA to blood clots. The control group was treated with tPA plus DMSO, the vehicle of TPEN. TPEN did not generate effective blood clot-lysis by itself (data not shown). However, when co-applied with tPA, TPEN 89
effectively promoted the overall blood clot-lysis (Figure 19). These results first
uncovered the potential enhancement of Zn2+ chelation during tPA-induced blood clot- lysis, and led to further investigation of the details on the inhibiting effect of Zn2+ during clot-lysis and thrombolysis.
Figure 19: The effect of TPEN in tPA-induced blood clot-lysis. Values are mean ± SEM.
The concentration of tPA is 0.05 mg/ml. The concentration of TPEN is 100 µM dissolved in DMSO. n = 19 in (tPA + TPEN) group and (tPA + DMSO) group. n = 3 in (saline +
DMSO) group. * p = 0.05.
4.3.2 Thrombolysis in vitro is measured by spectrophotometry
The method of measuring blood clot-lysis in centrifuge tubes was only able to quantify the outcomes of clot-lysis with different agents. It was not designed to capture the reaction activities throughout the process of clot-lysis. In order to quantify 90 thrombolytic reaction in a more detailed and sensitive manner, the spectrophotometer was used to measure the absorbance value during the clot-lysis. Customized cuvettes were used to host thrombolytic reaction for spectrophotometric measurements. Cuvettes contained solution with thrombolytic agents according to the treatment design. The upper chamber of the cuvette held the blood clot, and the lower chamber had the measuring window for spectrophotometry. (Detailed protocol in method.)
When blood clots were first transferred into cuvettes, samples were clear with low absorbance value (Figure 20A). As tPA was lysing clots, hemoglobin was released into clear solution, which gives the sample a red color and increased absorbance value (Figure
20B). As the blood clot was further lysed, the increasing hemoglobin concentration in the solution yielded an increasing growing absorbance value. The amount of blood clot-lysis could be observed by the total amount of hemoglobin in the solution.
The following investigation showed that spectrophotometric assay was sensitive method and was able to differentiate blood clot-lysis mediated by different doses of tPA, which is a prerequisite for using spectrophotometry to measure blood clot-lysis. Different doses of tPA (from 0.005 mg to 0.05 mg, as shown in Figure 20B, cuvette 1-3) resulted in different amount of hemoglobin release, which yield noticeable difference in the absorbance values. This observation supported the hypothesis that spectrophotometry can capture the various levels of blood clot-lysis, by measuring absorbance values. The higher absorbance value indicated more amount of thrombolysis. 91
Figure 20: The set-up of blood clot-lysis (thrombolysis) for spectrophotometric measurement. A-B. Thrombolysis from the beginning (A) to 60 minutes (B). Cuvette 1,
2, 3 have different doses of tPA: 0.005 mg, 0.01 mg, and 0.05 mg. Scale bar: 1 cm.
92
Thrombolysis was measured for 60 minutes in order to get detailed activity of tPA-induced thrombolysis. Steady increase of absorbance value was observed with 0.01 mg tPA and started at about 10 minutes after the initiation of thrombolysis and kept increasing during the period of observation. The thrombolysis of vehicle (saline) was also measured, which showed little change with low level of absorbance through the observation (Figure 21A).
The absorbance change of tPA-induced thrombolysis was measured every 5 minutes. The rate of the thrombolysis reaction was calculated as absorbance change over time. As shown in Figure 21B, the rate of tPA-induced thrombolysis started to increase after 10 minutes and reached the maximum at 20-25 minutes. The rate of thrombolysis gradually decreased through the rest of the reaction due to the depletion of plasminogen or loss of tPA activity. The vehicle of tPA (saline) did not yield effective thrombolysis, as expected.
93
Figure 21: The measurement of tPA-induced in vitro thrombolysis over time. The dose of tPA is 0.01 mg. A. The overall thrombolysis shown as increase of absorbance value at
580 nm wavelength. B. The reaction rate of tPA-induced thrombolysis. The rate of the thrombolysis reaction was calculated as absorbance (which is absorbance change at each measure point from the previous measure point) over time (5 minutes). Values are mean
± SEM. n=22 in tPA group, n=5 in saline group. 94
More detailed quantification is shown in Figure 22. Samples of blood clots were treated with different doses of tPA (from 0.005 mg to 0.1 mg). The absorbance increase was proportional to the overall thrombolysis, which was tPA dose-dependent.
Thrombolysis at 0.005 mg of tPA was low, which did not separate itself from saline until
40 minutes. Effect of thrombolysis at 0.1 mg of tPA was saturated and was not higher than thrombolysis yielded by 0.05 mg of tPA (Figure 22A).
At the end of 60 minutes, thrombolysis was tPA dose dependent until the tPA dose reached to 0.1 mg (Figure 22B). Results showed that at 580 nm wavelength, spectrophotometry was able to quantify the thrombolysis effect of tPA ranging from 0 mg to 0.1 mg. Results confirmed that spectrophotometric measurement was sensitive to different amount of thrombolysis, and was a reliable method to evaluate the therapeutic effect of tPA.
95
Figure 22: In vitro thrombolysis at different doses of tPA. A. Treatment of tPA induces thrombolysis, shown as absorbance increase at 580 nm wavelength. B. The overall thrombolysis at the end of measurement (50 minutes observation). Values are mean ±
SEM. n= 2-4 in each group.
The rate of thrombolysis was shown as tPA dose dependent. Low dose of tPA
(0.005 mg) generated slight thrombolysis (Figure 23A). The efficacy of the thrombolysis 96 reaction was proportional to the tPA dosage as well (Figure 23B). The treatment with tPA in 0.005 mg yield efficacy of 3 unit/min. The efficacy was promoted to 5 unit/min, when the dose of tPA was increased to 0.01 mg. The time taken to reach the maximum reaction rate was shortened from 60 minutes to 20 minutes (Figure 23A).
Further increasing the dose of tPA from 0.01 mg to 0.05 mg promoted the maximum rate from 5 unit/min to 8 unit/min. However, the time taken to reach the maximum rate was not further shortened. The treatment with tPA in 0.01 mg and 0.05 mg both reached the maximum rate of reaction at 20 minutes. Data support that spectrophotometric measurement was able to detect and evaluate the efficacy of tPA- induced in vitro blood clot-lysis. These results also provided a detailed picture of tPA- induced blood clot-lysis measured by spectrophotometry, which can be used as a fast method for the evaluation of future thrombolytic candidates.
97
Figure 23: The reaction rate of tPA-induced thrombolysis at different doses of tPA in vitro. A. The real-time reaction rate of thrombolysis. The reaction rate is calculated as absorbance change per minute. The absorbance value is measured by spectrophotometry at 580 nm wavelength. B. The maximum reaction rate (efficacy) of tPA-induced thrombolysis. Values are mean ± SEM. n= 2-4 in each group. 98
4.3.3 Inhibiting effect of zinc and iron on tPA-induced thrombolysis in vitro
High concentration of Zn2+ is packaged in α-granules in platelets. During thrombolysis, platelets in the blood clot are activated and release α-granules. The activation of platelets and the release of α-granules creates locally concentrated Zn2+. To
2+ investigate whether Zn affect thrombolysis, ZnCl2 was added together with tPA during tPA-induced thrombolysis. The application of 10 µM Zn2+ yielded mild inhibition of thrombolysis. The effect of reduction on thrombolysis increased in a dose-dependent manner and became significant at 100 µM and 200 µM of Zn2+. The overall thrombolysis
was reduced to 75% with the presence of 100 µM of Zn2+, and was inhibited by more
than 50% by the application of 200 µM of Zn2+ (Figure 24). 99
Figure 24: Zn2+ inhibits tPA-induced thrombolysis. The dose of tPA is 0.01 mg. A-C:
Thrombolysis of tPA comparing with tPA plus different concentrations of ZnCl2 (10 µM,
100 µM and 200 µM). n=22 in tPA group. n= 6—9 in each Zn2+ group. * p < 0.05, ** p <
0.01. 100
Similarly, locally high concentration of Fe3+ and Fe2+ will be produced by erythrocytes from the blood clot during thrombolysis. The involvement of iron in thrombolysis needs to be investigated. A method similar to Zn2+ was used to further understand iron and thrombolysis. FeCl3 in different concentrations was added together with tPA solution. The absorbance value was measured at 580 nm wavelength for 60 minutes to measure the overall thrombolysis. Fe3+ at 10 µM showed mild facilitation on tPA-induced thrombolysis, and the facilitation became statistically significant from 45 minutes of thrombolysis (Figure 25A).
At 100 µM, Fe3+ generated very significant inhibition of thrombolysis from 15 minutes of the reaction, but overall inhibition was not as effective as the inhibition observed with Zn2+ (100 µM) (Figure 25B). Further increasing the concentration of Fe3+
failed to reduce thrombolysis effectively (Figure 25C). The inhibition of thrombolysis
with Fe3+ was more significant in medium containing higher concentration of the ion,
compared to lower concentration. These results suggest that Fe3+ has inhibiting effect on
the tPA-induced blood clot-lysis.
101
Figure 25: Fe3+ inhibits tPA-induced thrombolysis. The dose of tPA is 0.01 mg. A-C:
Thrombolysis of tPA comparing with tPA plus different concentrations of FeCl3 (10 µM,
100 µM and 200 µM). n=22 in tPA group. n= 6—10 in each Fe3+ group. * p < 0.05, ** p
< 0.01. 102
Further investigation showed that Fe2+ at a low concentration (10 µM) did not alter tPA-induced thrombolysis (Figure 26A). Higher doses (100 µM and 200 µM) of
Fe2+ inhibited blood clot-lysis by 50%. The inhibiting effect of Fe2+ was saturated at 100
µM. Further increase of Fe2+ concentration did not correspondingly bring down the
overall thrombolysis of tPA (Figure 26B, C). When comparing Zn2+ with Fe3+ and Fe2+,
only Zn2+ yielded inhibiting effect on tPA-induced thrombolysis at 10 µM. At 100 µM, all three types of ions managed to yield significant reduction on thrombolysis at as early as 15 minute of the reaction, with Fe2+ generating the most robust inhibition. At 200 µM,
Zn2+ yielded the earliest significant inhibition (Figure 24, 25, 26). The results supported
that the inhibition of thrombolysis was contributed by different metal ions and might be
intervened from different aspects.
103
Figure 26: The inhibiting effect of Fe2+ on tPA-induced thrombolysis. The dose of tPA is
0.01 mg. A-C: Thrombolysis of tPA comparing with tPA plus different concentrations of
2+ FeCl2 (10 µM, 100 µM and 200 µM). n=22 in tPA group. n= 6—9 in each Fe group. * p
< 0.05, ** p < 0.01. 104
In a separated experiment, 200 µM of CaCl2 was tested in the same method used
to test zinc and iron. The application of Ca2+ did not affect tPA-induced thrombolysis
(data not shown). Taken together, both zinc and iron yielded significant reduction in tPA- induced thrombolysis. The reaction of Zn2+ was more sensitive as only zinc in small concentration (10 µM) produced mild but significant inhibition in tPA-induced thrombolysis. The optimal (maximum) inhibiting effect of zinc and iron were shown in figure 27. Zn2+, Fe2+ and Fe3+ all significantly attenuated tPA-induced thrombolysis, with the divalent ions being more powerful.
Figure 27: The maximal inhibiting effect of different ions on the overall amount of thrombolysis at the end of 60 minutes. The dose of tPA is 0.01 mg. The concentration of
Fe3+ is 100 µM. The dose of Zn2+ and Fe2+ is 200 µM. Values are mean ± SEM. The
absorbance values were measured at 580 nm wavelength, n = 22 in tPA alone group, n =
6-10 per group of other treatment. * p < 0.05. ** p < 0.01. 105
By calculating the reaction rate, we can further examine how zinc and iron altered
the rate of thrombolysis. Results show that Zn2+, at 10 µM, had significant inhibition
effect during the first 30 minutes of thrombolysis. The reaction rates in the groups of
Zn2+ and tPA co-application reached their maximum at about 40 minutes, compared to 20
minutes in the group with tPA alone. As revealed by the rate of thrombolysis over time in
Figure 28, the low dose (10 µM) of Zn2+ significantly reduced the slope of rate change.
However, it did not reduce maximum rate (efficacy) of tPA-induced thrombolysis. The observed delay of the maximum reaction rate suggests a competitive mechanism in which
Zn2+ might inhibit tPA activity. As the Zn2+ concentration increased to 100 µM and 200
µM, we observe a significant inhibition in the efficacy of tPA, and further delay of
maximum reaction rate (Figure 28).
106
Figure 28: The rate of thrombolytic activity in different concentrations of Zn2+. The
reaction rate was calculated by the change of absorbance value per minute. The dose of
tPA is 0.01 mg. A-C: Thrombolysis of tPA comparing with tPA plus different
concentrations of ZnCl2 (10 µM, 100 µM and 200 µM). n=22 in tPA group. n= 6—9 in each Zn2+ group. * p < 0.05, ** p < 0.01 107
On the other hand, when Fe3+ was co-applied with tPA, the results we observed
was a mixed story. At 10 µM Fe3+ showed a small increase in the rate of changes of tPA- induced thrombolysis, which was statistically significant at 25min, 40min, and 50min.
Although Fe3+, at 10 µM, did not alter the time needed for the reaction to achieve the
maximum rate, it did increase the amplitude of the maximum reaction rate (Figure 29A).
Fe3+ at 100 µM and 200 µM significantly inhibited the rate of thrombolysis (Figure 29B,
C). The efficacy of tPA was reduced in a similar and comparable manner with100 µM and 200 µM. Only Fe3+ at 100 µM significantly postponed the reaction (Figure 29B). The
mixed effect of Fe3+ in different concentrations suggested that Fe3+ might work through different mechanisms leading to both inhibition and promotion of thrombolysis, which depends on concentration of the ion.
108
Figure 29: The rate of thrombolytic activity in different concentrations of Fe3+. The dose
of tPA is 0.01 mg. A-C: Thrombolysis of tPA comparing with tPA plus different
concentrations of FeCl3 (10 µM, 100 µM and 200 µM). n=22 in tPA group. n= 6—10 in each Fe3+ group. * p < 0.05, ** p < 0.01.
109
Fe2+ at 10 µM did not affect the rate of thrombolysis in general, nor did it alter the
timing of the maximum reaction rate (Figure 30A). Fe2+ at higher concentrations significantly inhibited the rate of thrombolysis (Figure 30B). Comparing with the tPA group, the application of Fe2+ reduced the rate curve of tPA-induced thrombolysis to the
baseline. The powerful inhibition resulted in low amount of thrombolysis overall. Further
increasing Fe2+ concentration did not increase the inhibiting effect but still maintained the
reaction within an extremely low rate (Figure 30C). These data suggest that the inhibiting
effect of Fe2+ is saturated when the concentration reaches around 100 µM Fe2+.
110
Figure 30: The rate of thrombolytic activity in different concentrations of Fe2+. The dose
of tPA is 0.01 mg. A-C: Thrombolysis of tPA comparing with tPA plus different
concentrations of FeCl2 (10 µM, 100 µM and 200 µM). n=22 in tPA group. n= 6—9 in each Fe2+ group. * p < 0.05, ** p < 0.01. 111
Among all the ions studied, only Zn2+ at 10 µM, effectively attenuates the reaction by delaying the maximum rate, by more than 60%. Fe2+ and Fe3+ did not alter the timing of reaction efficacy (Figure 31).
Taken together, both zinc and iron affect the thrombolytic reaction of tPA. Zn2+
was the most sensitive inhibitor among the three, because it reduced the reaction at low
concentration. Fe3+ at low concentrations worked as a facilitator of thrombolysis. Fe3+ at
high concentration effectively inhibited thrombolysis. Divalent ions, both zinc and iron
had the most influence on the thrombolysis.
Figure 31: Time taken to reach the maximum rate of tPA-induced thrombolysis.
Treatment of 10 µM of the metal ion (ZnCl2, FeCl3 and FeCl2) was applied together with
tPA (0.01 mg) accordingly in each group. The rate of the thrombolysis reaction was
calculated as absorbance change per minute. Values are mean ± SEM. n = 22 in tPA
alone group, n = 6 per group of other treatments. ** p < 0.01. 112
4.3.4 Effect of ion chelation on tPA-induced thrombolysis in vitro
Given the results shown by this study, there are multiple ions involved in tPA induced thrombolysis. To further investigate zinc and iron involvement, EDTA, a chelator was used. This compound has been used as a reliable and high-affinity zinc and iron chelator. The stability constants of Zn2+, Fe2+ and Fe3+ are 16.5, 14.3 and 25.1.264
Blood clots were assigned into two groups. One group was treated with tPA alone.
Another group was treated with EDTA (5 mM) together with tPA. The third group was treated with EDTA (5 mM) without tPA.
While EDTA itself did not trigger thrombolysis, the EDTA significantly augmented the amount of thrombolysis when it was co-applied with tPA (Figure 32A).
The reaction rate with tPA and EDTA started to separate itself as being significantly higher than tPA-only group at 15 minutes (Figure 32B). Both treatments had a similar trend of rate change, with higher amplitude in (tPA + EDTA) group. Additionally, the efficacy (maximum rate) of thrombolysis was significantly higher when EDTA was co- treated with tPA. The treatment with (tPA + EDTA) yielded a significantly higher rate of thrombolysis from 15 minutes to 40 minutes. After 40 minutes, the curves of reaction rate in (tPA + EDTA) group and tPA alone group merged together.
113
Figure 32: EDTA, a chelating agent of metal ions, facilitates tPA-induced thrombolysis.
The dose of tPA is 0.01 mg. EDTA concentration is 5 mM. A. Quantified thrombolysis represented by change of optical density in 60 minutes. B. The rate of thrombolysis.
Values are mean ± SEM. n = 22 in tPA, n = 13 in (tPA + EDTA). * p < 0.05. ** p < 0.01.
114
4.3.5 Effect of Zn2+ chelation on tPA-induced fibrinolysis in vitro
To further focus on Zn2+ induced inhibition of blood clot-lysis, the fibrin clot was used. Blood samples were recalcified and immediately centrifuged (2000 rpm, 15 minutes, 20 °C). Then blood samples were transferred to 37 °C water bath for continuous coagulation for 3 hours. Using this method, the platelets and fibrin of the blood clot was preserved while the red blood cells were mostly centrifuged out from the blood clot, which minimized the potential effect of iron on blood clot-lysis during tPA reaction.
Those blood clots were called fibrin clot due to the absence of red blood cells. Fibrin clots were treated with tPA or tPA plus EDTA. The lysate was collected from each group and analyzed on a SDS-PAGE gel and labeled with D-dimer antibodies on western blots.
During blood clot-lysis, fibrin bundles are cleaved and digested by tPA into fibrin pieces. Fibrin D-dimer is one of the well detected pieces and has been used as a label of spontaneous thrombolysis in clinic. In this study, higher amount of fibrin D-dimer was detected in tPA plus EDTA group, in comparison to tPA only treatment. Results indicated that EDTA promoted fibrinolysis and thrombolysis through reducing Zn2+ level
(Figure 33).
115
Figure 33: Western blots of tPA-induced fibrinolysis. The dose of tPA is 0.01 mg.
EDTA concentration is 5 mM. A. Fibrin D-dimer is used as an indicator of the amount of fibrinolysis. B. Quantification of western blots. Increased fibrinolysis was observed in the group where EDTA was co-applied with tPA. Values are mean ± SEM. n = 2 in each treatment.
4.4 Effect of ion chelation on tPA-induced thrombolysis in vivo
EDTA is a high affinity chelator of zinc and iron but it also can combine with calcium. Given the fact that plasma contains high level of calcium that plays a critical role in hemostasis, we used EDTA’s analogue CaEDTA in the present study. CaEDTA is
EDTA saturated with calcium so that when it is administered it does not affect blood calcium levels.
The model of in vivo thrombosis was developed using the femoral artery of mice.
Photothrombosis was used to introduce blood clotting in the femoral artery by light exposure. Rose Bengal is a photosensitive chemical producing singlet oxygen under light 116 exposure. Singlet oxygen breaks down endothelial cells of the blood vessel and initiates thrombosis. The femoral artery runs superficially in the hind limb of mice (Figure 34A).
Rose Bengal was systematically delivered through the tail vein followed by 20 minutes of light exposure (light intensity: 1300 µW/cm2) on top of the femoral artery. The occlusion was considered successful if after light exposure there was a dark blood clot in the exposed location and emptiness of the artery downstream (Figure 34B). The dose of tPA was given according to body weight through 30 minutes of continuous intravenous perfusion which was preceded by a tPA bolus. Images of the femoral artery were acquired by Moticam 2500 camera every 10 minutes under a dissection microscope.
The blood clot in the femoral artery was initially dark, and gradually became lighter during the tPA treatment. We observed the reperfusion when blood had begun refilling in previously empty downstream region in the femoral artery (Figure 34C). The reperfusion was later re-confirmed by cutting the artery downstream of the occlusion and observing bleeding from the cut. Only these treatments were considered as successful reperfusion of the artery. Unsuccessful reperfusion was shown as a dark blood clot still occluding the vessel with emptiness remained in the artery (Figure 34B) and no bleeding when the femoral artery was cut.
117
Figure 34: Photothrombosis and thrombolysis in vivo. A. The femoral artery (shown by an arrow) of CD1 mouse. Scale bar: 1 cm. B. Photothrombotic occlusion (shown by an arrow) is formed in the femoral artery (outlined by dash lines), leaving downstream of the artery blood-free. C. Reperfusion of the femoral artery after tPA treatment. Scale bar: 1 mm.
We also examined the time course of tPA-induced thrombolysis by measuring the light transmission of thrombosis (blood clot) in the occluded femoral artery. Images of the occluded area of femoral artery were taken every 5 minutes. The change of light transmission in the blood clot was visualized and compared among all the cases from three groups. Representative sequenced images are shown in Figure 35A.
In general, in the cases of artery reperfusion which were mostly seen in tPA group and in ½ tPA + CaEDTA group, the light transmission of the blood clot was gradually increased as blood re-entered the empty artery indicating successful reperfusion. In the cases of continuous occlusion with no reperfusion, which were mostly seen in ½ tPA group, the occlusion region gradually became darker due to decreased light transmission.
The downstream region of the artery remained empty suggesting unsuccessful 118
reperfusion. Further quantification of the time course of reperfusion was then performed
using ImagePro software.
Sequenced images of each trial were measured with ImagePro. Change of light
transmission in the blood clot was measured as light intensity change, which was shown
as (F-F0)/F0 in arbitrary units. In the perfused cases, light transmission of the blood clot was gradually increased due to the gradually lysed blood clot. When the initial light transmission was set as the baseline, the blood clot being gradually digested generated positive change of light transmission. In contrast, in non-perfused cases, failure to digest the blood clot lead to decreased light transmission, which caused negative change of light transmission. We used the curve generated from the values of light transmission as an indicator of the rate of thrombolysis in vivo. The positive change of light transmission was label as a successful clot-lysis (Figure 35B). 119
Figure 35: Real-time thrombolysis in vivo. A. Representative sequenced images of tPA- induced thrombolysis in the femoral artery (outlined by dash lines). The region of interest
(ROI) for measurement of light intensity is schematically marked as ¢ in the first image of each group. Scale bar: 0.5 mm. B. The quantification of the blood clot-lysis. The ROI of sequenced images of the blood clot was analyzed by ImagePro and presented as the change of light intensity in every 5 minutes during tPA perfusion. The light intensity value of the blood clot at 0 min was set as zero. Cases with the femoral artery 120 successfully reperfused all generate a positive change of light transmission over time, indicating the blood clot being lysed gradually. Thrombosis from cases with the femoral artery not reperfused generates a negative change of light intensity due to unsuccessful lysis and the gradually darkened blood clot.
The results above suggest that the increase of light transmission in the occluded region can be an indicator of thrombolysis. The change of light transmission of the occlusion was then measured and was plotted overtime. When comparing tPA group and
(½ tPA + CaEDTA) group (both groups yielded the similar rate of successful reperfusion), the co-application of tPA and (½ tPA) together with CaEDTA showed fast increase of light transmission, suggesting faster thrombolysis, especially during the first
20 minutes (Figure 36). Thus, the result revealed that ion chelation not only improved thrombolytic outcomes of tPA, but also accelerated the thrombolysis process.
121
Figure 36: Change of light transmission of thrombosis in tPA group and (1/2 tPA +
CaEDTA) group. Data acquired from in vivo reperfused cases and were analyzed by
ImagePro. A rapid increase of light transmission is observed during the first 20 minutes in (1/2 tPA + CaEDTA) group, indicating a faster thrombolysis than tPA group. The full dose of tPA is 20 mg/kg. The dose of (1/2 tPA) is 10 mg/kg. Values are mean ± SEM. n =
7-9, * p < 0.05.
After photothrombotic treatment and blood clot formation, three different doses of thrombolysis agent tPA were administered through intravenous perfusion in separated experimental groups. One group, referred as tPA group, was given 20 mg/kg of tPA. 122
Another group was referred as ½ tPA group because only a half dose or 10 mg/kg of tPA was administered. In the third group, to investigate the effect of chelation on tPA-induced thrombolysis in vitro, the 10 mg/kg of tPA was co-applied with a chelating treatment
CaEDTA (30 mg/kg). It was therefore referred as ½ tPA + CaEDTA group. CaEDTA is
EDTA saturated with calcium so that when it is administered it does not affect blood calcium levels. CaEDTA when applied alone did not show thrombolytic effect. In tPA group, 2 mg/kg tPA resulted in 70% (7 out of 10) successful reperfusion rate after photothrombosis or occlusion. The rate of reperfusion was decreased to 20% (2 out of 10) when the dose of tPA was reduced into half (10 mg/kg) in the ½ tPA group. However, the insufficient reperfusion seen in ½ tPA group was significantly improved in the presence of CaEDTA. The co-application of tPA (1 mg/kg) and CaEDTA yielded 66.7% (8/12) reperfusion rate (Figure 37), indicating that CaEDTA improved the potency of tPA in tPA-induced thrombolysis. With the help of chelation, the necessary dose of tPA could be reduced for the same thrombolytic effectiveness.
123
Figure 37: The application of CaEDTA together with half dose of tPA facilitates thrombolysis, and achieves similar successful reperfusion as full dose of tPA in vivo. The bar graph shows that the percentage of the successful cases of reperfusion in the femoral artery with three tPA treatment groups. The dose of tPA is 20 mg/kg. The full dose of tPA is 20 mg/kg. The dose of (1/2 tPA) is 10 mg/kg. CaEDTA dose is 30 mg/kg. * p <
0.05 according to fisher exact test. n = 10-12
124
5. DISCUSSION
In this study, I investigated important role of Zn2+ overload and Zn2+ chelation in cerebral ischemia and thrombotic stroke. Zn2+ overload was observed in ischemic
cerebral damage. In order to determine the protective effect of Zn2+ chelation, I
developed adult zebrafish as a novel model of cerebral ischemia for high throughput tests
in vivo, and a model for thrombotic stroke to evaluate thrombolytic agents. Results
showed that Zn2+ chelation not only prevented ischemic cerebral damage and increased the recovery rate from hypoxic attack, but also promoted tPA-induced thrombolysis, including accelerating the thrombolytic process and reducing the necessary dose of tPA.
In light of these results, I have proposed a new strategy for treatment of thrombotic stroke, which includes using Zn2+ chelation as an assistant treatment together with tPA, in
order to increase therapeutic effects and minimize side effects and cytotoxicity of tPA.
5.1 The zebrafish is developed as a model of global cerebral ischemia
By developing adult zebrafish as a stroke model, I demonstrated that hypoxic
treatment effectively induced zebrafish brain damage. Under severe hypoxic conditions
(DO = 0.6-0.8 mg/L), zebrafish quickly lost motion except opercular movement with the
average hypoxia time of about 11 minutes (679.52 seconds) (table 2). The distribution
analysis of individual zebrafish revealed that hypoxia times for the majority of zebrafish
(>70%) were between10-12 minutes (Figure 6). Essentially, 55% of tested zebrafish did
not recover from hypoxia. These data indicate that zebrafish are sensitive to hypoxic
attack. About 45% of tested zebrafish survived hypoxic treatment and recovered by
regaining coordinated swimming. Through recovery following hypoxic treatment, all 125
surviving zebrafish experienced erratic movements such as unbalanced swimming before
sufficiently recovering their swimming ability (Figure 7).
Using fish in oxygen deficiency research is not entirely new. Many studies have
addressed the environmental impact of ecological hypoxia or anoxia killing fish
globally265. However, no study has reported the use of fish or zebrafish as a model for
hypoxic-ischemic brain damage, even though the zebrafish has been intensely used in
biological research, ranging from embryonic genetic modification to adult behavioral
studies. Because the zebrafish is freshwater fish that were originally found in shallow
streams and rice paddies, it is not surprising that zebrafish are not tolerant to hypoxic
treatment. As shown in this study, they were only able to sustain severe hypoxia for short
duration of time with mean duration of about 11 minutes, providing a prerequisite for
zebrafish being an ischemic model.
The custom-made hypoxia chamber allowed for the quick creation of an oxygen
deprivation environment, which was maintained at a steady non-fluctuating hypoxic state
throughout the testing period. Reducing the dissolved oxygen quickly, depleted
oxygenated blood in the zebrafish circulation, which is detrimental to brain function.
When dissolved oxygen reached below 0.8mg/L, the zebrafish became a model for
complete global cerebral ischemia that closely mimics the brain attack under the cardiac
arrest10, 266.
The fish gill is the primary regulatory interface between internal and external milieu. Because of the single circuit: heart-gills-body-heart, fish are regarded to be extremely susceptible bioindicators of environmental changes267, 268. Among ischemic 126 stroke models, the induction of global ischemia is compromised by the presence of the circle of Willis that gives collateral blood supply to the forebrain. The Mongolian gerbils are widely used as a model for global ischemia due to a species-specific incomplete circle of Willis. However, the gerbils might present various problems when used in models for global cerebral ischemia, as some animals do not have a totally incomplete circle of
Willis10, 266.
The endpoint of hypoxic treatment is determined as such: the zebrafish was motionless with occasional opercular movements for one minute. To determine an effective endpoint was a considerable task. Insufficient hypoxia or insufficient time in the hypoxia chamber allowed zebrafish to survive without brain damage, and on the other hand, over treatment of hypoxia resulted in zebrafish death, leaving no opportunity to use drugs to rescue the injured brain. The endpoint set in this study appears effective. We observed death in about 55% of zebrafish and 45% zebrafish survived following hypoxic treatments, implying that this dose of hypoxia is similar to LD50 (Figure 5, Table 2).
Tests of different hypoxia time also showed that this is a responsive animal model system to oxygen deprivation, and is suitable for use in developing therapeutic interventions for hypoxic-ischemic brain damage.
After hypoxic treatment, when zebrafish were recovering in normal oxygen water, we observed that some zebrafish were motionless, then regained swimming behavior but were unable to maintain balanced position. Data further showed a significant decrease in
TTC staining after zebrafish underwent hypoxic treatment. In normal tissue, dehydrogenases reduce TTC to formazan, which gives tissue red staining269. This method 127
has been widely used to demonstrate irreversibly damaged cerebral tissue in rats and
other rodents. To the best of our knowledge it is the first time that TTC staining has been
used in zebrafish. Due to the small size (about 2mm in diameter) of the zebrafish brain,
the density of the staining was quantified using spectrophotometry. Quantified TTC staining showed a significant difference in cell viability between healthy brain and 8-
minute hypoxic treated brain (Figure 9). These data indicate that hypoxic treatment
induces brain damage that can be detected and quantified by mortality rate, behavioral
changes, and histological staining. Results suggest that the model I present in this study
can be used as an alternative model to evaluate hypoxia induced brain damage and can be
possibly developed into a high throughput model.
5.2 Advantages of using zebrafish as an ischemia model
There is ongoing debate of the effectiveness of current stroke models271-275.
Experimental models of stroke are available in a variety of species, including primates, pigs, sheep, dogs, cats, Mongolian gerbils, rabbits, rats and mice. Whether zebrafish can be used to be a model for ischemic brain damage remains to be seen since there is no literature available about hypoxic-ischemic brain damage in zebrafish. My results suggest that the zebrafish is a reliable and reproducible model appropriate for use in the study of hypoxic-ischemic brain damage. Hypoxic brain damage in zebrafish evaluated by TTC staining closely resembles hypoxic-ischemic brain damage documented in other animal models or species. The zebrafish, as a relative simple animal, allows studies of ischemic, especially global ischemic, damages under well-controlled, noninvasive, reproducible conditions. 128
Besides its simplicity as an animal model, and its susceptibility to ischemic brain
damage, other advantages of this model include that it is not invasive and doesn’t
mechanically injure vasculature and tissues. Contemporary models for the study of
pathophysiology and therapies for cerebral ischemia, including both focal and global
ischemia models and they have limitations. One of limitations is that injuring vasculature
or brain structures is unavoidable in some circumstances, which may contribute to the
failure in making a reliable model, increase animal mortality, and create difficulty in
evaluating data266, 270. Another unexpected effect of tissue injuries is that it may trigger the local or global stress responses that may pre-condition the targeted regions to hypoxic/ischemic insults. The zebrafish model presented in this study has therefore the advantage of being noninvasive and technically easier and faster than other stroke models.
Another unique advantage of this zebrafish system is that drugs can be directly added into the water to accelerate brain restoration after hypoxic treatment. The zebrafish can easily take dissolved chemicals with water into its body through its gills merely by breathing. Zebrafish circulation is a single circuit: heart—gills–body–heart. Chemicals are typically added to the aquatic environment in which they live, allowing for easy experimental manipulation and observation. In this study, the Zn2+ chelator DEDTC was administrated by adding into water. DEDTC is both lipophilic and hydrophilic276, 277,
which allows it to cross the cell membrane and blood brain barrier. Zn2+ staining after
DEDTC treatment shows obvious reduction of Zn2+ signal, suggesting a successful drug
delivery (Figure 13). 129
Additional advantages of developing a translational system in zebrafish include
the ability to create genetically mosaic animals, efficient in vivo tests for genetic
interactions, conserved cellular architecture of the central nervous system, and tractable
behavior278, 279. Zebrafish are useful vertebrates who have a completed genome synthesized. Forward and reverse screenings have been successfully applied in zebrafish.
Recent breakthrough evidence has shown the feasibility and successful use of zebrafish for high throughput behavioral-based drug screening280, 281. These will help us develop and refine the argument that the zebrafish is a useful animal model for studying brain damages resulting from hypoxic-ischemic stroke.
5.3 Advantages of adult zebrafish being a thrombotic stroke model
I demonstrate that photothrombotic treatment effectively and reproducibly induces brain damage in zebrafish. Photothrombosis has been a well-accepted method to reproduce brain ischemia in rodent models. We are the first group to use zebrafish to develop a focal cerebral ischemia model.
To induce effective and reproducible brain damage via photothrombosis, both the dose of Rose Bengal and light exposure, as well as the time of exposure has to be strictly controlled. One of the major findings of this study is that using adult zebrafish as a model of focal cerebral ischemia is feasible via the photothrombotic method. Brain injury of zebrafish can be observed as unbalanced swimming due to the damage of optic tectum113,
126. We observed two characteristically abnormal swimming patterns of circling and
rotating in zebrafish with brain injuries (Figure 10A, B). Behavioral changes were closely
correlated to the dose of photothrombotic treatment (light intensity and duration of light 130
exposure) (table 4). TTC staining also showed significantly reduced staining comparing
to that of healthy controls (Figure 11A). The application of tPA resulted in both
significant increase of TTC staining and significantly greater rate of recovery (Figure 11,
table 5). Those results support that the brain damage is caused by occlusion of blood
vessels in the zebrafish brain.
There are growing efforts to develop zebrafish as a model system in neurological
studies ranging from genetic to behavioral evaluations116, 121, 282. Adult zebrafish as a model to study focal ischemic brain damage has not been fully explored at the present.
There are several reasons that the zebrafish model helps the research of thrombotic ischemic brain damage and ultimately contributes to the advancement of therapeutic drug development. First, my study indicates that the brain of zebrafish is sensitive to thrombotic ischemic damage. As shown here (Table 3, 4, Figure 11, 12), photothrombosis induced brain injury and death of zebrafish occurred in a short time.
Second, the optic tectum of zebrafish communicates with the visual input and the body movement150, and is required for optomotor response while swimming127. As a result, consistent abnormal swimming patterns such as rotating and circling can be observed after local ischemia of the optic tectum, which makes zebrafish a reliable model for observing behavioral changes after brain damage (Figure 10). Delayed behavioral changes are observed in zebrafish 24 hours after photothrombosis, which make the evaluation and measurements practical for delayed brain injury and recovery.
Another advantage of the zebrafish is the fully sequenced genome and the conservation of clotting factors. Studies have shown that zebrafish preserve most factors 131
and regulators of blood coagulation, which are structurally and functionally the same as
in humans156, 157, 283, 284. Because of availability of well-characterized mutants and conservation of blood coagulation, it is currently practical and superior to use zebrafish for studies of hemostasis and thrombosis.
Activase (tPA) has been the only FDA approved drug for thrombotic cerebral ischemia. We demonstrate in this study that photothrombotic zebrafish respond well to tPA treatment (Figure 11), making zebrafish a feasible model for investigating candidates of thrombolytic agents and comparing them to the effect of tPA. Application of tPA reduced brain damage and the mortality rate, as well as improved behavioral recovery
(Table 5) zebrafish, which provides standard criteria for future evaluation. We propose zebrafish as a reliable alternative model for thrombotic stroke. This model can benefit not only the screening of neuroprotective candidates, but also studies to improve tPA efficacy.
5.4 The in vivo neuroprotective effect of Zn2+ chelation in oxygen deprivation
Besides demonstrating that the zebrafish is a useful model for the screening of anti-stroke treatments, another major finding is that Zn2+ chelation with DEDTC increases zebrafish survival rate after hypoxic treatment and enhances the tolerance of zebrafish to hypoxia. The results support previous observations that rising intracellular
Zn2+ during hypoxic-ischemic treatment plays a critical role in neuronal damage and
brain function187, 188. Reducing intracellular Zn2+ significantly increased neuronal
survival3, 254, 285. 132
Zn2+ has been found to be released from metalloproteins and other storages during
oxidative stress235, 286. Zn2+ itself is a strong inducer of oxidative stress by promoting mitochondrial and extra-mitochondrial production of reactive oxygen species, and promoting neuronal death191, 287. The application of DEDTC in this study reduced intracellular Zn2+ accumulation and increased zebrafish survival (Figure 13, 14). The latter was probably achieved by reducing oxidative stress that was triggered by Zn2+.
Therefore, this study supports that the increased level of intracellular Zn2+ is a critical
factor in brain damage caused by hypoxic stress.
Brain damage was evaluated with TTC staining, a common method used in other
mammal models. In zebrafish that were treated with DEDTC and hypoxic treatment we
observed improvement of TTC staining that was similar to the control. Hypoxic treatment
significantly reduced TTC staining. The reduction in TTC staining was reversed when
DEDTC was applied (Figure 16). The elevated intracellular Zn2+ is correlated with mitochondrial dysfunction, loss of mitochondrial defenses, and increased production of reactive oxygen species184, 191, 288. TTC is enzymatically reduced to a red formazan
product by dehydrogenases, which are most abundant in mitochondria. Zn2+ chelation, by
removing excessive Zn2+ during hypoxia, may minimize mitochondrial dysfunction, and
consequently preserved TTC reduction to a red formazan.
The application of a Zn2+ chelator to prevent damages caused by Zn2+ overload is
under active investigation as a treatment for stroke254, 289. In this study, the application of
the Zn2+ chelator DEDTC improved the survival and tolerance under the hypoxic- ischemic treatment in the zebrafish model (Figure 15). Zn2+ is required for normal 133
biological processes, the advantage for using a moderate chelator is to maintain the level
of Zn2+ in the optimal range, thereby preventing the brain damage by excessive Zn2+
while avoiding an unwanted Zn2+ deficiency187.
At present, despite clear demonstrations of numerous agents that can prevent the
cascade of events leading to ischemic neuronal death in animal models, it is not known
whether any particular neuroprotectant is more effective than others to improve the
outcome of a stroke in humans 258, 290-292. Therefore, the effective strategy may be a combined treatment that targets the multiple events that are related to Zn2+
dyshomeostasis.
5.5 Spectrophotometric measurement of thrombolysis
The first method used in this study to measure blood clot-lysis was to measure
clot weight in the centrifuge tubes before and after treatment with clot-lysis. The
difference of clot weight was considered as overall amount of clot-lysis. The method is
useful especially in screenings, when a large number of clot-lysis candidates need to be
tested for optimal clot-lysis outcomes. It is also a method suited for large sample volume,
in order to overcome the potential error from fluid aspirating and weight measurement.
The method is not designed for sensitive measurement of sample volume smaller than
100 µl. While the procedure is straightforward, it cannot provide kinetic information of
blood clot-lysis. Therefore, spectrophotometry was adopted and modified for capturing in
more details blood clot-lysis in a more sensitive manner.
The spectrophotometric method has been well accepted in blood quantification. It
has been used to detect hemoglobin since 1960s293. Spectrophotometric assays can 134 quantify intracellular hemorrhage by measuring absorbance of cyanomethemoglobin294.
Spectrophotometric measurement of euglobulin has been used for fibrinolysis assays of blood plasma295. In this study, we used measurement of hemoglobin as an indicator of blood clot-lysis. Optimal wavelength is used for minimizing standard error of absorbance. The wavelengths from 400 nm to 600 nm were examined and the one of the peaks of absorbance value for hemoglobin was at 580 nm (data not shown). Clot-lysis is quantified by measuring absorbance value of each sample at 580 nm.
When blood clot is being lysed, proteolysis of fibrin is releasing red blood cells and hemoglobin into the clear solution and yields increasing absorbance value. Lysis of the blood clot, showing as increased absorbance value, became obvious within 15 minutes and continued to increase during one-hour measurement. The rate of clot-lysis was calculated as absorbance change per minute, which reached maximum at 25 minutes of clot-lysis and gradually decreased after that. The overall thrombolysis generated by different doses of tPA is well separated by measuring absorbance (Figure 22).
The plot of rate changes during thrombolysis provides more details throughout the thrombolysis reaction, such as the maximum rate, and the time needed to achieve maximum rate (Figure 21). The overall tPA-induced thrombolysis, as well as the efficacy which is indicated by the maximum reaction rate was significantly reduced by zinc and iron (Figure 24-31). Results have revealed that both zinc and iron play significant roles in thrombolysis with divalent ions being most potent. 135
5.6 Zn2+ in blood dynamic and its inhibiting effect in tPA-induced thrombolysis
High concentration of Zn2+ is packaged in α-granules of platelets. During thrombolysis, mechanical stimulation triggers exocytosis of platelets. The release of granules from platelets produces locally high concentration of zinc296-300. In this study, blood clots used in vitro were made from whole blood after 3-hours of incubation at
37°C. I tested different doses of Zn2+ up to 200 µM. Zn2+ attenuates thrombolysis by reducing efficacy of tPA in a dose-dependent manner. Zn2+ started to show inhibition at
10 µM by postponing the maximum rate of tPA. At 100 µM, Zn2+ further delayed the maximum rate and significantly reduced the total amount of thrombolysis. When Zn2+
concentration was high (200 µM), efficacy (maximum rate) of thrombolysis was
significantly inhibited (Figure 24, 28). These data suggest that zinc suppresses tPA-
mediated thrombolysis. The mechanisms may include the following different aspects.
First, Zn2+ prevents thrombolysis by activating coagulation. It has been shown
that zinc plays important roles in several steps of blood coagulation. For example, it is
well known that Zn2+ triggers platelets aggregation. Even though physiological Zn2+ in
plasma does not activate coagulation pathways, Zn2+ is critical for the initial step of the
intrinsic coagulation pathways. Zn2+ activates FXII and potentiates blood clotting when
local zinc concentration is 10-30 folds higher than plasma301, 302. Zn2+ is also required for the interactions between FXI and platelets, and FXI-supported platelets adhesion in hemostasis303. When it comes to intensifying polymerization of fibrin oligomer, Zn2+ is
an inducer stronger than Ca2+ 233. Previous studies show that Zn2+ promotes 136
polymerization of fibrin oligomer several folds more than Ca2+, and keeps increasing as
the concentration of Zn2+ goes up above physiological levels304.
Second, Zn2+ prevents thrombolysis by inhibiting fibrin clearance. Zn2+ binding and neutralizing the effect of anticoagulation of heparin233, Zn2+ has been found to bind
to fibrinogen and fibrin. This binding inhibits the binding of decorin to fibrinogen.
Decorin changes fibrin structure by binding with it, and makes the fibrin thinner and
easier to be lysed by tPA. By preventing decorin binding, Zn2+ abolishes the enhancement effect of decorin on tPA-induced thrombolysis305.
Additionally, Zn2+ binds to fibrinogen and fibrin. This binding strengthens fibrin
bundles, and amplifies the diameter of fibrin, which makes fibrin harder to be degraded.
The results of this study showed that Zn2+, in the low concentration (10 µM) postpones the maximum reaction rate of tPA-induced thrombosis, without reducing it (Figure 28A).
This results support the hypothesis that Zn2+ inhibits tPA-induced thrombolysis by competitively binding to fibrin with plasmin. As the Zn2+ concentration increases, the maximum rate is reduced, suggesting other inhibiting mechanisms are involved as well.
Inhibiting mechanisms may include Zn2+ binding to tPA and directly inhibiting the conversion of plasminogen to plasmin.
Dissociated Zn2+ during thrombolysis binds to tPA and can reduce tPA activity and suppress the conversion of plasminogen to plasmin9, 70, 306-309. Previous studies using
56Zn have shown that Zn2+ binds to tPA in a concentration-dependent manner306. Latest studies have found that zinc binds to tPA and plasmin with high affinity (with Kd value of 0.2 µM), and reduces the tPA-induced activation of plasminogen310. Results in this 137
study also show that Zn2+ (10 µM) postpones the maximum reaction rate of tPA, suggesting a competitive binding between tPA and Zn2+. Additionally, Zn2+ attenuates
plasmin-induced fibrinolysis in a dose-dependent manner in recent studies310, which is consistent with the finding discussed above from whole blood clots. However, the Zn2+
binding domain of tPA remains unknown.
Different methods were used to confirm the importance of Zn2+ in thrombolysis.
Fibrin clots were prepared to eliminate the potential interference of red blood cells and
eliminate effect of Fe2+ and Fe3+. Centrifuging blood samples during blood coagulation is
an accepted method to produce platelets-rich fibrin clots in clinical setting, with the
therapeutic application on wound healing311-313. The western blot assay shows that the co-
application of EDTA with tPA generated higher thrombolysis than tPA treatment alone,
in the absence of red blood cells. The western blot assay is not used as the main
quantitative assay in this study, because of the fact that fibrin and fibrinogen, together
with fibrin degradation pieces share similar protein structure. This leads to non-specific
binding of antibodies. However, the assay still supported the involvement of Zn2+ in inhibition of tPA-induced thrombolysis (Figure 33). The proposed mechanisms are illustrated in Figure 38.
138
Figure 38: Zn2+ as an inhibitor of blood clot-lysis. Zn2+ inhibits fibrin degradation through three aspects: promoting aggregation of platelets (red arrow), reducing tPA- mediated activation of plasmin (green dash line), and binding with fibrin to prevent fibrin degradation (green dash line). Zn2+ also prevents overall blood clot-lysis through potentiating blood coagulation.
5.7 Iron in blood dynamic and thrombolysis
Erythrocyte contains 2-3 g iron, with hemoglobin as one of the major sites of storage. Iron has been proposed to be an important factor in blood coagulation. Fe3+
generates hydroxyl radicals through the following reaction: Fe3+ + HO– → Fe2+ + HO.
Previous studies in purified system as well as in whole blood have shown that fibrinogen can be converted to fibrin-like polymer in the presence of hydroxyl radicals252. It is also suggested that Fe3+ strengthens fibrin and make it more resistant to proteolysis314. Studies
of relationship between Fe2+ and coagulation are not as substantial as that of Fe3+. During thrombolysis, the release of hemoglobin contributes to locally high concentration of Fe3+ 139
and Fe2+, which leads to the importance of investigating the possible effect of iron on tPA
and fibrinolysis.
In vitro results showed no effective inhibition on thrombolysis when adding 10
µM of Fe3+ or Fe2+. Fe3+ was observed to mildly promote thrombolysis at 10 µM (Figure
25). The possible explanation is that increase of iron may promote plasmin activity at cell surface315. When increasing iron (Fe2+ and Fe3+) concentration, the inhibition effect of iron overcame the promotion effect by suppressing fibrinolytic enzymes and accelerating thrombosis (Figure 25, 26)316, 317. In our study, Fe3+ was observed to postpone the
maximum rate of thrombolysis at 100 µM. And this effect was replaced by slight
reduction of maximum velocity at 200 µM. Fe2+ showed more severe inhibition by reducing rate of thrombolysis reaction than Fe3+ at 100 µM and 200 µM (Figure 29, 30).
That could be explained because of iron binding to proteins, when it happens, the bond
distance generated by trivalent iron (Fe3+) is longer than that generated by divalent iron
(Fe2+)318. Therefore, Fe2+ binding with proteins is more stable. The hypothesized mechanism is that iron blocks fibrinolysis pathway by either binding and inhibiting tPA like Zn2+, or blocking the pathway of fibrin degradation. The hypothesis remains to be tested in future studies.
5.8 Metal ion chelation with EDTA and CaEDTA
EDTA is a cell impermeable zinc and iron chelator. The stability constants of
Zn2+, Fe2+ and Fe3+ are 16.5, 14.3 and 25.1264. Our results show that EDTA promotes
thrombolysis by augmenting efficacy of tPA-mediated thrombolysis. The efficacy of
thrombolysis, being represented by the maximum rate, is increased by 55% with EDTA 140
(Figure 32). Although with relative low affinity, we cannot rule out the possibility that
EDTA binds to calcium while binding to zinc and iron (the stability constant of calcium
is 10.96). When adding extra Zn2+ together with tPA treatment, both the amount of clot-
lysis and the rate of thrombolysis are significantly reduced. Adding extra Ca2+ (200 µM), on the other hand, does not reduce thrombolysis at all (data are not shown). I conclude that the enhancement of thrombolysis by EDTA is because of Zn2+ and Fe3+/Fe2+ chelation, not Ca2+ drop.
Interestingly, even though Fe3+ has a much higher binding affinity to EDTA than
Zn2+, Zn2+ is able to displace Fe3+-EDTA. The evidence is that Zn2+ reduces FeEDTA- mediated oxidation by 90% when Zn2+ is added in 1:1 molar ratio to Fe3+ 264. The mechanism of displacement of Fe3+ from EDTA by Zn2+ is not clear. It may include hydrolysis-aided dissociation of Fe3+ or Fe3+ as a redox-active ion, is reduced to Fe2+ and
outcompeted by Zn2+ at binding EDTA264, 319. The important part is that Zn2+ is able to bind EDTA even when EDTA is preoccupied by Fe3+. It further supports our hypothesis
that Zn2+ chelation plays a critical role in improvement of thrombolysis.
In vivo study, keeping the homeostasis of Ca2+ is important because of the large
amount of Ca2+ both in cells and in blood plasma. CaEDTA was used instead of EDTA to
minimize calcium disturbance. Results revealed a significant effect on thrombolysis when
chelation was combined with tPA compared to tPA used alone (Figure 37). Half dose of
tPA plus CaEDTA achieved similar reperfusion rate compared to full dose of tPA (Figure
37), suggesting CaEDTA increased the potency of tPA-mediated thrombolysis. The
measurement of light transmission during thrombosis showed that half dose of tPA 141
together with CaEDTA lysed the blood clot faster than full dose of tPA (Figure 36). That
finding is consistent with EDTA increasing efficacy of thrombolysis in vitro. Data
suggest that by co-applying CaEDTA with tPA, the necessary dose of tPA is reduced
with improved efficacy.
CaEDTA combined with tPA may lead to less side effects and better thrombolytic
outcomes. As a severe side effect in thrombolytic treatment, hemorrhage is tPA dose-
dependent320. Studies have shown that reducing tPA dose (from 1 mg/kg to 0.5 mg/kg)
shortens the primary bleeding time by 60%321. Secondly, the application of tPA during stroke increases permeability of blood-brain barrier, thereby causes brain edema322.
Previous studies have shown that tPA can induce brain edema without plasminogen or
matrix metallopeptidases323, and in tPA-deficient mice the brain edema was reduced324.
Additionally cytotoxicity of tPA contributes to neuronal death325, 326. Therefore, by
reducing the necessary dose of tPA, ion chelation can be a promising treatment to reduce
side effects of thrombolysis.
Furthermore, ion chelation can benefit the neurovascular unit by binding to free
iron. It has been well established that accumulated free iron can injure endothelial cells
by producing free radicals327, 328. A growing body of evidence shows that zinc increase contributes to neuronal death329-332. Therefore, it is reasonable to hypothesize that ion
chelation can also improve the outcome of thrombolysis by reducing the toxicity of
dissociated zinc and iron.
Results of this study suggest that the application of EDTA/CaEDTA-mediated ion
chelation is promising to reduce tissue damage from oxygen-glucose-deprivation by 142
promoting reperfusion efficacy, and decrease the severe side effects by increasing tPA
potency. Specifically in brain ischemia, Zn2+ chelation not only protects neurons by limiting Zn2+ overload which induces ischemic injuries, but also promotes successful
restoration of blood flow. It can be developed as a new therapeutic strategy combining
with tPA treatment for a better outcome of thrombotic stroke.
5.9 Summary of the study
It is known that changes in ionic concentration have a fundamental effect on
numerous physiological and pathological processes related to ischemic cell death and
blood coagulation. I established zebrafish stroke models and used them together with
mouse models to further investigate the effect of ions (especially Zn2+). The adult
zebrafish has shown consistent ischemic damage during thrombotic stroke and is
responsive to tPA therapy. This further supports that zebrafish is a promising model
animal for high throughput studies. Given the results of the present study on mice and the
newly developed zebrafish model, I have found that zinc, at local high concentration, not
only enhances ischemic cell death, but also prevents fibrinolysis of tPA. Zn2+, together with iron (Fe3+ and Fe2+), inhibits tPA-induced thrombolysis by postponing the maximum
effect or decreasing tPA efficacy, depending on the ionic concentration. In vivo studies
confirm that Zn2+ chelation, more specifically, rescue ischemic brain damage and reduces
overall mortality rate. Furthermore, effective Zn2+ chelation can assist tPA for a higher rate of reperfusion, and reduces the necessary dose of tPA treatment. Future investigations with the current results can lead to an assistant therapy combined with tPA for better thrombolytic results and protection of the brain during or after stroke. 143
Part of this study has been published in the following journals: 1) Yu X, Li YV.
Zebrafish (Danio rerio) developed as an alternative animal model for focal ischemic stroke. Acta Neurochir Suppl. 2016;121:115-119. 2) Yu X, Li YV. Neuroprotective effect of zinc chelator DEDTC in a zebrafish (Danio rerio) model of hypoxic brain injury.
Zebrafish. 2013;10:30-35. 3) Yu X, Li YV. Zebrafish as an alternative model for hypoxic-ischemic brain damage. Int J Physiol Pathophysiol Pharmacol. 2011;3:88-96.
144
REFERENCES
1. Caplan LR. Stroke classification: A personal view. Stroke. 2011;42:S3-6
2. Grau AJ, Weimar C, Buggle F, Heinrich A, Goertler M, Neumaier S, et al. Risk
factors, outcome, and treatment in subtypes of ischemic stroke: The german
stroke data bank. Stroke. 2001;32:2559-2566
3. Stork CJ, Li YV. Rising zinc: A significant cause of ischemic neuronal death in
the ca1 region of rat hippocampus. J Cereb Blood Flow Metab. 2009;29:1399-
1408
4. Brott, T. Thrombolysis for stroke. Archives of neurology. 1996;53:1305-1306.
5. Kleindorfer D, Lindsell CJ, Brass L, Koroshetz W, Broderick JP. National us
estimates of recombinant tissue plasminogen activator use: Icd-9 codes
substantially underestimate. Stroke. 2008;39:924-928
6. Fang MC, Cutler DM, Rosen AB. Trends in thrombolytic use for ischemic stroke
in the united states. J Hosp Med. 2010;5:406-409
7. Xu J, Kenneth D. National vital statistics reports deaths: Preliminary data for
2010. Natl Vital Stat Rep. 2012;60
8. Wood-Dauphinee SL, Williams JI, Shapiro SH. Examining outcome measures in
a clinical study of stroke. Stroke. 1990;21:731-739
9. Vu TT, Fredenburgh JC, Weitz JI. Zinc: An important cofactor in haemostasis
and thrombosis. Thrombo Haemost. 2013;109:421-430
10. Bacigaluppi M, Comi G, Hermann DM. Animal models of ischemic stroke. Part
two: Modeling cerebral ischemia. Open Neurol J. 2010;4:34-38 145
11. Brierley JB, Graham DI, Adams JH, Simpsom JA. Neocortical death after cardiac
arrest: A clinical, neurophysiological, and neuropathological report of two cases.
The Lancet. 1971;298:560-565
12. Colbourne F, Auer RN, Sutherland GR. Behavioral testing does not exacerbate
ischemic CA1 damage in gerbils. Stroke. 1998;1967-70
13. Krause GS, Kumar K, White BC, Aust SD, Wiegenstein JG. Ischemia,
resuscitation, and reperfusion: Mechanisms of tissue injury and prospects for
protection. Am Heart J. 1986;111:768-780.
14. White BC, Sullivan JM, DeGracia DJ, O’Neil BJ, Neumar RW, Grossman LI, et
al. Brain ischemia and reperfusion: Molecular mechanisms of neuronal injury. J
Neurol Sci. 2000;179:1-33
15. Kerr JF, Wyllie AH, Currie AR. Apoptosis: A basic biological phenomenon with
wide-ranging implications in tissue kinetics. Br J Cancer. 1972;26:239
16. Niquet J, Baldwin RA, Allen SG, Fujikawa DG, Wasterlain CG. Hypoxic
neuronal necrosis: Protein synthesis-independent activation of a cell death
program. Proc Natl Acad Sci U S A. 2003;100:2825-2830
17. Portera‐Cailliau C, Price DL, Martin LJ. Excitotoxic neuronal death in the
immature brain is an apoptosis‐necrosis morphological continuum. J Comp
Neurol. 1997;378:10-87
18. Savill J, Fadok V, Henson P, Haslett C. Phagocyte recognition of cells undergoing
apoptosis. Immunol today. 1993;14:131-136 146
19. Neumar RW. Molecular mechanisms of ischemic neuronal injury. Ann Emerg
Med. 2000;36:483-506
20. Taatjes DJ, Sobel BE, Budd RC. Morphological and cytochemical determination
of cell death by apoptosis. Histochem Cell Biol. 2008;129:33-43
21. Häcker G. The morphology of apoptosis. Cell Tissue Res. 2000;301:5-17
22. Trump BE, Berezesky IK, Chang SH, Phelps PC. The pathways of cell death:
Oncosis, apoptosis, and necrosis. Toxicol Pathol. 1997;25:82-88
23. Wyllie A, Morris R, Smith A, Dunlop D. Chromatin cleavage in apoptosis:
Association with condensed chromatin morphology and dependence on
macromolecular synthesis. J Pathol. 1984;142:67-77
24. Zamai L, Falcieri E, Zauli G, Cataldi A, Vitale M. Optimal detection of apoptosis
by flow cytometry depends on cell morphology. Cytometry. 1993;14:891-897
25. Kristián T, Siesjö BK. Calcium in ischemic cell death. Stroke. 1998;29:705-718
26. Lipton P. Ischemic cell death in brain neurons. Physiol Rev. 1999;79:1431-1568
27. Reimer K, Jennings R. The" wavefront phenomenon" of myocardial ischemic cell
death. Ii. Transmural progression of necrosis within the framework of ischemic
bed size (myocardium at risk) and collateral flow. Lab Invest. 1979;40:633-644
28. Kametsu Y, Osuga S, Hakim AM. Apoptosis occurs in the penumbra zone during
short-duration focal ischemia in the rat. J Cereb Blood Flow Metab. 2003;23:416-
422
29. Kristian T, Siesjo BK. Calcium in ischemic cell death. Stroke. 1998;29:705-718 147
30. Tominaga T, Kure S, Narisawa K, Yoshimoto T. Endonuclease activation
following focal ischemic injury in the rat brain. Brain research. 1993;608:21-26
31. Sun M, Zhao Y, Gu Y, Xu C. Inhibition of nnos reduces ischemic cell death
through down-regulating calpain and caspase-3 after experimental stroke.
Neurochemistry international. 2009;54:339-346
32. Delledonne M, Xia Y, Dixon RA, Lamb C. Nitric oxide functions as a signal in
plant disease resistance. Nature. 1998;394:585-588
33. Nathan C, Xie Q-w. Nitric oxide synthases: Roles, tolls, and controls. Cell.
1994;78:915-918
34. Vandelle E, Poinssot B, Wendehenne D, Bentéjac M, Pugin A. Integrated
signaling network involving calcium, nitric oxide, and active oxygen species but
not mitogen-activated protein kinases in bcpg1-elicited grapevine defenses. Mol
Plant Microbe Interact. 2006;19:429-440
35. Scimeca J-C, Servant MJ, Dyer J-O, Meloche S. Essential role of calcium in the
regulation of map kinase phosphatase-1 expression. Oncogene. 1997;15:717-725
36. Whitmarsh A, Davis R. Transcription factor ap-1 regulation by mitogen-activated
protein kinase signal transduction pathways. J Mol Med. 1996;74:589-607
37. Clarke a, Desikan R, Hurst RD, Hancock JT, Neill SJ. No way back: Nitric oxide
and programmed cell death in arabidopsis thaliana suspension cultures. Plant J.
2000;24:667-677
38. Lam E. Controlled cell death, plant survival and development. Nat Rev Mol Cell
Biol. 2004;5:305-315 148
39. Leist M, Volbracht C, Kühnle S, Fava E, Ferrando-May E, Nicotera P.
Caspase-mediated apoptosis in neuronal excitotoxicity triggered by nitric oxide.
Molecular Medicine. 1997;3:750
40. Kristián T, Gertsch J, Bates TE, Siesjö BK. Characteristics of the calcium‐
triggered mitochondrial permeability transition in nonsynaptic brain
mitochondria. J Neurochem. 2000;74:1999-2009
41. Petit PX, Goubern M, Diolez P, Susin SA, Zamzami N, Kroemer G. Disruption of
the outer mitochondrial membrane as a result of large amplitude swelling: The
impact of irreversible permeability transition. FEBS letters. 1998;426:111-116
42. Pivovarova NB, Nguyen HV, Winters CA, Brantner CA, Smith CL, Andrews SB.
Excitotoxic calcium overload in a subpopulation of mitochondria triggers delayed
death in hippocampal neurons. J Neurosci. 2004;24:5611-5622
43. Arts IS, Gennaris A, Collet JF. Reducing systems protecting the bacterial cell
envelope from oxidative damage. FEBS letters. 2015;589:1559-1568
44. Jomova K, Valko M. Importance of iron chelation in free radical-induced
oxidative stress and human disease. Curr Pharm Des. 2011;17:3460-73.
45. Pigeolet E, Corbisier P, Houbion A, Lambert D, Michiels C, Raes M, et al.
Glutathione peroxidase, superoxide dismutase, and catalase inactivation by
peroxides and oxygen derived free radicals. Mech Ageing Dev. 1990;51:283-297.
46. Bristow RG, Hill RP. Hypoxia and metabolism: Hypoxia, DNA repair and genetic
instability. Nature Reviews Cancer. 2008;8:180-192 149
47. Shrivastav M, De Haro LP, Nickoloff JA. Regulation of DNA double-strand
break repair pathway choice. Cell research. 2008;18:134-147
48. Yakes FM, Van Houten B. Mitochondrial DNA damage is more extensive and
persists longer than nuclear DNA damage in human cells following oxidative
stress. Proc Natl Acad Sci U S A. 1997;94:514-519
49. Orrenius S, Gogvadze V, Zhivotovsky B. Mitochondrial oxidative stress:
Implications for cell death. Annu Rev Pharmacol Toxicol. 2007;47:143-183
50. Zorov DB, Filburn CR, Klotz L-O, Zweier JL, Sollott SJ. Reactive oxygen
species (ros-induced) ros release a new phenomenon accompanying induction of
the mitochondrial permeability transition in cardiac myocytes. J Exp Med.
2000;192:1001-1014
51. Zorov DB, Juhaszova M, Sollott SJ. Mitochondrial ros-induced ros release: An
update and review. Biochim Biophys Acta. 2006;1757:509-517
52. Crompton M. The mitochondrial permeability transition pore and its role in cell
death. Biochem J.. 1999;341 Pt 2:233-249
53. Bernardi P, Scorrano L, Colonna R, Petronilli V, Di Lisa F. Mitochondria and cell
death. Eur J Biochem. 1999;264:687-701
54. Mattson MP, Kroemer G. Mitochondria in cell death: Novel targets for
neuroprotection and cardioprotection. Trends Mol Med. 2003;9:196-205
55. Moll U, Marchenko N, Zhang X. P53 and nur77/tr3–transcription factors that
directly target mitochondria for cell death induction. Oncogene. 2006;25:4725-
4743 150
56. Rizzuto R, Giorgi C, Romagnoli A, Pinton P. Ca2+ signaling, mitochondria and
cell death. Curr Mol Med. 2008;8:119-130
57. Tait SW, Green DR. Mitochondria and cell death: Outer membrane
permeabilization and beyond. Nat Rev Mol Cell Biol. 2010;11:621-632
58. Misra MK, Sarwat M, Bhakuni P, Tuteja R, Tuteja N. Oxidative stress and
ischemic myocardial syndromes. Medical Science Review. 2009;15:209-219
59. Pedersen PL. Mitochondrial events in the life and death of animal cells: A brief
overview. J Bioenerg Biomembr. 1999;31:291-304
60. Kluck RM, Bossy-Wetzel E, Green DR, Newmeyer DD. The release of
cytochrome c from mitochondria: A primary site for bcl-2 regulation of apoptosis.
Science. 1997;275:1132-1136
61. Kroemer G, Zamzami N, Susin SA. Mitochondrial control of apoptosis. Immunol
Today. 1997;18:44-51
62. del Zoppo GJ, Poeck K, Pessin MS, Wolpert SM, Furlan AJ, Ferbert A, et al.
Recombinant tissue plasminogen activator in acute thrombotic and embolic
stroke. Ann Neurol.. 1992;32:78-86
63. Hacke W, Kaste M, Fieschi C, Toni D, Lesaffre E, Von Kummer R, et al.
Intravenous thrombolysis with recombinant tissue plasminogen activator for acute
hemispheric stroke: The european cooperative acute stroke study (ecass). JAMA.
1995;274:1017-1025
64. Topol EJ, Califf RM, George BS, Kereiakes DJ, Abbottsmith CW, Candela RJ, et
al. A randomized trial of immediate versus delayed elective angioplasty after 151
intravenous tissue plasminogen activator in acute myocardial infarction. N Engl J
Med. 1987;317:581-588
65. Wardlaw JM, Murray V, Berge E, del Zoppo G, Sandercock P, Lindley RL, et al.
Recombinant tissue plasminogen activator for acute ischaemic stroke: An updated
systematic review and meta-analysis. The Lancet. 2012;379:2364-2372
66. Wilcox R, Olsson C, Skene A, Von Der Lippe G, Jensen G, Hampton J, et al.
Trial of tissue plasminogen activator for mortality reduction in acute myocardial
infarction: Anglo-scandinavian study of early thrombolysis (asset). The Lancet.
1988;332:525-530
67. Kierszenbaum AL, Tres L. Histology and cell biology: An introduction to
pathology. Elsevier Health Sciences; 2015.
68. Fang MC, Cutler DM, Rosen AB. Trends in thrombolytic use for ischemic stroke
in the United States. J Hosp Med. 2010;5:406-409
69. García-Yébenes I, Sobrado M, Zarruk JG, Castellanos M, Pérez de la Ossa N,
Dávalos A, et al. A mouse model of hemorrhagic transformation by delayed tissue
plasminogen activator administration after in situ thromboembolic stroke. Stroke.
2011;42:196-203
70. Kawata H, Uesugi Y, Soeda T, Takemoto Y, Sung J-H, Umaki K, et al. A new
drug delivery system for intravenous coronary thrombolysis with thrombus
targeting and stealth activity recoverable by ultrasound. J Am Coll Cardiol.
2012;60:2550-2557 152
71. Kametsu Y, Osuga S, Hakim AM. Apoptosis occurs in the penumbra zone during
short-duration focal ischemia in the rat. J Cereb Blood Flow Metab. 2003:416-
422
72. Richard Green A, Odergren T, Ashwood T. Animal models of stroke: Do they
have value for discovering neuroprotective agents? Trends Pharmacol Sci.
2003;24:402-408
73. Muir KW, Lees KR. Clinical experience with excitatory amino acid antagonist
drugs. Stroke. 1995;26:503-513
74. Carmichael ST. Rodent models of focal stroke: Size, mechanism, and purpose.
NeuroRx. 2005;2:396-409
75. Pulsinelli WA, Brierley JB. A new model of bilateral hemispheric ischemia in the
unanesthetized rat. Stroke. 1979;10:267-272
76. Chen M, Ona VO, Li M, Ferrante RJ, Fink KB, Zhu S, et al. Minocycline inhibits
caspase-1 and caspase-3 expression and delays mortality in a transgenic mouse
model of huntington disease. Nat Med. 2000;6:797-801
77. Nakano S, Kato H, Kogure K. Neuronal damage in the rat hippocampus in a new
model of repeated reversible transient cerebral ischemia. Brain Res.
1989;490:178-180
78. Pulsinelli WA, Waldman S, Rawlinson D, Plum F. Moderate hyperglycemia
augments ischemic brain damage a neuropathologic study in the rat. Neurology.
1982;32:1239-1239 153
79. Xu D, Bureau Y, McIntyre DC, Nicholson DW, Liston P, Zhu Y, et al.
Attenuation of ischemia-induced cellular and behavioral deficits by x
chromosome-linked inhibitor of apoptosis protein overexpression in the rat
hippocampus. J Neurosci. 1999;19:5026-5033
80. Belayev L, Ginsberg MD, Alonso OF, Singer JT, Zhao W, Busto R. Bilateral
ischemic tolerance of rat hippocampus induced by prior unilateral transient focal
ischemia: Relationship to c‐fos mrna expression. Neuroreport. 1996;8:55-59
81. Ordy J, Wengenack T, Bialobok P, Coleman P, Rodier P, Baggs R, et al. Selective
vulnerability and early progression of hippocampal ca1 pyramidal cell
degeneration and gfap-positive astrocyte reactivity in the rat four-vessel occlusion
model of transient global ischemia. Exp Neurol. 1993;119:128-139
82. Sheng H, Laskowitz DT, Pearlstein RD, Warner DS. Characterization of a
recovery global cerebral ischemia model in the mouse. J Neurosci Methods.
1999;88:103-109
83. Wellons III JC, Sheng H, Laskowitz DT, Mackensen GB, Pearlstein RD, Warner
DS. A comparison of strain-related susceptibility in two murine recovery models
of global cerebral ischemia. Brain Res. 2000;868:14-21
84. Cuomo O, Rispoli V, Leo A, Politi GB, Vinciguerra A, di Renzo G, et al. The
antiepileptic drug levetiracetam suppresses non-convulsive seizure activity and
reduces ischemic brain damage in rats subjected to permanent middle cerebral
artery occlusion. PLoS One. 2013;8:e80852 154
85. Kim HJ, Rowe M, Ren M, Hong J-S, Chen P-S, Chuang D-M. Histone
deacetylase inhibitors exhibit anti-inflammatory and neuroprotective effects in a
rat permanent ischemic model of stroke: Multiple mechanisms of action. J
Pharmacol Exp Ther. 2007;321:892-901
86. Wang X, Yue T-L, Young PR, Barone FC, Feuerstein GZ. Expression of
interleukin-6, c-fos, and zif268 mrnas in rat ischemic cortex. J Cereb Blood Flow
Metab. 1995;15:166-171
87. Wen Y-D, Sheng R, Zhang L-S, Han R, Zhang X, Zhang X-D, et al. Neuronal
injury in rat model of permanent focal cerebral ischemia is associated with
activation of autophagic and lysosomal pathways. Autophagy. 2008;4:762-769
88. Yang Z, Zhong L, Zhong S, Xian R, Yuan B. Hypoxia induces microglia
autophagy and neural inflammation injury in focal cerebral ischemia model. Exp
Mol Pathol. 2015;98:219-224
89. Robinson RG. Differential behavioral and biochemical effects of right and left
hemispheric cerebral infarction in the rat. Science. 1979;205;707-710
90. Belayev L, Alonso OF, Busto R, Zhao W, Ginsberg MD. Middle cerebral artery
occlusion in the rat by intraluminal suture neurological and pathological
evaluation of an improved model. Stroke. 1996;27:1616-1623
91. Memezawa H, Smith M-L, Siesjö B. Penumbral tissues salvaged by reperfusion
following middle cerebral artery occlusion in rats. Stroke. 1992;23:552-559
92. Schmid-Elsaesser R, Zausinger S, Hungerhuber E, Baethmann A, Reulen H-J. A
critical reevaluation of the intraluminal thread model of focal cerebral ischemia 155
evidence of inadvertent premature reperfusion and subarachnoid hemorrhage in
rats by laser-doppler flowmetry. Stroke. 1998;29:2162-2170
93. Dittmar M, Spruss T, Schuierer G, Horn M. External carotid artery territory
ischemia impairs outcome in the endovascular filament model of middle cerebral
artery occlusion in rats. Stroke. 2003;34:2252-2257
94. Chen T-Y, Goyagi T, Toung TJ, Kirsch JR, Hurn PD, Koehler RC, et al.
Prolonged opportunity for ischemic neuroprotection with selective κ-opioid
receptor agonist in rats. Stroke. 2004;35:1180-1185
95. Yamamoto M, Tamura a, Kirino T, Shimizu M, Sano K. Behavioral changes after
focal cerebral ischemia by left middle cerebral artery occlusion in rats. Brain Res.
1988;452:323-328
96. Schroeter M, Jander S, Stoll G. Non-invasive induction of focal cerebral ischemia
in mice by photothrombosis of cortical microvessels: Characterization of
inflammatory responses. J Neurosci Methods. 2002;117:43-49
97. Bacigaluppi M, Comi G. Animal models of ischemic stroke. Part one: Modeling
risk factors animal models of ischemic stroke. Part two: Modeling cerebral
ischemia. Open Neurol J.. 2010;4:26-33
98. Colbourne F, Auer Rn, Sutherland GR. Behavioral testing does not exacerbate
ischemic ca1 damage in gerbils. Sroke.1998;29:1967-1970
99. Schaffer CB, Friedman B, Nishimura N, Schroeder LF, Tsai PS, Ebner FF, Lyden
PD, et al. Two-photon imaging of cortical surface microvessels reveals a robust
redistribution in blood flow after vascular occlusion. PLoS biol. 2006;4:e26 156
100. Watson BD, Dietrich WD, Busto R, Wachtel MS, Ginsberg MD. Induction of
reproducible brain infarction by photochemically initiated thrombosis. Ann
Neurol. 1985;17:497-504
101. Dietrich WD, Ginsberg MD, Busto R, Watson BD. Photochemically induced
cortical infarction in the rat. 2. Acute and subacute alterations in local glucose
utilization. J Cereb Blood Flow Metab. 1986;6:195-202
102. Braun JS, Jander S, Schroeter M, Witte OW, Stoll G. Spatiotemporal relationship
of apoptotic cell death to lymphomonocytic infiltration in photochemically
induced focal ischemia of the rat cerebral cortex. Acta Neuropathol. 1996;92:255-
263
103. Kim GW, Sugawara T, Chan PH. Involvement of oxidative stress and caspase-3
in cortical infarction after photothrombotic ischemia in mice. J Cereb Blood Flow
Metab. 2000;20:1690-1701
104. Jander S, Schroeter M, Stoll G. Role of nmda receptor signaling in the regulation
of inflammatory gene expression after focal brain ischemia. J Neuroimmunol.
2000;109:181-187
105. Schroeter M, Jander S, Huitinga I, Witte OW, Stoll G. Phagocytic response in
photochemically induced infarction of rat cerebral cortex the role of resident
microglia. Stroke. 1997;28:382-386
106. Lee J-K, Park M-S, Kim Y-S, Moon K-S, Joo S-P, Kim T-S, et al.
Photochemically induced cerebral ischemia in a mouse model. Surg Neurol.
2007;67:620-625 157
107. Kuroiwa T, Xi G, Hua Y, Nagaraja TN, Fenstermacher JD, Keep RF.
Development of a rat model of photothrombotic ischemia and infarction within
the caudoputamen. Stroke. 2009;40:248-253
108. Dietrich W, Watson B, Busto R, Ginsberg M. Metabolic plasticity following
cortical infarction: A 2-deoxyglucose study in adult rats. Cerebrovascular
diseases. Raven Press New York; 1987:285-295.
109. Que M, Schiene K, Witte OW, Zilles K. Widespread up-regulation of n-methyl-d-
aspartate receptors after focal photothrombotic lesion in rat brain. Neurosci Lett.
1999;273:77-80
110. Traystman RJ. Animal models of focal and global cerebral ischemia. ILAR J.
2003;44:85-95
111. Jopling C, Sleep E Fau - Raya M, Raya M Fau - Marti M, Marti M Fau - Raya A,
Raya A Fau - Izpisua Belmonte JC, Izpisua Belmonte JC. Zebrafish heart
regeneration occurs by cardiomyocyte dedifferentiation and proliferation. Nature.
2010;464:606-609
112. Guo S. Linking genes to brain, behavior and neurological diseases: What can we
learn from zebrafish? Genes Brain Behav. 2004;3:63-74
113. Laale HW. The biology and use of zebrafish, brachydanio rerio in fisheries
research. J Fish Biol. 1977;10:121-173
114. Sterba G. Freshwater fishes of the world. Cosmo Publications. 1967 158
115. Dasmahapatra AK, Doucet HL, Bhattacharyya C, Carvan MJ. Developmental
expression of alcohol dehydrogenase (adh3) in zebrafish (danio rerio). Biochem
Biophys Res Commun. 2001;286:1082-1086
116. Hill AJ, Teraoka H, Heideman W, Peterson RE. Zebrafish as a model vertebrate
for investigating chemical toxicity. Toxicol Sci. 2005;86:6-19
117. Kishimoto N, Shimizu K, Sawamoto K. Neuronal regeneration in a zebrafish
model of adult brain injury. Dis Model Mech. 2012;5:200-209
118. McGrath P, Li CQ. Zebrafish: A predictive model for assessing drug-induced
toxicity. Drug Discov Today. 2008;13:394-401
119. Norton W, Bally-Cuif L. Adult zebrafish as a model organism for behavioural
genetics. BMC Neurosci. 2010;11:90
120. Panula P, Chen YC, Priyadarshini M, Kudo H, Semenova S, Sundvik M, et al.
The comparative neuroanatomy and neurochemistry of zebrafish cns systems of
relevance to human neuropsychiatric diseases. Neurobiol Dis. 2010;40:46-57
121. Parng C, Ton C, Lin YX, Roy NM, McGrath P. A zebrafish assay for identifying
neuroprotectants in vivo. Neurotoxicol Teratol. 2006;28:509-516
122. Baumgart EV, Barbosa JS, Bally-Cuif L, Gotz M, Ninkovic J. Stab wound injury
of the zebrafish telencephalon: A model for comparative analysis of reactive
gliosis. Glia. 2012;60:343-357
123. Barros TP, Alderton WK, Reynolds HM, Roach AG, Berghmans S. Zebrafish: An
emerging technology for in vivo pharmacological assessment to identify potential
safety liabilities in early drug discovery. Br J pharmacol. 2008;154:1400-1413 159
124. Sprague J, Bayraktaroglu L, Clements D, Conlin T, Fashena D, Frazer K, et al.
The zebrafish information network: The zebrafish model organism database.
Nucleic acids research. 2006;34:D581-585
125. Santana S, Rico EP, Burgos JS. Can zebrafish be used as animal model to study
alzheimer's disease? Am J Neurodegener Dis. 2012;1:32-48
126. Tandon K, Sharma SC. On the degeneration and regeneration of optic nerve fibres
with return of vision indanio rerio. Proc Indian As Sc. 1964;60:287-292
127. Springer AD, Easter SS, Agranoff BW. The role of the optic tectum in various
visually mediated behaviors of goldfish. Brain Res. 1977;128:393-404
128. Li L, Dowling JE. A dominant form of inherited retinal degeneration caused by a
non-photoreceptor cell-specific mutation. Proc Natl Acad Sci U S A.
1997;94:11645-11650
129. Baulac S, Lu H, Strahle J, Yang T, Goldberg MS, Shen J, et al. Increased dj-1
expression under oxidative stress and in alzheimer's disease brains. Mol
Neurodegener. 2009;4:12
130. Stewart A, Riehl R, Wong K, Green J, Cosgrove J, Vollmer K, et al. Behavioral
effects of mdma ('ecstasy') on adult zebrafish. Behav Pharmacol. 2011;22:275-
280
131. Stewart A, Wu N, Cachat J, Hart P, Gaikwad S, Wong K, et al. Pharmacological
modulation of anxiety-like phenotypes in adult zebrafish behavioral models. Prog
Neuropsychopharmacol Biol Psychiatry. 2011;35:1421-1431 160
132. Zon LI, Peterson RT. In vivo drug discovery in the zebrafish. Nat Rev Drug
Discov. 2005;4:35-44
133. Miller JD, Neely MN. Zebrafish as a model host for streptococcal pathogenesis.
Acta Trop. 2004;91:53-68
134. Paquet D, Bhat R, Sydow A, Mandelkow EM, Berg S, Hellberg S, et al. A
zebrafish model of tauopathy allows in vivo imaging of neuronal cell death and
drug evaluation. J Clin Invest. 2009;119:1382-1395
135. Poss KD, Keating MT, Nechiporuk A. Tales of regeneration in zebrafish. Dev
Dyn. 2003;226:202-210
136. Poss KD, Wilson LG, Keating MT. Heart regeneration in zebrafish. Science.
2002;298:2188-2190
137. Panula P, Sallinen V, Sundvik M, Kolehmainen J, Torkko V, Tiittula A, et al.
Modulatory neurotransmitter systems and behavior: Towards zebrafish models of
neurodegenerative diseases. Zebrafish. 2006;3:235-247
138. Chen AT, Zon LI. Zebrafish blood stem cells. J Cell Biochem. 2009;108:35-42
139. Jagadeeswaran P, Sheehan J, Craig F, Troyer D. Identification and
characterization of zebrafish thrombocytes. Br J Haematol. 1999;107:731-738
140. Thattaliyath B, Cykowski M, Jagadeeswaran P. Young thrombocytes initiate the
formation of arterial thrombi in zebrafish. Blood. 2005;106:118-124
141. Gregory M, Hanumanthaiah R, Jagadeeswaran P. Genetic analysis of hemostasis
and thrombosis using vascular occlusion. Blood Cells Mol Dis. 2002;29:286-295 161
142. Jagadeeswaran P. Zebrafish: A tool to study hemostasis and thrombosis. Curr
Opin Hematol. 2005;12:149-152
143. Jagadeeswaran P, Gregory M, Day K, Cykowski M, Thattaliyath B. Zebrafish: A
genetic model for hemostasis and thrombosis. J Thromb Haemost. 2005;3:46-53
144. Jagadeeswaran P, Liu YC. Developmental expression of thrombin in zebrafish
embryos: A novel model to study hemostasis. Blood Cells Mol Dis.1997;23:147-
156
145. Jagadeeswaran P, Liu YC. A hemophilia model in zebrafish: Analysis of
hemostasis. Blood Cells Mol Dis.1997;23:52-57
146. Jagadeeswaran P, Liu YC, Sheehan JP. Analysis of hemostasis in the zebrafish.
Method Cell Biol. 1999;59:337-358
147. Jagadeeswaran P, Sheehan JP. Analysis of blood coagulation in the zebrafish.
Blood Cells Mol Dis. 1999;25:239-249
148. Lang M, Gihr G, Gawaz M, Müller I. Hemostasis in danio rerio: Is the zebrafish a
useful model for platelet research? J Thromb Haemost. 2010;8:1159-1169
149. Rees BB, Sudradjat FA, Love JW. Acclimation to hypoxia increases survival time
of zebrafish, danio rerio, during lethal hypoxia. J Exp Zool. 2001;289:266-272
150. Papoutsoglou SE. The role of the brain in farmed fish. Rev Aquacult. 2012;4:1-10
151. Rico EP, Rosemberg DB, Seibt KJ, Capiotti KM, Da Silva RS, Bonan CD.
Zebrafish neurotransmitter systems as potential pharmacological and
toxicological targets. Neurotoxicol Teratol. 2011;33:608-617 162
152. Maximino C, de Brito TM, da Silva Batista AW, Herculano AM, Morato S,
Gouveia A. Measuring anxiety in zebrafish: A critical review. Behav Brain Res.
2010;214:157-171
153. Bowman TV, Zon LI. Swimming into the future of drug discovery: In vivo
chemical screens in zebrafish. ACS Chem Biol. 2010;5:159-161
154. Spitsbergen J, Kent M. The state of the art of the zebrafish model for toxicology
and toxicologic pathology research-advantages and current limitations. Toxicol
Pathol. 2003;31:62-87
155. Buchner DA, Su F, Yamaoka JS, Kamei M, Shavit JA, Barthel LK, et al. Pak2a
mutations cause cerebral hemorrhage in redhead zebrafish. Proc Natl Acad Sci U
S A. 2007;104:13996-14001
156. Liu Y, Kretz CA, Maeder ML, Richter CE, Tsao P, Vo AH, et al. Targeted
mutagenesis of zebrafish antithrombin iii triggers disseminated intravascular
coagulation and thrombosis, revealing insight into function. Blood. 2014;124:142-
150
157. Ghosh A, Vo A, Twiss BK, Kretz CA, Jozwiak MA, Montgomery RR, et al.
Characterization of zebrafish von willebrand factor reveals conservation of
domain structure, multimerization, and intracellular storage. Adv Hematol.
2012;2012
158. Sigel H. Metal ions in biological systems: Volume 15: Zinc and its role in biology
and nutrition. CRC Press. 1983. 163
159. Berg JM, Shi Y. The galvanization of biology: A growing appreciation for the
roles of zinc. Science. 1996;271:1081
160. López‐García C, Varea E, Palop JJ, Nacher J, Ramirez C, Ponsoda X, et al.
Cytochemical techniques for zinc and heavy metals localization in nerve cells.
Microsc Res Tech. 2002;56:318-331
161. Coleman JE. Zinc proteins : Enzymes, storage replication proteins. Annu Rev
Biochem. 1992:897-946
162. Vallee BL, Coleman JE, Auld DS. Zinc fingers, zinc clusters, and zinc twists in
DNA-binding protein domains. Proc Natl Acad Sci U S A. 1991;88:999-1003
163. Vallee BL, Falchuk KH. The biochemical basis of zinc physiology. Physiol Rev.
1993;73:79-118
164. Fraker PJ, Telford WG. A reappraisal of the role of zinc in life and death
decisions of cells. Exp Biol Med. 1997;215:229-236
165. Yamasaki S, Sakata-Sogawa K, Hasegawa A, Suzuki T, Kabu K, Sato E, et al.
Zinc is a novel intracellular second messenger. J Cell Biol. 2007;177:637-645
166. Prasad AS. Zinc in growth and development and spectrum of human zinc
deficiency. J Am Coll Nutr. 1988;7:377-384
167. Prasad AS. Discovery of human zinc deficiency and studies in an experimental
human model. J Am Coll Nutr. 1991;53:403-412
168. Beyersmann D, Haase H. Functions of zinc in signaling, proliferation and
differentiation of mammalian cells. Biometals. 2001;14:331-341 164
169. Medvedeva YV, Lin B, Shuttleworth CW, Weiss JH. Intracellular zn2+
accumulation contributes to synaptic failure, mitochondrial depolarization, and
cell death in an acute slice oxygen–glucose deprivation model of ischemia. J
Neurosci. 2009;29:1105-1114
170. MCNULTY T, TAYLOR C. Extracellular heavy-metal ions stimulate ca2+
mobilization in hepatocytes. Biochem. J. 1999;339:555-561
171. Maret W. Crosstalk of the group iia and iib metals calcium and zinc in cellular
signaling. Proc Natl Acad Sci U S A. 2001;98:12325-12327
172. Cai L, Li X-K, Song Y, Cherian MG. Essentiality, toxicology and chelation
therapy of zinc and copper. Curr Med Chem. 2005;12:2753-2763
173. Wudarczyk J, Dębska G, Lenartowicz E. Zinc as an inducer of the membrane
permeability transition in rat liver mitochondria. Arch Biochem Biophys.
1999;363:1-8
174. Gupta S, Maggon K, Venkitasubramanian T. Effect of zinc on tricarboxylic acid
cycle intermediates and enzymes in relation to aflatoxin biosynthesis. J Gen
Microbiol. 1977;99:43-48
175. Sheline CT, Behrens MM, Choi DW. Zinc-induced cortical neuronal death:
Contribution of energy failure attributable to loss of nad+ and inhibition of
glycolysis. J Neurosci. 2000;20:3139-3146
176. Guo D, Du Y, Wu Q, Jiang W, Bi H. Disrupted calcium homeostasis is involved
in elevated zinc ion-induced photoreceptor cell death. Arch Biochem Biophys.
2014;560:44-51 165
177. Kim Y-H, Kim E, Gwag B, Sohn S, Koh J-Y. Zinc-induced cortical neuronal
death with features of apoptosis and necrosis: Mediation by free radicals.
Neuroscience. 1999;89:175-182
178. Manev H, Kharlamov E, Uz T, Mason RP, Cagnoli CM. Characterization of zinc-
induced neuronal death in primary cultures of rat cerebellar granule cells. Exp
Neurol. 1997;146:171-178
179. Sargazi M, Shenkin A, Roberts NB. Zinc induced damage to kidney proximal
tubular cells: Studies on chemical speciation leading to a mechanism of damage. J
Trace Elem Med Biol. 2013;27:242-248
180. Truong-Tran AQ, Carter J, Ruffin RE, Zalewski PD. The role of zinc in caspase
activation and apoptotic cell death. Biometals. 2001;14:315-330
181. Kim Y-H, Koh J-Y. The role of nadph oxidase and neuronal nitric oxide synthase
in zinc-induced poly(adp-ribose) polymerase activation and cell death in cortical
culture. Exp Neurol. 2002;177:407-418
182. Dineley KE, Votyakova TV, Reynolds IJ. Zinc inhibition of cellular energy
production: Implications for mitochondria and neurodegeneration. J Neurochem.
2003;85:563-570
183. Vander Jagt T, Connor J, Weiss J, Shuttleworth C. Intracellular zn 2+ increases
contribute to the progression of excitotoxic ca 2+ increases in apical dendrites of
ca1 pyramidal neurons. Neuroscience. 2009;159:104-114 166
184. Gazaryan IG, Krasinskaya IP, Kristal BS, Brown AM. Zinc irreversibly damages
major enzymes of energy production and antioxidant defense prior to
mitochondrial permeability transition. J Biol Chem. 2007;282:24373-24380
185. Jiang D, Sullivan PG, Sensi SL, Steward O, Weiss JH. Zn(2+) induces
permeability transition pore opening and release of pro-apoptotic peptides from
neuronal mitochondria. J Biol Chem. 2001;276:47524-47529
186. Li YV. Metal ion in stroke. Springer science abd business media. 2012:167-189
187. Frederickson CJ, Koh J-Y, Bush AI. The neurobiology of zinc in health and
disease. Nature reviews. Neuroscience. 2005;6:449-462
188. Sensi SL, Paoletti P, Bush AI, Sekler I. Zinc in the physiology and pathology of
the cns. Nature reviews. Neuroscience. 2009;10:780-791
189. Shahbaz AU, Zhao T, Zhao W, Johnson PL, Ahokas Ra, Bhattacharya SK, et al.
Calcium and zinc dyshomeostasis during isoproterenol-induced acute stressor
state. Am J Physiol Heart Circ Physiol. 2011;300:H636-644
190. Noh KM, Koh JY. Induction and activation by zinc of nadph oxidase in cultured
cortical neurons and astrocytes. J Neurosci. 2000;20:RC111
191. Bossy-Wetzel E, Talantova MV, Lee WD, Schölzke MN, Harrop A, Mathews E,
et al. Crosstalk between nitric oxide and zinc pathways to neuronal cell death
involving mitochondrial dysfunction and p38-activated k+ channels. Neuron.
2004;41:351-365
192. Cuajungco MP, Lees GJ. Nitric oxide generators produce accumulation of
chelatable zinc in hippocampal neuronal perikarya. Brain Res. 1998;799:118-129 167
193. Kunzmann A, Dedoussis G, Jajte J, Malavolta M, Mocchegiani E, Bürkle A.
Effect of zinc on cellular poly (adp-ribosyl) ation capacity. Exp Gerontol.
2008;43:409-414
194. Galasso SL, Dyck RH. The role of zinc in cerebral ischemia. Mol Med.
2007;13:380
195. Lee J-M, Zipfel GJ, Choi DW. The changing landscape of ischaemic brain injury
mechanisms. Nature. 1999;399:A7-A14
196. Tønder N, Johansen FF, Frederickson C, Zimmer J, Diemer N. Possible role of
zinc in the selective degeneration of dentate hilar neurons after cerebral ischemia
in the adult rat. Neurosci Lett. 1990;109:247-252
197. Astrup J, Siesjö BK, Symon L. Thresholds in cerebral ischemia-the ischemic
penumbra. Stroke. 1981;12:723-725
198. Chen H, Yoshioka H, Kim GS, Jung JE, Okami N, Sakata H, et al. Oxidative
stress in ischemic brain damage: Mechanisms of cell death and potential
molecular targets for neuroprotection. Antioxid Redox Signal. 2011;14:1505-1517
199. Dirnagl U, Iadecola C, Moskowitz MA. Pathobiology of ischaemic stroke: An
integrated view. Trends Neurosci. 1999;22:391-397
200. Manzanero S, Santro T, Arumugam TV. Neuronal oxidative stress in acute
ischemic stroke: Sources and contribution to cell injury. Neurochem Int.
2013;62:712-718 168
201. Puyal J, Ginet V, Clarke PG. Multiple interacting cell death mechanisms in the
mediation of excitotoxicity and ischemic brain damage: A challenge for
neuroprotection. Prog Neurobiol. 2013;105:24-48
202. Sanderson TH, Reynolds CA, Kumar R, Przyklenk K, Hüttemann M. Molecular
mechanisms of ischemia–reperfusion injury in brain: Pivotal role of the
mitochondrial membrane potential in reactive oxygen species generation. Mol
Neurobiol. 2013;47:9-23
203. SIESJÖ BK. Mechanisms of ischemic brain damage. Crit Care Med.
1988;16:954-963
204. Kim B-E, Wang F, Dufner-Beattie J, Andrews GK, Eide DJ, Petris MJ. Zn2+-
stimulated endocytosis of the mzip4 zinc transporter regulates its location at the
plasma membrane. J Biol Chem. 2004;279:4523-4530
205. Liuzzi JP, Cousins RJ. Mammalian zinc transporters. Annu Rev Nutr.
2004;24:151-172
206. Palmiter RD, Findley SD. Cloning and functional characterization of a
mammalian zinc transporter that confers resistance to zinc. EMBO J. 1995;14:639
207. Atar D, Backx PH, Appel MM, Gao WD, Marban E. Excitation-transcription
coupling mediated by zinc influx through voltage-dependent calcium channels. J
Biol Chem. 1995;270:2473-2477
208. Büsselberg D, Michael D, Evans ML, Carpenter DO, Haas HL. Zinc (Zn2+)
blocks voltage gated calcium channels in cultured rat dorsal root ganglion cells.
Brain Res. 1992;593:77-81 169
209. Harrison N, Gibbons S. Zn 2+: An endogenous modulator of ligand-and voltage-
gated ion channels. Neuropharmacology. 1994;33:935-952
210. Weiss JH, Hartley DM, Koh J-y, Choi DW. Ampa receptor activation potentiates
zinc neurotoxicity. Neuron. 1993;10:43-49
211. Weiss JH, Sensi SL, Koh JY. Zn 2+: A novel ionic mediator of neural injury in
brain disease. Trends Pharmacol Sci. 2000;21:395-401
212. Colvin RA. Zinc inhibits ca2+ transport by rat brain Na+/Ca2+ exchanger.
Neuroreport. 1998;9:3091-3096
213. Colvin RA, Davis N, Nipper RW, Carter PA. Evidence for a zinc/proton
antiporter in rat brain. Neurochem Int. 2000;36:539-547
214. Blaudez D, Chalot M. Characterization of the er-located zinc transporter znt1 and
identification of a vesicular zinc storage compartment in hebeloma
cylindrosporum. Fungal Genet Biol. 2011;48:496-503
215. Ellis CD, Wang F, MacDiarmid CW, Clark S, Lyons T, Eide DJ. Zinc and the
msc2 zinc transporter protein are required for endoplasmic reticulum function. J
Cell Biol. 2004;166:325-335
216. He K, Aizenman E. Erk signaling leads to mitochondrial dysfunction in
extracellular zinc‐induced neurotoxicity. J Neurochem. 2010;114:452-461
217. Bishop GM, Dringen R, Robinson SR. Zinc stimulates the production of toxic
reactive oxygen species (ROS) and inhibits glutathione reductase in astrocytes.
Free Radic Biol Med. 2007;42:1222-1230 170
218. Bossy-Wetzel E, Talantova MV, Lee WD, Schölzke MN, Harrop A, Mathews E,
et al. Crosstalk between nitric oxide and zinc pathways to neuronal cell death
involving mitochondrial dysfunction and p38-activated K+ channels. Neuron.
2004;41:351-365
219. Dineley KE, Richards LL, Votyakova TV, Reynolds IJ. Zinc causes loss of
membrane potential and elevates reactive oxygen species in rat brain
mitochondria. Mitochondrion. 2005;5:55-65
220. Donadelli M, Dalla Pozza E, Scupoli M, Costanzo C, Scarpa A, Palmieri M.
Intracellular zinc increase inhibits p53−/− pancreatic adenocarcinoma cell growth
by ros/aif-mediated apoptosis. Biochim Biophys Acta. 2009;1793:273-280
221. Biggs R, Macfarlane G. Human blood coagulation and its disorders. Blackwell
Scientific Publications; 1962.
222. Sharathkumar AA, Pipe SW. Bleeding disorders. Pediatr Rev. 2008;29:121
223. Furie B, Furie BC. The molecular basis of blood coagulation. Cell.1988;53:505-
518
224. Davie EW. A brief historical review of the waterfall/cascade of blood
coagulation. J Biol Chem. 2003;278:50819-50832
225. Davie EW, Ratnoff OD. Waterfall sequence for intrinsic blood clotting. Science.
1964;145:1310-1312
226. Davie EW, Fujikawa K, Kisiel W. The coagulation cascade: Initiation,
maintenance, and regulation. Biochemistry. 1991;30:10363-10370 171
227. Lowe GD. Virchow’s triad revisited: Abnormal flow. Pathophysiol Haemost
Thromb. 2003;33:455-457
228. Chung I, Lip GY. Virchow’s triad revisited: Blood constituents. Pathophysiol
Haemost Thromb. 2003;33:449-454
229. Kleinegris M-C, ten Cate-Hoek AJ, Ten Cate H. Coagulation and the vessel wall
in thrombosis and atherosclerosis. Pol Arch Med Wewn. 2012;122:557-566
230. Blann AD, Lip GY. Virchow's triad revisited: The importance of soluble
coagulation factors, the endothelium, and platelets. Thromb Res. 2001;101:321-
327
231. Hackley BM, Smith JC, Halsted JA. A simplified method for plasma zinc
determination by atomic absorption spectrophotometry. Clin Chem. 1968;14:1-5
232. Marx G1, Korner G, Mou X, Gorodetsky R. Packaging zinc, fibrinogen, and
factor XIII in plaitelet a-granules. J Cell Physiol. 1993;437442:1-6
233. Tubek S, Grzanka P, Tubek I. Role of zinc in hemostasis: A review. Biol Trace
Elem Res. 2008;121:1-8
234. Masuoka J, Hegenauer J, Van Dyke B, Saltman P. Intrinsic stoichiometric
equilibrium constants for the binding of zinc (ii) and copper (ii) to the high
affinity site of serum albumin. J Biol Chem. 1993;268:21533-21537
235. Stork CJ, Li YV. Zinc release from thapsigargin/ip3-sensitive stores in cultured
cortical neurons. J Mol Signal. 2010;5:5 172
236. Wudarczyk J, Debska G, Lenartowicz E. Zinc as an inducer of the membrane
permeability transition in rat liver mitochondria. Arch Biochem Biophys.
1999;363:1-8
237. Mann K, Whelihan M, Butenas S, Orfeo T. Citrate anticoagulation and the
dynamics of thrombin generation. J Thromb Haemost. 2007;5:2055-2061
238. Greengard JS, Griffin JH. Receptors for high molecular weight kininogen on
stimulated washed human platelets. Biochemistry. 1984;23:6863-6869
239. Bernardo M, Day D, Olson S, Shore J. Surface-independent acceleration of factor
xii activation by zinc ions. I. Kinetic characterization of the metal ion rate
enhancement. J Biol Chem. 1993;268:12468-12476
240. Bernardo M, Day D, Halvorson H, Olson S, Shore J. Surface-independent
acceleration of factor xii activation by zinc ions. II. Direct binding and
fluorescence studies. J Biol Chem. 1993;268:12477-12483
241. Røjkjær R, Schousboe I. The surface‐dependent autoactivation mechanism of
factor xii. Eur J Biochem. 1997;243:160-166
242. Røjkjær R, Schousboe I. Partial identification of the zn2+‐binding sites in factor
xii and its activation derivatives. Eur J Biochem. 1997;247:491-496
243. Eldor A, Yarom R, Marx G. Zinc-induced platelet aggregation is mediated by the
fibrinogen receptor and is not accompanied by release or by thromboxane
synthesis. Blood. 1985;66:213-219 173
244. Kowalska M, Juliano D, Trybulec M, Lu W, Niewiarowski S. Zinc ions potentiate
adenosine diphosphate-induced platelet aggregation by activation of protein
kinase c. The Journal of laboratory and clinical medicine. 1994;123:102-109
245. Ganz T. Systemic iron homeostasis. Physiol Rev. 2013;93:1721-1741
246. Marx G. Zinc binding to fibrinogen and fibrin. Arch Biochem Biophys.
1988;266:285-288
247. Marx G, Hopmeier P, Gurfel D. Zinc alters fibrin ultrastructure. Thromb
Haemostat. 1987;57:73-76
248. Trybulec M, Kowalska MA, McLane MA, Silver L, Lu W, Niewiarowski S.
Exposure of platelet fibrinogen receptors by zinc ions: Role of protein kinase c.
Exp Biol Med. 1993;203:108-116
249. Gordon PR, Woodruff CW, Anderson HL, O'Dell B. Effect of acute zinc
deprivation on plasma zinc and platelet aggregation in adult males. Am J Clin
Nutr. 1982;35:113-119
250. Emery MP, Browning JD, O'Dell BL. Impaired hemostasis and platelet function
in rats fed low zinc diets based on egg white protein. J Nutr. 1990;120:1062-1067
251. Emery MP, O'Dell BL. Low zinc status in rats impairs calcium uptake and
aggregation of platelets stimulated by fluoride. Exp Biol Med. 1993;203:480-484
252. Lipinski B, Pretorius E. Novel pathway of iron ‑ induced blood coagulation:
Implications for diabetes mellitus and its complications. Pol Arch Med Wewn.
2012;122:115 174
253. Frostig RD, Lieke EE, Ts'o DY, Grinvald A. Cortical functional architecture and
local coupling between neuronal activity and the microcirculation revealed by in
vivo high-resolution optical imaging of intrinsic signals. Proc Natl Acad Sci U S
A. 1990;87:6082-6086
254. Diener H-C, Schneider D, Lampl Y, Bornstein NM, Kozak A, Rosenberg G. Dp-
b99, a membrane-activated metal ion chelator, as neuroprotective therapy in
ischemic stroke. Stroke. 2008;39:1774-1778
255. Blanusa M, Varnai VM, Piasek M, Kostial K. Chelators as antidotes of metal
toxicity: Therapeutic and experimental aspects. Curr Med Chem. 2005;12:2771-
2794
256. Stork CJ, Li YV. Measuring cell viability with membrane impermeable zinc
fluorescent indicator. J Neurosci Methods. 2006;155:180-186
257. Stork CJ, Li YV. Intracellular zinc elevation measured with a "calcium-specific"
indicator during ischemia and reperfusion in rat hippocampus: A question on
calcium overload. J Neurosci. 2006;26:10430-10437
258. O'Collins VE, Macleod MR, Donnan GA, Horky LL, van der Worp BH, Howells
DW. 1,026 experimental treatments in acute stroke. Ann Neurol. 2006;59:467-477
259. Alvarez-Buylla A, Ling C-Y, Kirn JR. Cresyl violet: A red fluorescent nissl stain.
J Neurosci Methods. 1990;33:129-133
260. Preston E, Webster J. Spectrophotometric measurement of experimental brain
injury. J Neurosci Methods. 2000;94:187-192 175
261. Prasad S, Kashyap RS, Deopujari JY, Purohit HJ, Taori GM, Daginawala HF.
Development of an in vitro model to study clot lysis activity of thrombolytic
drugs. Thromb J. 2006;4:1-4
262. Zhang HF, Maslov K, Sivaramakrishnan M, Stoica G, Wang LV. Imaging of
hemoglobin oxygen saturation variations in single vessels in vivo using
photoacoustic microscopy. Appl Phys Lett. 2007;90:053901
263. Frederickson C, Hernandez M, Goik S, Morton J, McGinty J. Loss of zinc
staining from hippocampal mossy fibers during kainic acid induced seizures: A
histofluorescence study. Brain Res. 1988;446:383-386
264. Hutcheson RM, Engelmann MD, Cheng IF. Voltammetric studies of Zn and Fe
complexes of edta: Evidence for the push mechanism. Biometals. 2005:43-51
265. Hobbs J, McDonald C. Increased seawater temperature and decreased dissolved
oxygen triggers fish kill at the cocos (keeling) islands, indian ocean. J Fish Biol.
2010;77:1219-1229
266. Woodruff TM, Thundyil J, Tang S-C, Sobey CG, Taylor SM, Arumugam TV.
Pathophysiology, treatment, and animal and cellular models of human ischemic
stroke. Mol Neurodegener. 2011;6:11
267. Allner B, von der Gönna S, Griebeler E-M, Nikutowski N, Weltin A,
Stahlschmidt-Allner P. Reproductive functions of wild fish as bioindicators of
reproductive toxicants in the aquatic environment. Environ Sci Pollut R.
2010;17:505-518 176
268. Kolok AS. Sublethal identification of susceptible individuals: Using swim
performance to identify susceptible fish while keeping them alive. Ecotoxicology.
2001;10:205-209
269. Lippold H. Quantitative succinic dehydrogenases histochemistry. Histochemistry.
1982;76:381-405
270. Kirino T, Sano K. Selective vulnerability in the gerbil hippocampus following
transient ischemia. Acta neuropathol. 1984;62:201-208
271. Durukan A, Tatlisumak T. Animal models of ischemic stroke. Handb Clin
Neurol.. 2008;92:43-66
272. Kaste M. Use of animal models has not contributed to development of acute
stroke therapies pro. Stroke. 2005;36:2323-2324
273. Fisher M, Tatlisumak T. Use of animal models has not contributed to
development of acute stroke therapies con. Stroke. 2005;36:2324-2325
274. Mergenthaler P, Dirnagl U, Meisel A. Pathophysiology of stroke: Lessons from
animal models. Metab Brain Dis. 2004;19:151-167
275. Green AR, Odergren T, Ashwood T. Animal models of stroke: Do they have
value for discovering neuroprotective agents? Trends Pharmacol Sci.
2003;24:402-408
276. Hilder E, Haddad P. Separation of dithiocarbamate metal complexes by micellar
electrokinetic chromatography. Analyst. 1998;123:2865-2870
277. Ogawa E, Kodama H. Effects of disulfiram treatment in patients with menkes
disease and occipital horn syndrome. J Trace Elem Med Biol. 2012;26:102-104 177
278. Allison WT. Preface: Zebrafish models of neurology. Biochim Biophys Acta.
2011;1812:333-334
279. Liu S, Leach SD. Zebrafish models for cancer. Annu Rev Pathol-Mech 2011;6:71-
93
280. Kokel D, Bryan J, Laggner C, White R, Cheung CYJ, Mateus R, et al. Rapid
behavior-based identification of neuroactive small molecules in the zebrafish. Nat
Chem Biol. 2010;6:231-237
281. Rihel J, Prober DA, Arvanites A, Lam K, Zimmerman S, Jang S, et al. Zebrafish
behavioral profiling links drugs to biological targets and rest/wake regulation.
Science. 2010;327:348-351
282. Carvan Iii MJ, Sonntag DM, Cmar CB, Cook RS, Curran MA, Miller GL.
Oxidative stress in zebrafish cells: Potential utility of transgenic zebrafish as a
deployable sentinel for site hazard ranking. Sci Total Environ. 2001;274:183-196
283. Weyand AC, Shavit JA. Zebrafish as a model system for the study of hemostasis
and thrombosis. Curr Opin Hematol. 2014;21:418-422
284. Vo AH, Swaroop A, Liu Y, Norris ZG, Shavit JA. Loss of fibrinogen in zebrafish
results in symptoms consistent with human hypofibrinogenemia. PloS One.
2013;8:e74682
285. Shuttleworth CW, Weiss JH. Zinc: New clues to diverse roles in brain ischemia.
Trends Pharmacol Sci. 2011;32:480-486 178
286. Lee J-Y, Kim J-H, Palmiter RD, Koh J-Y. Zinc released from metallothionein-iii
may contribute to hippocampal ca1 and thalamic neuronal death following acute
brain injury. Exp Neurol. 2003;184:337-347
287. Frazzini V, Rockabrand E, Mocchegiani E, Sensi S. Oxidative stress and brain
aging: Is zinc the link? Biogerontology. 2006;7:307-314
288. Bonanni L, Chachar M, Jover-Mengual T, Li H, Jones A, Yokota H, et al. Zinc-
dependent multi-conductance channel activity in mitochondria isolated from
ischemic brain. J Neurosci. 2006;26:6851-6862
289. Wang T, Zheng W, Xu H, Zhou J-M, Wang Z-Y. Clioquinol inhibits zinc-
triggered caspase activation in the hippocampal ca1 region of a global ischemic
gerbil model. PloS One. 2010;5:e11888
290. White BC, Grossman LI, O'Neil BJ, DeGracia DJ, Neumar RW, Rafols JA, et al.
Global brain ischemia and reperfusion. Ann Emerg Med. 1996;27:588-594
291. Marler JR. Ninds clinical trials in stroke lessons learned and future directions.
Stroke. 2007;38:3302-3307
292. Saver JL, Albers GW, Dunn B, Johnston KC, Fisher M. Stroke therapy academic
industry roundtable (stair) recommendations for extended window acute stroke
therapy trials. Stroke. 2009;40:2594-2600
293. Chiamori N, Henry RJ, Golub OJ. Studies on the determination of bile pigments
ii. Spectrophotometric determination of bilirubin and hemoglobin in serum. Clin
Chim Acta. 1961;6:1-6 179
294. Choudhri TF, Hoh BL, Solomon RA, Connolly ES, Pinsky DJ. Use of a
spectrophotometric hemoglobin assay to objectively quantify intracerebral
hemorrhage in mice. Stroke. 1997;28:2296-2302
295. Smith Aa, Jacobson LJ, Miller BI, Hathaway WE, Manco-Johnson MJ. A new
euglobulin clot lysis assay for global fibrinolysis. Thromb Res. 2003;112:329-337
296. Apodaca G. Modulation of membrane traffic by mechanical stimuli. Renal
Physiol. 2002;282:179-190
297. Beigi R, Kobatake E, Aizawa M, Dubyak GR. Detection of local ATP release
from activated platelets using cell surface-attached firefly luciferase. Am J
Physiol. 1999;276:267-278
298. Enomoto K, Furuya K, Yamagishi S, Oka T, Maeno T. The increase in the
intracellular Ca2+ concentration induced by mechanical stimulation is propagated
via release of pyrophosphorylated nucleotides in mammary epithelial cells.
Pflugers Arch. 1994:533-542
299. Hamill OP, Martinac B. Molecular basis of mechanotransduction in living cells.
Physiol Rev. 2001;81:685-741
300. Lemons PP, Chen D, Whiteheart SW. Molecular mechanisms of platelet
exocytosis: Requirements for alpha-granule release. Biochem Biophys Res
Commun. 2000;267:875-880
301. Mahdi F. Factor xii interacts with the multiprotein assembly of urokinase
plasminogen activator receptor, gc1qr, and cytokeratin 1 on endothelial cell
membranes. Blood. 2002;99:3585-359 180
302. Stavrou E, Schmaier AH. Factor xii: What does it contribute to our understanding
of the physiology and pathophysiology of hemostasis and thrombosis. Thromb
Res. 2010;125:210-215
303. White-Adams TC, Berny MA, Tucker EI, Gertz JM, Gailani D, Urbanus RT, et al.
Identification of coagulation factor xi as a ligand for platelet apolipoprotein e
receptor 2 (apoer2). Arterioscler Thromb Vasc Biol. 2009;29:1602-1607
304. Marx G. Divalent cations induce protofibril gelation. Am J Hematol.
1988;27:104-109
305. Dugan Ta, Yang VW-C, McQuillan DJ, Höök M. Decorin modulates fibrin
assembly and structure. J Biol Chem. 2006;281:38208-38216
306. Siddiq MM, Tsirka SE. Modulation of zinc toxicity by tissue plasminogen
activator. Mol Cell Neurosci. 2004;25:162-171
307. Chavakis BT, May AE, Preissner KT, Kanse SM. Molecular mechanisms of zinc-
dependent leukocyte adhesion involving the urokinase receptor and beta2-
integrins. Blood. 1999;93:2976-2983
308. Hwang IY, Sun ES, An JH, Im H, Lee SH, Lee JY, et al. Zinc-triggered induction
of tissue plasminogen activator by brain-derived neurotrophic factor and
metalloproteinases. J Neurochem. 2011;118:855-863
309. Kim YH, Park JH, Hong SH, Koh JY. Nonproteolytic neuroprotection by human
recombinant tissue plasminogen activator. Sciences. 1999;284:647-651 181
310. Henderson SJ, Stafford AR, Leslie BA, Kim PY, Vaezzadeh N, Ni R, et al. Zinc
delays clot lysis by attenuating plasminogen activation and plasmin-mediated
fibrin degradation. Thromb Haemost. 2015;113:1278-1288
311. Ehrenfest DMD, Rasmusson L, Albrektsson T. Classification of platelet
concentrates: From pure platelet-rich plasma (p-prp) to leucocyte-and platelet-rich
fibrin (l-prf). Trends Biotechnol. 2009;27:158-167
312. Su CY, Kuo YP, Tseng YH, Su C-H, Burnouf T. In vitro release of growth factors
from platelet-rich fibrin (prf): A proposal to optimize the clinical applications of
prf. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2009;108:56-61
313. Foster TE, Puskas BL, Mandelbaum BR, Gerhardt MB, Rodeo SA. Platelet-rich
plasma from basic science to clinical applications. Am J Sports Med.
2009;37:2259-2272
314. Lipinski B, Pretorius E. Iron-induced fibrin in cardiovascular disease. Curr
Neurovasc Res. 2013:269-274
315. Hasegawa T, Sorensen L, Ooi H, Marshall BC. Surface plasmin generation
transcriptional and post-transcriptional effects on u-pa and pai-1 expression. Am J
Respir Cell Mol Biol. 1999;21:275-282.
316. Day SM, Duquaine D, Mundada LV, Menon RG, Khan BV, Rajagopalan S, et al.
Chronic iron administration increases vascular oxidative stress and accelerates
arterial thrombosis. Circulation. 2003;107:2601-2606 182
317. Park SE, Li MH, Kim JS, Sapkota K, Kim JE, Choi BS, et al. Purification and
characterization of a fibrinolytic protease from a culture supernatant of
flammulina velutipes mycelia. Biosci Biotechnol Biochem. 2014;71:2214-2222
318. Putnis A. An Introduction to Mineral Sciences. Cambridge University Press;
1992:116.
319. Karck M, Tanaka S, Berenshtein E, Sturm C, Haverich A, Chevion M. The push-
and-pull mechanism to scavenge redox-active transition metals: A novel concept
in myocardial protection. J Thorac Cardiovasc Surg. 2001;121:1169-1178
320. Burggraf D, Martens HK, Dichgans M, Hamann GF. Rt-pa causes a dose-
dependent increase in the extravasation of cellular and non-cellular blood
elements after focal cerebral ischemia. Brain Res. 2007;1164:55-62
321. Stewart D, Kong M, Novokhatny V, Jesmok G, Marder VJ. Distinct dose-
dependent effects of plasmin and tpa on coagulation and hemorrhage. Blood.
2003;101:3002-3007
322. Yepes M, Roussel BD, Ali C, Vivien D. Tissue-type plasminogen activator in the
ischemic brain: More than a thrombolytic. Trends Neurosci. 2009;32:48-55
323. Yepes M, Sandkvist M, Moore EG, Bugge TH, Strickland DK, Lawrence DA.
Tissue-type plasminogen activator induces opening of the blood-brain barrier via
the LDL receptor–related protein. J Clin Invest. 2003;112:1533-1540
324. Thiex R, Mayfrank L, Rohde V, Gilsbach JM, Tsirka SA. The role of endogenous
versus exogenous tpa on edema formation in murine ICH. Exp Neurol.
2004;189:25-32 183
325. Nicole O, Docagne F, Ali C, Margaill I, Carmeliet P, MacKenzie ET, et al. The
proteolytic activity of tissue-plasminogen activator enhances nmda receptor-
mediated signaling. Nat Med. 2001;7:59-64
326. Siao CJ, Tsirka SE. Tissue plasminogen activator mediates microglial activation
via its finger domain through annexin II. J Neurosci. 2002;22:3352-3358
327. Alessio AM, Beltrame MP, Nascimento MCF, Vicente CP, de Godoy JaP, Silva
JCRS, et al. Circulating progenitor and mature endothelial cells in deep vein
thrombosis. Int J Med Med Sci. 2013;10:1746-1754
328. Wu L, Du Y, Lok J, Lo EH, Xing C. Lipocalin-2 enhances angiogenesis in rat
brain endothelial cells via reactive oxygen species and iron-dependent
mechanisms. J Neurochem. 2014:1-7
329. Canzoniero LM, Turetsky DM, Choi DW. Measurement of intracellular free zinc
concentrations accompanying zinc-induced neuronal death. Journal Neurosci.
1999;19:RC31
330. Choi BY, Kim JH, Kim HJ, Lee BE, Kim IY, Sohn M, et al. Zinc chelation
reduces traumatic brain injury-induced neurogenesis in the subgranular zone of
the hippocampal dentate gyrus. J Trace Elem Med Biol. 2014;28:474-481
331. Medvedeva YV, Lin B, Shuttleworth CW, Weiss JH. Intracellular Zn2+
accumulation contributes to synaptic failure, mitochondrial depolarization, and
cell death in an acute slice oxygen–glucose deprivation model of ischemia. J
Neurosci. 2009;29:1105-1114 184
332. Weiss JH, Hartley DM, Koh JY, Choi DW. Ampa receptor activation potentiates
zinc neuroioxicity. Neuron. 1993;10:43-49
! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !
Thesis and Dissertation Services ! !