role of the haptoglobin-cd163 scavenging pathway...
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ROLE OF THE HAPTOGLOBIN-CD163 SCAVENGING PATHWAY FOLLOWING HEMORRHAGIC STROKE
By
JENNA L. LECLERC
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
2016
© 2016 Jenna L. Leclerc
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ACKNOWLEDGMENTS
I would like to thank my mentor Dr. Sylvain Doré for providing me unique and
tailored guidance and a thriving environment throughout my PhD studies. Over these
years, the many great times in the lab and abroad at various national and international
conferences have been most influential, and I am grateful for the friendship. The
knowledge I have gained regarding experimental design, leadership, administration, and
mentoring will certainly continue to further my professional development as a clinician-
scientist. A special acknowledgment goes to Dr. Spiros Blackburn, an endovascular
neurosurgeon who has provided his own unique guidance, friendship, and a distinct
clinical continuity during this time. A special thanks also goes to my committee
members, Drs. David Borchelt, Brian Hoh, and Alfred Lewin. The diverse backgrounds
and associated input towards this work has definitely facilitated moving this research
forward. With this team in place, it has truly been an amazing journey, and I thank
everyone for their time and longitudinal input, from both a personal and professional
perspective. Last, I would like to thank all the awesome people I have had the
opportunity to work with in the College of Medicine, Departments of Anesthesiology and
Neuroscience, and notably in the Center for Translational Research in
Neurodegenerative Disease, for their kindness and willingness to help with the many
various aspects of this work on a day-to-day basis. My friends and family, especially my
husband, deserve an utmost acknowledgment for their continued support throughout
this time.
This work was in part supported by my individual NIH fellowship NS086441.
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TABLE OF CONTENTS page
ACKNOWLEDGMENTS .................................................................................................. 3
LIST OF TABLES ............................................................................................................ 7
LIST OF FIGURES .......................................................................................................... 8
ABSTRACT ................................................................................................................... 10
CHAPTER
1 HEMORRHAGIC STROKE AND THE PUTATIVE ROLE OF THE HAPTOGLOBIN-CD163 SCAVENGING PATHWAY .............................................. 12
Stroke Epidemiology ............................................................................................... 12
Pathophysiology of Subarachnoid Hemorrhage ...................................................... 13 Pathophysiology of Intracerebral Hemorrhage ........................................................ 16 Role of Hemoglobin, Heme, and Iron Metabolism in Hemorrhagic Stroke .............. 18
The Haptoglobin-CD163 Scavenging Pathway ....................................................... 21 Specific Aims .......................................................................................................... 24
2 HAPTOGLOBIN PHENOTYPE PREDICTS THE DEVELOPMENT OF FOCAL AND GLOBAL CEREBRAL VASOSPASM AND MAY INFLUENCE OUTCOMES AFTER ANEURYSMAL SUBARACHNOID HEMORRHAGE ............ 27
Introduction ............................................................................................................. 27 Methods .................................................................................................................. 28
Clinical Data and Biospecimen Collection ........................................................ 28 Radiographic Vasospasm ................................................................................. 29
Clinical Deterioration from Delayed Cerebral Ischemia .................................... 29 Hp Typing ......................................................................................................... 30 Statistical Analyses .......................................................................................... 31
Results .................................................................................................................... 31
Radiographic Vasospasm ................................................................................. 32 Delayed Cerebral Ischemia .............................................................................. 33 Functional Outcomes ....................................................................................... 33
Mortality ............................................................................................................ 34 Discussion .............................................................................................................. 34
3 INCREASED BRAIN HAPTOGLOBIN LEVELS IMPROVES OUTCOMES FOLLOWING EXPERIMENTAL INTRACEREBRAL HEMORRHAGE .................... 48
Introduction ............................................................................................................. 48 Methods .................................................................................................................. 49
Mice .................................................................................................................. 49
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rAAV1 Construction and Preparation ............................................................... 49 rAAV1 Injection ................................................................................................. 51 Randomization, Exclusion, Blinding ................................................................. 51
Neuronal-Glial Mixed Primary Cultures and rAAV1 Transduction .................... 52 Collagenase ICH Model.................................................................................... 53 Autologous Whole Blood ICH Model ................................................................ 54 Functional Outcomes ....................................................................................... 55 Tissue and Biospecimen Harvesting ................................................................ 56
Western Blotting ............................................................................................... 57 Histology and Quantification ............................................................................. 57 Statistics ........................................................................................................... 59
Results .................................................................................................................... 60
Characterization of rAAV1 Expression ............................................................. 60 ICH-Induced Brain Injury and Functional Outcomes ........................................ 60 Hemoglobin ...................................................................................................... 61
Heme Oxygenase 1 .......................................................................................... 61
Perls’ Iron ......................................................................................................... 62 Lipid Peroxidation ............................................................................................. 62 BBB Integrity .................................................................................................... 63
Angiogenesis/Neovascularization ..................................................................... 63 Astrogliosis ....................................................................................................... 63
Microgliosis ....................................................................................................... 64 Discussion .............................................................................................................. 65
4 CD163 HAS DISTINCT TEMPORAL INFLUENCES ON INTRACEREBRAL HEMORRAHAGE OUTCOMES .............................................................................. 91
Introduction ............................................................................................................. 91
Methods .................................................................................................................. 92 Mice .................................................................................................................. 92
ICH Model ........................................................................................................ 92 Functional Outcomes ....................................................................................... 93 Histology and Quantification ............................................................................. 93
Statistics ........................................................................................................... 94 Results .................................................................................................................... 95
Mortality ............................................................................................................ 95 ICH-induced Brain Damage .............................................................................. 95
Functional Outcomes ....................................................................................... 96 Hemoglobin ...................................................................................................... 96 Heme Oxygenase 1 and Iron ............................................................................ 96 Blood-Brain Barrier Integrity ............................................................................. 97 Astrogliosis ....................................................................................................... 97
Angiogenesis/Neovascularization ..................................................................... 99 Discussion .............................................................................................................. 99
5 SUMMARY AND CONCLUSIONS ........................................................................ 111
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Summary .............................................................................................................. 111 Discussion ............................................................................................................ 111
LIST OF REFERENCES ............................................................................................. 120
BIOGRAPHICAL SKETCH .......................................................................................... 139
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LIST OF TABLES
Table page 2-1 Demographics, patient characteristics, and subarachnoid hemorrhage
severity stratified by Hp phenotype ..................................................................... 43
2-2 Multivariate analysis of radiographic vasospasm and delayed cerebral ischemia ............................................................................................................. 44
2-3 Covariate results for multivariate analysis of radiographic vasospasm and delayed cerebral ischemia .................................................................................. 45
2-4 Multivariate analysis of functional outcomes and mortality ................................. 46
2-5 Covariate results for multivariate analysis of functional outcomes and mortality .............................................................................................................. 47
3-1 Details of the antibodies used for Western blotting and/or immunohistochemistry ........................................................................................ 90
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LIST OF FIGURES
Figure page 2-1 Demonstration of Hp typing methods ................................................................. 41
2-2 A prototypical example of a 34 year old female with aSAH ................................ 42
3-1 Demonstration of lesion volume quantification methods ..................................... 71
3-2 In vitro characterization of rAAV1 expression ..................................................... 72
3-3 In vivo characterization of rAAV1 expression ..................................................... 74
3-4 High local levels of haptoglobin reduces collagenase ICH-induced brain injury ................................................................................................................... 76
3-5 Haptoglobin therapy improves functional outcomes following collagenase-induced ICH ........................................................................................................ 77
3-6 High local levels of haptoglobin reduces ICH-induced brain injury in the autologous whole blood model ........................................................................... 78
3-7 High local levels of haptoglobin reduces the amount of hemoglobin after ICH ... 79
3-8 Haptoglobin therapy decreases heme oxygenase 1 expression after ICH ......... 80
3-9 High local levels of haptoglobin increases Perls’ iron content after collagenase-induced ICH ................................................................................... 81
3-10 High local levels of haptoglobin increases Perls’ iron content after ICH in the autologous whole blood model ........................................................................... 82
3-11 Haptoglobin therapy reduces lipid peroxidation after ICH ................................... 83
3-12 High local levels of haptoglobin improves blood-brain barrier integrity after ICH ..................................................................................................................... 84
3-13 Haptoglobin therapy reduces angiogenesis/neovascularization after ICH .......... 85
3-14 High local levels of haptoglobin reduces VEGF expression following ICH ......... 86
3-15 Effect of haptoglobin therapy on astrogliosis after ICH ....................................... 87
3-16 Haptoglobin therapy increases microgliosis following collagenase-induced ICH ..................................................................................................................... 88
3-17 Effect of haptoglobin therapy on microgliosis after ICH in the autologous whole blood model .............................................................................................. 89
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4-1 CD163 deficiency temporally influences ICH-induced brain damage ............... 104
4-2 CD163 deficiency temporally influences functional outcomes after ICH ........... 105
4-3 CD163 deficiency reduces BBB dysfunction and Hb content ........................... 107
4-4 Effect of CD163 deficiency on HO1 and Perls’ iron .......................................... 108
4-5 CD163 deficiency temporally influences astrogliosis ........................................ 109
4-6 Effect of CD163 deficiency on angiogenesis/neovascularization ...................... 110
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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
ROLE OF THE HAPTOGLOBIN-CD163 SCAVENGING PATHWAY FOLLOWING
HEMORRHAGIC STROKE
By
Jenna L. Leclerc
August 2016
Chair: Sylvain Doré Major: Medical Sciences – Neuroscience
Hemorrhagic strokes are acute debilitating neurological insults, yet no effective
treatments exist. Brain injury and poor outcomes are precipitated by hemolytic events
that release massive quantities of cytotoxic hemoglobin (Hb) into the extracellular
space. The haptoglobin (Hp)-CD163 scavenging system represents the most upstream
defense mechanism against Hb. Hp immediately and irreversibly binds extracellular Hb,
which directly abrogates its intrinsic proxidant/proinflammatory properties and prevents
the formation of neurotoxic Hb degradation products. Hp may be present in the brain in
miniscule quantities, and although Hp enters the brain as part of the bleed, the
combined levels are insufficient to combat the supraphysiologic Hb levels seen following
hemorrhagic stroke. CD163 is a receptor that safely clears Hp-Hb complexes and
uncomplexed Hb, and also has potent anti-inflammatory effects that are particularly
important during the resolution phase of tissue injury.
Despite the vital role for the Hp-CD163 scavenging pathway following systemic
intravascular or extravascular hemolysis, a paucity of literature exists regarding similar
central paradigms, a surprising notion given the putative therapeutic implications of
targeting this pathway. The present work was designed to further characterize the
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contribution of the Hp-CD163 scavenging pathway following hemorrhagic stroke, a
central form of extravascular hemolysis. Viral and genetic approaches were utilized in
two complimentary preclinical hemorrhagic stroke models, collagenase-induced
spontaneous bleeding and autologous whole blood injection. Additionally, biospecimens
from a clinical hemorrhagic stroke population were analyzed.
The findings of this work reveal that following hemorrhagic stroke, i) Hp
phenotype is clinically an independent risk factor for the development of cerebral
vasospasm, poor outcomes and mortality, ii) high local levels of Hp improve anatomical
and functional outcomes in two preclinical models, and iii) CD163 has distinct temporal
influences on experimental outcomes, with acute deleterious effects, but delayed
beneficial properties. Collectively, these results demonstrate the importance of the Hp-
CD163 scavenging pathway following hemorrhagic stroke and establish the potential of
therapeutically targeting this pathway. Although additional studies are needed to further
characterize this pathway in this setting, the results presented here are promising and
are expected to apply to the various other conditions in which blood is released within
the brain.
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CHAPTER 1 HEMORRHAGIC STROKE AND THE PUTATIVE ROLE OF THE HAPTOGLOBIN-
CD163 SCAVENGING PATHWAY
Stroke Epidemiology
Stroke is the fourth most common cause of death in the United States and the
leading cause of long-term severe disability.1 Each year, ~800,000 people experience a
new or recurrent stroke, which equates to someone having a stroke every 40 seconds
on average in the United States.2 In 2013, ~1 of 20 deaths in the United States was due
to a stroke, and someone dies of stroke approximately every four minutes.2 The overall
prevalence of stroke is 2.6%, and age, sex and ethnic risk differences exist among the
various stroke subtypes.2
Strokes are segmented into two main categories: ischemic and hemorrhagic.
Approximately 87% of strokes are ischemic and 13% are hemorrhagic, with 10% and
3% of the latter representing intracerebral hemorrhage (ICH) and subarachnoid
hemorrhage (SAH), respectively.3 Ischemic stroke occurs when a local thrombus or
embolus occludes a cerebral vessel and obstructs blood flow to the brain, depriving the
brain of oxygen and nutrients. On the other hand, hemorrhagic stroke occurs when a
cerebral artery ruptures resulting in the release of blood into the brain. The distinction
between ICH and SAH is based on the location of intracranial bleeding, either
intraparenchymal or within the subarachnoid space, respectively.
In a recent systematic review of population-based studies, the worldwide
incidence of SAH ranged from 2-16 per 100,000 persons.4 Such wide variation is due to
regional differences in SAH occurrence rate. For example, certain isolated populations
like Finland report rates at 22.5 per 100,000 persons, while the incidence in China is
recorded at 2.0 cases per 100,000 persons.5 In the United States, the incidence of SAH
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is estimated at 9 per 100,000 persons per year.6, 7 SAH carries a large toll in terms of
productive life-years lost because it has an earlier mean age of onset and is associated
with high disability and morbidity rates when compared to other types of stroke.8 Sex
plays a distinct role, where SAH more often occurs in females, and younger age and
female sex is associated with increased risk for key SAH-associated secondary
complications that have strong implications on patient outcomes.9-11
ICH affects 16-33 per 100,000 people worldwide each year.12-19 The incidence of
ICH is lowest and highest in whites and asians, respectively, with blacks having an
intermediate incidence.14 Age is an important risk factor and outcome predictor for ICH,
with the risk doubling every decade after the age of 35,20, 21 and elderly patients having
been reported to have worse functional outcomes than their younger counterparts.22 In
addition to older age and black ethnicity, hypertension, high alcohol intake, and lower
cholesterol, LDL cholesterol, and triglycerides have been identified as risk factors for
ICH.20, 21 In contrast to SAH, sex does not seem to impact the risk for ICH.16
The actual rate of SAH and ICH may be higher, since death can occur prior to
hospital admission, and thus some cases go undocumented if no autopsy is performed.
The distinct difference in SAH and ICH incidence across different geographic regions is
likely attributed to certain genetic and/or behavioral risk factors intrinsic to these areas.
Pathophysiology of Subarachnoid Hemorrhage
Intracranial aneurysms, most commonly found within arteries of the Circle of
Willis, affect 2-5% of the population and rupture of these aneurysms accounts for 85%
of all SAH cases.23, 24 Ten and five percent of the remaining SAH cases are accounted
for by the relatively benign non-aneurysmal perimesencephalic hemorrhage and rare
causes such as drug use, vascular malformations, arterial dissections, mycotic
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aneurysms and other inflammatory or non-inflammatory lesions of cerebral arteries,
respectively.24 Overall mortality has been estimated to be as high as two-thirds,25 and
although around 12-15% of people die instantly from the bleed and in-hospital mortality
is estimated at 20%,6, 26 delayed mortality and morbidity is high and largely has been
linked to cerebral vasospasm (CV)27 and early brain injury.28
Those that survive the initial bleed are at risk for a multitude of secondary insults
including rebleeding, hydrocephalus, and CV-induced delayed ischemic deficits.29 Other
than rebleeding, which occurs in less than 7% SAH patients,30 CV is the leading cause
of morbidity and mortality following SAH.27 Poor outcome, due to SAH, occurs in 50 to
75% of patients, and this is attributed to secondary ischemia in approximately 30% of
patients.31 This delayed cerebral ischemia (DCI) in SAH patients has been attributed to
the anatomic narrowing of arteries in the cerebral vasculature.32 The reason certain
people develop CV and symptomatic ischemia following SAH while others remain
asymptomatic with minimal CV remains an enigma.
Acute SAH is a complex and multifaceted disorder that plays out over days to
weeks.29 Aneurysm rupture results in the release of blood into the subarachnoid space
where several main arteries supplying the brain are located. The presence of red blood
cells (RBCs), and their main cellular component, hemoglobin (Hb), in close proximity to
these major cerebral vessels has been suggested as the primary instigator of CV,
resulting DCI and/or infarction, and poor outcomes in SAH patients.33-36 This correlation
is strengthened by the known association between the volume of blood in the
subarachnoid space and the severity of angiographic vasospasm8 and a study involving
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monkeys where removal of the blood clot was shown to reverse angiographic
vasospasm.34
CV has its onset around day 3 after SAH, peaks on days 6-8, and can last 2-3
weeks.8 Within 24h following SAH, an intense polymorphonuclear cell infiltration of the
meninges is seen.33 Phagocytosis and lysis of RBCs occurs by 16-32h, peaks around
day 7, but continues for days, with clumps of intact RBCs still enmeshed in the
arachnoid for up to 35 days after SAH.33 It has previously been established that
changes in Hb concentrations within the subarachnoid space tend to mirror the
evolution of CV, though the mechanisms by which free Hb causes delayed arterial
narrowing are multiple and poorly understood.37-39 Possibilities include neuronal
apoptosis, scavenging or decreased production of nitric oxide, increased endothelin 1
levels, direct oxidative stress on smooth muscle cells, free radical production and lipid
peroxidation of cell membranes, modification of potassium and calcium channels, and
differential up-regulation of genes.38
Most patients with SAH are critically ill and require prolonged intensive care unit
stay,29 mainly due to the requirement for extended monitoring for the development of
CV, resulting in disproportionately high costs. Because of this relationship between CV,
cerebral ischemia and/or infarction, and poor outcome, significant efforts have been
made to establish treatments that decrease the incidence of CV after SAH. However,
recent drug trials have had disappointing results.40, 41 Currently, medications and
hemodynamic maneuvers are used as standard of care for the treatment of CV and to
improve outcome after SAH.42, 43 However, these treatments have either a very limited
efficacy and are initiated after the cascade of symptomatic ischemia is realized.
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Pathophysiology of Intracerebral Hemorrhage
ICH is most commonly a sequelae of hypertensive vasculopathy, and secondly
from underlying cerebral amyloid angiopathy (CAA), but can also result from various
other etiologies such as trauma, vascular malformations, and medical therapies like
anticoagulants.44-49 Persistent high blood pressure preferentially damages the tortuous
small penetrator vessels that branch off major intracerebral arteries since there is no
intermediate size vessel to stepwise reduce the pressure from the larger parent
vessel.50-52 Accordingly, spontaneous hypertensive ICH most frequently occurs in the
caudate and putamen (lenticulostriate penetrators off the proximal segment of the
middle cerebral artery), thalamus (thalamostriate penetrators off proximal segments of
the posterior cerebral arteries), and pons and midbrain (basilar artery penetrators).
Whereas, CAA-ICH presents as lobar hemorrhage (cortical/subcortical).53, 54 Subclinical
cerebral microbleeds are relatively common in patients with ICH, suggesting that they
are a marker of bleeding-prone vessels diseased by amyloid deposition or persistent
high blood pressure.55-57 Furthermore, the location of these microbleeds usually
correlates with the etiology-dependent ICH location.
Primary damage occurs at the time of insult and is due to pressure from the
hematoma that causes tissue compression and physiomechanical disruption of neurons
and glia.12, 58 In addition to the clot volume, perilesional edema also contributes to mass
effects and increased intracranial pressure, resulting in reduced cerebral perfusion
pressure and ischemic injury, and in severe cases, herniation and death.59 In those that
survive the initial bleed, secondary injury occurs hours to days later and is largely due to
the presence of blood components and their breakdown products that precipitate many
parallel-operating neurotoxic processes leading to irreversible brain damage and poor
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outcomes.58 These processes include oxidative stress, inflammation, blood-brain barrier
(BBB) breakdown, edema, oligemia, mitochondrial dysfunction, excitotoxicity, spreading
depression, and cell death.12, 58, 60 Between 35-52% of patients with ICH will not survive
the first 30 days, and only 20% of patients regain functional independence at 6 months
post-bleed.61, 62 Currently, no treatments exist, and the only available interventions
include supportive care, and in some select cases, invasive surgery to evacuate
hematomas, which has produced disappointing results.63
In a subset of patients, hemorrhage enlargement occurs and further exacerbates
primary and secondary damage. Hemorrhage expansion has been associated with
neurological deterioration and significantly increased mortality and morbidity in several
retrospective and prospective studies.64-70 With the initial hemorrhage and subsequent
expansion, surrounding vessels are stretched and are more prone to rupture, resulting
in the recruitment of new bleeding sites that further contribute to clot enlargement.
However, hemorrhage enlargement only occurs in a subset of patients, and although
the exact mechanisms are not yet defined, BBB dysfunction and inflammatory-mediated
dysregulation of hemostasis have been implicated.71, 72 In a prospective study, 38% of
patients experienced a hematoma volume increase of >33% over the first 24h.73
In vivo studies have delineated the respective contribution of purely mass effects
and tissue compression from that of the toxicity associated with the presence of blood
components and their breakdown products. First, increased intracranial pressure,
reduced cerebral blood flow, and some brain damage is seen with microballon inflation
in the basal ganglia of rats.74 However, worse damage and edema is seen following
autologous blood injection as compared to an oil-wax mixture.75 In humans, mass
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effects cause neurological deficits, but little edema is seen within the acute period prior
to the start of hemolytic events. Compared to whole packed erythrocytes, infusion of
lysed RBCs results in significantly more brain damage, BBB dysfunction, and edema
within 24h,76-79 directly showing that RBC contents are highly toxic. Erythrocytes are
frequently termed ‘sacs of Hb’ due to the 250 million molecules of Hb they contain,
which far exceeds the quantities of other cellular contents. Indeed, infusions of Hb
and/or its breakdown products results in significant brain damage.80, 81 Hemolytic events
begin around 24h post-ICH, and continue for days.77, 82-86 Hemolysis and the liberation
of large quantities of Hb is associated with delayed brain edema in a bleed volume
dose-dependent manner, and bleed volume is an important predictor of outcomes
following ICH.87 Consequently, therapeutic paradigms aimed at detoxifying and
improving the clearance of blood products that precipitate secondary damage and poor
outcomes would represent a clinically relevant treatment strategy for ICH.
Role of Hemoglobin, Heme, and Iron Metabolism in Hemorrhagic Stroke
A common pathologic mechanism leading to secondary brain injury and poor
outcomes following hemorrhagic stroke is that of the toxicity associated with blood
components and their breakdown products. RBCs represent ~45% of blood by volume,
and Hb is overwhelmingly the main cellular component of erythrocytes. RBCs are
initially stable, but soon after the hemorrhage they begin to lose their oxygen and de-
stabilize, resulting in oxyhemoglobin conversion to deoxyhemoglobin. Hemolysis after
SAH and ICH follows a similar time course, beginning around 24h post-bleed, resulting
in the release of large quantities of deoxyhemoglobin into the CSF compartment or
parenchyma, respectively. Extracorpuscular Hb is a potent neurotoxin and major
contributor to brain injury following hemorrhagic stroke due to its ability to consume
19
nitric oxide and serve as a Fenton reagent, ultimately resulting in the production of
highly reactive superoxide and hydroxyl radicals.60, 88-91 These radicals impose strong
pro-oxidative insults towards nearby viable cells causing further breakdown of the BBB,
edema, inflammation, and neuronal apoptosis.89, 92 The Hp-CD163 scavenging pathway
is the endogenous system responsible for the immediate detoxification and receptor-
mediated endocytosis of Hb, a pathway that is the main focus of this dissertation, and
accordingly, is detailed in a subsequent section.
All components of the Hb/heme degradation pathway are present in the central
nervous system (CNS), and a plethora of studies have shown that this pathway is active
following hemorrhagic stroke. If not cleared by the Hp-CD163 pathway,
deoxyhemoglobin is spontaneously and nonenzymatically oxidized to methemoglobin as
the heme-iron is converted from ferrous to ferric form.93 The toxic heme moieties quickly
separate from methemoglobin, particularly in the presence of nitric oxide and reactive
oxygen species, which exist at sites of inflammation such as the injured brain area after
ICH or subarachnoid space after SAH.94 Free heme has several routes of toxicity
including the generation of superoxide and hydroxyl radicals, release of redox-active
iron, depletion of cellular stores of NADPH and glutathione, peroxidation of membrane
lipids, and sensitization of cells to subsequent noxious stimuli.94, 95 In an analogous
fashion to the Hp-CD163 pathway, the hemopexin-CD91 scavenging pathway is the
endogenous high-affinity system responsible for the immediate detoxification and
receptor-mediated endocytosis and clearance of toxic extracellular heme. However, it
should be noted that albumin also possesses moderate heme affinity and is the most
abundant plasma protein, although this albumin-bound heme is transferred to
20
hemopexin if present.93, 96 Thus, albumin could also serve as a secondary heme-trap
with overload of the hemopexin pathway since it can be internalized by neurons and
glia.93
Heme is metabolized by intracellular heme oxygenase (HO) enzymes, of which
there are two primary isozymes, HO1 and HO2. HO1 is highly inducible, notably by
heme its substrate and oxidative stress, among many other factors, and can be
expressed by many brain cell types.97 HO2 is constitutively expressed at high levels in
the brain.98 Heme degradation by HO enzymes is a multi-step process beginning with
the oxidation of heme, followed by ring-opening and release of carbon monoxide (CO),
ending with formation of biliverdin and iron (Fe2+). Biliverdin is rapidly converted to
bilirubin by biliverdin reductase.99 HO enzymes also have various other functions,
including serving as chaperones and facilitating cellular iron efflux.93
The heme degradation products CO, biliverdin/bilirubin, and iron have their own
physiological or pathological effects. First, CO could act as a gaseous messenger within
the cell or on adjacent cells and thus at low concentrations is considered
neuroprotective mostly via acting through guanylate cyclase to produce anti-oxidant and
anti-inflammatory effects.93, 97 Whereas, high CO concentrations are classically toxic
(carbon monoxide poisoning) and can exacerbate mitochondrial free radical
generation.100 Iron precipitates free-radical induced damage to biological molecules
including lipids, proteins, and DNA.101 To render Fe2+ non-toxic, it is either exported
where it binds soluble extracellular transferrin or it enters an intracellular labile iron pool
where it is oxidized to Fe3+ and stored by ferritin, thereby eliciting anti-inflammatory
responses.97 With transferrin saturation, citrate and ascorbate are expected to
21
participate in iron transport.93 Biliverdin and bilirubin are molecules known to have anti-
oxidant properties through their ability to scavenge ROS.102 Biliverdin and bilirubin may
also bind albumin, thereby preventing the harmful oxidation of albumin-bound lipids103.
However, at high concentrations, biliverdin and bilirubin are toxic. Additionally, bilirubin
may spontaneously oxidize generating bilirubin oxidation products, which have
previously been shown to have direct vasoconstrictive and neurotoxic properties.104, 105
In summary, the Hp-CD163 and Hpx-CD91 scavenging pathways function to
reduce the pro-oxidative, pro-inflammatory and cytotoxic effects that extracorpuscular
Hb and heme, respectively, impose on nearby cells. These systems effectively deliver
any extracellular toxic Hb/heme moieties to an intracellular compartment for degradation
by HO enzymes, such that the safe storage and/or redistribution of Hb degradation
products can be accomplished following intravascular or extravascular hemolysis.
The Haptoglobin-CD163 Scavenging Pathway
The Hp-CD163 scavenging system is the primary defense mechanism in the
body against the injurious effects of extracorpuscular Hb, and thus also represents the
most upstream modulator of the deleterious properties of Hb degradation products. Hp
is an acute phase glycoprotein that is predominately produced by the liver and secreted
into the plasma at high concentrations (0.3-3.0 mg/mL).97 Extracorpuscular Hb is
present with physiologic intravascular hemolysis that accounts for 10-20% of the normal
turnover of RBCs and with pathologic extravascular hemolysis that occurs following
internal bleeding. This extracorpuscular Hb is immediately and essentially irreversibly
bound by Hp that is present in the blood stream in the case of intravascular hemolysis,
or Hp that enters the space as part of an internal bleed in the case of extravascular
hemolysis. The Hp-Hb complex is endocytosed by cells of the monocyte lineage
22
through scavenger receptor CD163,106 resulting in the safe degradation of Hb. With the
many known systemic conditions that can result in severe hemolytic episodes, Hp is
depleted since it is not recycled following endocytosis of the Hp-Hb complex, and it
takes approximately 5-7d for the Hp levels to return to baseline.107, 108 As such,
serum/plasma levels of Hp are a highly sensitive and specific biomarker of acute
hemolytic events that is routinely used as a clinical diagnostic. With
hypohaptoglobinemia, free Hb and its toxic breakdown products, heme and iron, are
free to impose their strong cytotoxic effects, injuring nearby cells.
Hp attenuates extracorpuscular Hb toxicity initially through direct binding and
secondarily by facilitating its safe clearance by CD163. Several in vitro and in vivo
studies suggest that formation of the Hp-Hb complex is both redox protective and
improves Hb clearance by CD163.35, 97, 109-112 Further, recent crystal structure analyses
of the Hp-Hb complex show the reactive iron and pro-oxidative tyrosine residues are
buried in the complex near the Hp-Hb interface.113, 114 In humans, two Hp alleles exist,
resulting in three possible genotypes: Hp1-1, Hp2-1, or Hp2-2. Hp1-1 is the most
bioactive form, permitting the highest affinity binding to Hb, enhanced redox protection,
and fastest clearance of Hp-Hb complexes by the scavenger receptor.36, 115, 116 Not
surprisingly, the Hp2-2 phenotype has been correlated with increased risk for several
human disorders, including cardiovascular diseases.
Additional Hp-independent mechanisms of extracorpuscular Hb detoxification
have been reported.117, 118 These mechanisms all rely on either membrane-associated
CD163 (mCD163) or the soluble form. When Hp levels are depleted, mCD163 can
directly bind and internalize Hb, thus serving as its own fail-safe Hb scavenger
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receptor.118 The soluble form of CD163 (sCD163) is generated by TACE/ADAM17 and
neutrophil elastase-mediated ectodomain cleavage of mCD163.97 It is well known that
during inflammation and macrophage activation, sCD163 levels rise acutely due to
proteolytic cleavage near the cell membrane of mCD163 positive cells.119-121 It was
recently shown with surface plasma resonance and in vitro methods that sCD163 is
capable of binding Hp-Hb complexes with high affinity.113, 122 Another recent finding
demonstrated that sCD163 is able to interact directly with free Hb and IgG, forming a
sCD163-Hb-IgG complex that is readily endocytosed by monocytes/macrophages.117
Under conditions of severe hemolysis, when Hp levels are depleted, these Hp-
independent pathways could be particularly important in detoxifying free Hb.123
While the Hp-CD163 scavenging pathway is well-characterized in the periphery,
a paucity of literature exists regarding the role of this system centrally under normal or
pathologic conditions. The production of Hp in the brain is controversial, where some
have reported that Hp is synthesized by some brain cell types107 and others state Hp is
not synthesized in the CNS but is detectable within the CSF with a pattern suggestive of
leakage across the BBB.124 In either case, the low CNS Hp levels leave the brain
vulnerable to even small amounts of extravascular hemolysis. The expression of
mCD163 in the brain is restricted to primarily perivascular macrophages, but has also
been seen on choroid plexus and meningeal macrophages.125-129 Induction of Hp
expression by oligodendrocytes has been reported following “ICH-like” conditions,
although this conclusion requires further investigation.107, 108 In human post-mortem
samples and experimental models, CD163-positive macrophages/microglia have been
24
shown to accumulate within and surrounding the lesions following acute hemorrhagic or
non-hemorrhagic CNS pathology.125-129
Beyond the canonical role for the Hp-CD163 scavenging pathway in facilitating
the safe degradation of Hb, thereby preventing the damaging consequences of
extracorpuscular Hb, a few other relevant but relatively unexplored functions have been
described for Hp, mCD163, and sCD163. Hp has been reported to have angiogenic
properties and an overall anti-inflammatory effect.130, 131 Both mCD163 and sCD163
have been implicated as regulators of the pro-inflammatory cytokine TWEAK, and
mCD163 and sCD163 have also been suggested to modulate angiogenesis and T cell-
proliferation, respectively.97, 117, 132-134
Specific Aims
The specific aims of my dissertation work are to further understand the
mechanisms by which toxic free Hb is managed following hemorrhagic stroke and to
delineate the contribution of the haptoglobin-CD163 scavenging pathway in attenuating
this toxicity and improving outcomes. Such a mechanistic understanding is crucial for
additional molecular characterization of hemorrhagic stroke pathophysiology and for
subsequent design of innovative treatments for these acute neurological disorders that
currently have no effective therapies. The working hypotheses, as supported by the
aforementioned studies, are that Hp and/or CD163 are neuroprotective following
hemorrhagic stroke in few overlapping ways: 1) by sequestering extracellular pro-
oxidant and pro-inflammatory Hb, 2) by mediating its safe degradation, and 3) by
positively modulating the neuroinflammatory response. To address these hypotheses,
viral and genetic approaches were utilized in preclinical hemorrhagic stroke models and
25
the predictive and prognostic potential of Hp as a biomarker in a clinical hemorrhagic
stroke population was evaluated.
The first step was to identify whether Hp played a significant role following clinical
hemorrhagic stroke. At the time this work began in July of 2013, there were several
previous clinical studies demonstrating a key role for Hp phenotype in predicting
outcomes of various peripheral disorders associated with hemolysis and oxidative
stress-driven pathology, such as atherosclerosis, cardiovascular disease, diabetic
complications, and other pathologies associated with a significant vascular
component.135-144 There were also a few previous clinical studies aimed at correlating
Hp phenotype with the incidence of CV and outcomes after SAH. However, these
studies demonstrated conflicting results, likely resulting from methodological variations,
diverse patient populations, and limited data.145-147 Despite this past research
investigating the contribution of Hp type in SAH, several issues were unaddressed.
Building upon these studies, we comprehensively evaluated whether Hp phenotype is
an independent risk factor for CV, clinical deterioration as a result of CV-induced DCI,
poor functional outcomes, and mortality after SAH. This work bridges between the
previous ones, filling in some gaps, and also provides some novel findings aimed to
better understand the role of Hp phenotype in predicting CV, DCI, mortality and poor
outcomes after SAH. This study is detailed in Chapter 2 of this dissertation.
After establishing that Hp does indeed play a substantial predictive and
prognostic role following clinical SAH, the next step was to begin understanding the
mechanisms of neuroprotection and extend the findings to other types of hemorrhagic
stroke. A previous in vivo study had shown that Hp2-2 mice had increased vasospasm
26
and inflammatory infiltrates in the subarachnoid space, and reduced activity.148 One
other study had suggested that Hp is protective using non-specific techniques to deplete
and upregulate the expression of Hp mostly in a model of “ICH-like” brain injury.107 No
studies had directly evaluated whether high local levels of specifically Hp were
neuroprotective following ICH. Here, it is shown that adeno-associated viral-mediated
Hp overexpression locally in the brain reduces ICH-induced brain injury and improves
functional outcomes in two models of ICH. This study is detailed in Chapter 3 of this
dissertation.
The last step was to evaluate the role of the Hb scavenger receptor, CD163,
following hemorrhagic stroke. CD163-positive macrophages/microglia had previously
been shown to accumulate in the brain with time post-bleed and in other types of acute
neuropathology,125-129, 149, 150 yet no studies had directly evaluated the role of the CD163
after ICH. Here, CD163 is shown to have distinct temporal influences on ICH outcomes.
Acutely, the presence of CD163-positive macrophages is deleterious where CD163-/-
mice have improved function and reduced mortality. The opposite is true chronically,
where CD163-/- mice are worse and have increased mortality. This study is detailed in
Chapter 4 of this dissertation.
27
CHAPTER 2 HAPTOGLOBIN PHENOTYPE PREDICTS THE DEVELOPMENT OF FOCAL AND GLOBAL CEREBRAL VASOSPASM AND MAY INFLUENCE OUTCOMES AFTER
ANEURYSMAL SUBARACHNOID HEMORRHAGE
Introduction
Aneurysmal subarachnoid hemorrhage (aSAH) affects approximately 30,000
people per year in the United States, with 30-day mortality rates as high as 50%.24, 151
Only 20-25% of survivors regain their original functional capacity due to chronic
cognitive impairments and physical disabilities.145 Cerebral vasospasm (CV) is a
frequent complication, and this prolonged vasoconstriction may lead to delayed cerebral
ischemia (DCI), a known contributor to poor functional outcomes following aSAH.151
While various hypotheses have been put forward to explain the development of
aSAH-related CV, the presence of red blood cells, hemoglobin (Hb), and Hb breakdown
products within close proximity to major cerebral vessels have been strongly implicated
in the pathogenesis.33, 102, 124, 152 Haptoglobin (Hp) is an acute-phase protein with a
primary function of binding free Hb.130 Formation of this Hp-Hb complex directly
detoxifies Hb and mediates its safe clearance.35, 97, 112 There are two Hp alleles in the
human population, Hp1 and Hp2, allowing for three possible Hp genotypes: Hp1-1, Hp2-
1, and Hp2-2. The Hp2-2 protein has been reported to have a reduced ability to bind
and detoxify free Hb, and impairs the safe clearance of the Hp-Hb complex.153
Therefore, we hypothesized that the Hp2-2 phenotype may negatively contribute to
aSAH outcomes by mediating a greater degree of Hb-mediated oxidative and
inflammatory brain injury.
Previous clinical studies aimed at correlating Hp phenotype with the incidence of
CV and aSAH outcomes have demonstrated different results, likely resulting from
28
methodological variations, diverse patient populations, and limited data.145-147 Despite
this past research investigating whether Hp genotype is predictive of CV and aSAH
outcomes, several issues remain unaddressed. The purpose of this study was to
comprehensively evaluate whether Hp phenotype is an independent risk factor for CV,
clinical deterioration as a result of CV-induced DCI, poor functional outcomes, and
mortality after aSAH.
Methods
Following institutional review board approval, 74 patients with aSAH were
enrolled at the University of Florida between November 2006 and December 2013.
Patients over the age of 18 with a ruptured intracranial aneurysm were included.
Patients with non-aneurysmal SAH were excluded.
Clinical Data and Biospecimen Collection
Biospecimens were collected as part of two separate protocols: 62 patients are
from a previous prospective study for identifying biomarkers in SAH, and an additional
12 patients are from our ongoing sample biorepository for studying brain injuries. Blood
was obtained from an arterial line or by intravenous puncture and processed for storage
of serum. Patient demographics, including age, sex, and race, were collected as part of
enrollment for both protocols. For the prospective study, treatment type (clipping,
coiling), aneurysm size and location, and inpatient notes were collected as part of the
study. For the biorepository patients, these data were abstracted through a
retrospective chart review.
The initial clinical presentation and severity of aSAH were determined by the
following scales: World Federation of Neurological Surgeons (WFNS), Glasgow Coma
Scale (GCS), Fisher Grade and Hunt-Hess grade. For the majority of patients, WFNS,
29
GCS, and Hunt-Hess grade were collected prospectively by the treating physician. An
endovascular neurosurgeon, blinded to Hp phenotyping results, reviewed charts and
imaging in order to fill in any missing data and to determine Fisher Grade.154
Outcomes were assessed using the modified Rankin Scale (mRS) and Glasgow
Outcome Scale Extended (GOSE). For the prospective study, these data were collected
at discharge, 6 weeks and 1 year. For the biorepository, mRS scores were recorded at
discharge and last clinic follow-up.
Radiographic Vasospasm
To assess for radiographic CV, computed tomography angiography (CTA) and
cerebral angiography imaging was reviewed by an endovascular neurosurgeon blinded
to Hp phenotype and aSAH clinical course. Cerebral angiography was used to grade
the degree of CV when available, and CTA was used when angiography was not
performed. Imaging performed closest to post-bleed day seven was used to grade CV
as mild (<33% narrowing), moderate (33%-66% narrowing) or severe (>66%
narrowing). A CV grade was assigned bilaterally to the supraclinoid carotid, proximal
MCA (M1), distal MCA (M2), proximal ACA (A1), distal ACA (A2), vertebral, proximal
PCA (P1), distal PCA (P2), and for the basilar artery. Each of these arteries were
assigned a score of 0, 1, 2, or 3 corresponding to absent, mild, moderate, or severe CV,
respectively. We defined the term “global” vasospasm corresponding to the sum of the
CV values for the 17 cerebral arteries evaluated for each patient.
Clinical Deterioration from Delayed Cerebral Ischemia
Clinical deterioration as a result of CV-induced DCI was defined on the basis of
acute mental status changes after excluding for other causes (metabolic,
hydrocephalus, fever, infection, seizure). Clinical improvement after initiation of
30
hypertensive therapy, intra-arterial treatment of CV with verapamil, balloon angioplasty,
or brain imaging demonstrating ischemia was used for confirmation of DCI.
Hp Typing
The Hp type of aSAH patients was determined using a modified previously
described method based on detecting the α1- and α2-chain size differences of the
denatured Hp protein.155 A serum sample from each patient was diluted 75-fold, mixed
with an equal volume of 2X sample buffer (Bio-Rad, Hercules, CA), and boiled at 95˚C
for 5min. Ten microliters was loaded onto a 12% polyacrylamide gel and
electrophoresed at 100V for 10min, followed by 150V for 50min. Samples were
transferred to polyvinylidene fluoride membranes, which were blocked for 1h at room
temperature with 0.5% casein in Tris-buffered saline containing 0.01% Tween-20
(TBST). Membranes were incubated overnight at 4˚C with polyclonal rabbit anti-human
Hp (Dako, Carpinteria, CA) diluted 1:7,500 in blocking buffer supplemented with 0.2%
Tween-20. After four washes in TBST, the membranes were incubated for 1h at room
temperature with peroxidase labeled goat anti-rabbit IgG (Vector Labs, Burlingame, CA)
diluted 1:10,000 in blocking buffer supplemented with 0.2% Tween-20 and 0.01% SDS.
Following four washes in TBST, chemiluminescence was visualized using SuperSignal
West Pico substrate (Thermo Scientific, Waltham, MA) with a FluorChem E detection
system (ProteinSimple, San Jose, CA). Hp phenotyping was performed without
knowledge of aSAH clinical course. Serum samples from controls of known Hp type
were incorporated in all analyses. The type for these controls was determined by two
separate methods, including the one described above, with 100% match. Figure 2-1
provides a representative example of the Hp typing methods developed here, showing
both these controls and examples of aSAH patients of all Hp types.
31
Statistical Analyses
All statistical analyses were performed by a biostatistician using the R statistical
software package (V.3.0.2, Vienna, Austria). In order to compare outcomes across the
Hp phenotype groups, logistic regression was used for the dichotomous outcomes (DCI
and mortality), linear regression for the continuous scale outcomes (global CV, GOSE
and mRS), and negative binomial regression for the count outcomes (number of vessels
with a specific CV value). The logistic regression models included only age as a
covariate due to a limited number of patients with DCI and mortality in each of the Hp
phenotype groups. For the linear regression and negative binomial regression
multivariate models, age, GCS, WFNS, Fisher grade, Hunt-Hess grade, aneurysm size,
and treatment type were included as covariates. The Hp2-2 group was compared to the
Hp1-1/Hp2-1 group because of a relatively small number of patients in the Hp1-1 group
(n=11), and similar functional profile for Hp1-1 and Hp2-1.153 A two-sided p value less
than 0.05 was considered significant for all analyses.
Results
Of the 74 aSAH patients in this study, 11 were found to be Hp1-1 (14.9%), 39
Hp2-1 (52.7%), and 24 Hp2-2 (32.4%), which is in agreement with previously reported
Hp allele frequencies in this geographic region.153 Figure 2-1 shows an example of the
Hp typing. Demographics, patient characteristics, and the severity of aSAH for each of
the Hp phenotype groups are listed in Table 2-1. Overall, this cohort was predominately
female (73.0%) and Caucasian (77.0%), with a mean age (±SD) of 54.7±15.3 (range
20-88 years). The Hp phenotype groups did not show any significant differences
between age, gender, race, GCS, WFNS, Fisher Grade, Hunt-Hess Grade, or aneurysm
size (Table 2-1). Hp2-2 patients did tend to receive clipping more often than Hp1-1/2-1
32
patients (70.8% vs. 48%, respectively, p=0.083). Figure 2-2 provides a typical case
example of a 34-year-old female patient included in this study, who developed moderate
and severe CV bilaterally in multiple vessels, DCI, and poor outcome.
Radiographic Vasospasm
For the 17 cerebral arteries evaluated, the mean number of vessels (±SD) with
mild, moderate, and severe CV was 5.1±3.3, 2.8±2.4, 1.5±2.6, respectively. Hp2-2
phenotype was associated with 1.8 (CI=[1.12, 2.98], p=0.014) and 3.7 (CI=[1.33, 11.5],
p=0.008) times the number of vessels with moderate and severe CV, respectively
(Table 2-2). We did not find a significant relationship between Hp2-2 phenotype and the
number of vessels with mild CV (CI=[0.536, 1.38], p=0.531; Table 2-2). The overall
global CV, corresponding to the sum of the individual CV values for each of the 17
arteries (±SD), was 15.2±9.6. Hp2-2 phenotype was significantly associated with
increased global CV, with a 6.5 higher total CV value (CI=[1.39, 11.9], p=0.014; Table 2-
2).
As part of these analyses, we also found that age, aneurysm size, and treatment
type were associated with focal and global CV. It is estimated that the number of
vessels with moderate or severe CV decreases by 2% (CI=[0.80%, 3.8%], p=0.003) and
8% (CI=[4.6%, 11.3%], p<0.0001), respectively, for each additional year of age (Table
2-3). Similarly, older age was also associated with less global CV, with a 0.41 lower
total CV value for each additional year (CI=[-0.535, -0.215], p<0.0001; Table 2-3).
Larger aneurysms tended to be correlated with less severe (CI=[1.10%, 24.6%],
p=0.062; Table 2-3) and global CV (CI=[-1.66, 0.033], p=0.043; Table 2-3). Likewise,
patients who received coiling had more vessels with severe (CI=[2.13, 18.0], p=0.001;
Table 2-3) and global CV (CI=[0.953, 11.3]; p=0.047; Table 2-3). We did not find a
33
significant association between radiographic CV and aSAH severity identified by GCS,
WFNS, Fisher Grade, or Hunt-Hess Grade (Table 2-3).
Delayed Cerebral Ischemia
CV-induced DCI occurred in 22 of 74 (29.7%) of aSAH patients in this study. We
found that 10 of 24 (41.7%) Hp2-2 individuals developed DCI, as compared to 12 of 50
(24.0%) Hp1-1/2-1 patients. Logistic regression controlling for age did not show a
significant association between Hp2-2 phenotype and DCI (OR=2.1, CI=[0.706, 6.00],
p=0.180; Table 2-2). Age was not associated with the incidence of DCI (Table 2-3).
Functional Outcomes
The overall mean (±SD) mRS scores at discharge, 6 weeks, and 1 year after
aSAH were 3.9±1.3, 3.1±1.7, and 2.5±2.0, respectively. Likewise, the overall mean
(±SD) GOSE scores at discharge, 6 weeks, and 1 year post-aSAH were 3.1±1.5,
3.9±2.0, and 5.2±2.5, respectively. A multivariate analysis of Hp phenotype and
functional outcomes (mRS and GOSE) controlling for age, GCS, WFNS, Fisher Grade,
Hunt-Hess Grade, aneurysm size, and treatment type (clipping vs. coiling)
demonstrated a strong trend towards Hp2-2 phenotype and worse functional outcomes
(Table 2-4). Hp2-2 individuals had mRS scores 0.84 and 1.20 higher at 6 weeks (CI=[-
0.090, 1.78], p=0.076) and 1 year (CI=[-0.006, 2.38], p=0.051), respectively. Similarly,
Hp2-2 individuals had GOSE scores 0.74 and 1.45 lower at discharge (CI=[-1.60,
0.121], p=0.091) and 1 year (CI=[-2.92, 0.030], p=0.055), respectively. For each unit
increase on GCS, mRS and GOSE scores at 1 year were 0.25 lower (CI=[-0.538,
0.034], p=0.082; Table 2-5) and 0.39 higher (CI=[0.033, 0.746], p=0.033; Table 2-5),
respectively. We did not find any other significant predictors of outcomes identified by
the mRS or GOSE scores at discharge, 6 weeks, or 1 year (Table 2-5).
34
Mortality
The overall mortality rate was 11 out of 74 (14.9%). We found that 6 of 24
(25.0%) Hp2-2 individuals died, as compared to only 5 of 50 (10.0%) Hp1-1/2-1
patients. Logistic regression controlling for age showed a trend towards increased
mortality for the Hp2-2 individuals (OR=3.3, CI=[0.871, 13.3], p=0.079; Table 2-4). Age
was not associated with mortality (Table 2-5).
Discussion
Cerebral vasospasm (CV) has long been regarded as a key contributor to poor
outcomes after aSAH, mainly due to the resulting DCI and cerebral infarction that may
occur.156 The purpose of this study was to determine whether individuals with the Hp2-2
phenotype had increased risk for CV, DCI, mortality, and poor outcomes following
aSAH. We found that Hp2-2 individuals had significantly more vessels with moderate
and severe focal radiographic CV, and given the design of this study, we are the first to
show that Hp2-2 phenotype is predictive of global CV. We also observed strong trends
towards Hp2-2 phenotype and poor outcomes as identified by both mRS and GOSE
scales at discharge, 6 week, and 1 year post-bleed. Additionally, we found a significant
relationship between Hp2-2 phenotype and increased incidence of mortality.
After aSAH, hemolysis within the subarachnoid space releases massive amounts
of Hb, a molecule that we believe has strongly pro-oxidative and pro-inflammatory
properties when not confined within a red blood cell. We and others have been
speculating that free Hb would be the major instigator of CV through a multifactorial
mechanism involving the generation of reactive intermediates that cause endothelial cell
damage, depletion of the vasodilator nitric oxide, and proliferation of smooth muscle
cells, a combination that ultimately leads to sustained vasoconstriction and DCI.33, 102,
35
152, 157 In a phenotype-dependent manner, the redox potential and clearance of Hb is
directly reduced and improved by Hp, respectively, where the Hp2-2 protein has been
suggested to have less overall protective abilities.35, 36, 97, 112, 115, 116
While there have been previous studies investigating the role of Hp phenotype in
aSAH,145-147 there are a few critical differences between these studies, and as
compared to the one described here, which could possibly explain the varied results: 1)
subjects of different ethnicity, 2) different clinical management approaches, and 3)
variations in study methodologies. This study forms a bridge between the previous
ones, filling in some gaps, and also provides some novel findings aimed to better
understand the role of Hp phenotype in predicting CV, DCI, mortality and poor
outcomes after aSAH.
Indeed, previous studies have varied in the methods used for CV determination,
both in the type of imaging modality used, number of arteries evaluated, and criteria
used for determination of CV. Here, we evaluated a large number of vessels for each
patient, and graded the CV as mild, moderate or severe instead of dichotomizing to
“yes” or “no”. In this way, we obtained a comprehensive view of CV in each of the
patients, including an analysis of both the distribution and severity. This approach also
allowed us to evaluate CV from a global standpoint. In the minority of patients, these
determinations were done using CTA imaging, rather than the gold standard cerebral
angiography. However, previous studies have shown good correlation between CTA
and angiography measurement of arterial diameters.158-160 If there was a discrepancy
between CTA and angiography, CTA tended to overestimate the degree of CV. Here,
the majority of patients with CTA imaging were Hp1-1 or Hp2-1, and thus overestimation
36
in this case would lead to less observed differences; although, Hp2-2 individuals still
had significantly more focal moderate (p=0.017) and severe (p=0.009) CV, and more
global CV (p=0.014). Furthermore, these findings suggest there is less of a clinical need
to do invasive imaging studies in Hp1-1/Hp2-1 patients, indirectly substantiating our
findings of Hp2-2 phenotype and increased risk for CV. For the two previous studies
that looked at Hp phenotype and CV after aSAH: Ohnishi et al. found an association
between Hp2-2 phenotype and increased risk for angiographical CV, while Borosody et
al. found no such link.146, 147 The latter study included 32 patients with aSAH, and
angiography data was not available for all subjects. However, with their more abundant
transcranial Doppler ultrasonography data, they did find an association between the
presence of the Hp2 allele and increased incidence of CV.
Our results showing that Hp2-2 phenotype is an independent risk factor for global
CV is of particular interest, since patients who develop CV and DCI requiring
endovascular treatment may later develop CV and DCI in a different arterial distribution
that was not affected or treated in the first episode.161, 162 As Tekle et al. proposed,
these events suggest that some patients may have a greater overall propensity for
developing CV and DCI after aSAH.162 We have shown that Hp2-2 phenotype is
predictive of global CV, though it is important to note that while a greater percentage of
Hp2-2 patients experienced DCI (41.7% versus 24%), this trend did not reach statistical
significance (p=0.180). This finding is likely due to our small sample size – if the
difference between groups is truly 17.7 percentage points, our study had only 24%
power to detect it. Ohnishi et. al. were essentially the only other group to analyze the
37
incidence DCI in the context of Hp phenotype, where they similarly found the Hp2-2
group trending towards increased risk.146
As part of our multivariate analysis, we also found that other factors were
independent predictors of CV. Younger age was associated with more focal moderate
(p=0.003) and severe CV (p<0.0001), and more global CV (p<0.0001), which correlates
well with previous studies.9-11 Fisher grade is a classification of the amount and
distribution of subarachnoid blood on admission CT scans after aneurysm rupture, and
is a well-known predictor of CV.154, 163, 164 Our current study was underpowered to
evaluate Fisher grade as a risk factor for CV, as only two of the seven Fisher 2 patients
had imaging available to evaluate vasospasm. While we were not able to reliably draw
statistical conclusions, two other factors suggest less CV risk for the Fisher 2 patients in
this cohort: 1) none of the seven Fisher 2 patients had DCI, and 2) the lack of CV
diagnostic imaging studies performed in these patients imply there was no clinical need.
With respect to aneurysm size and treatment modality, previous studies have shown
conflicting evidence regarding the risk for CV.165-170 Here, we found a correlation
between coiling and increased CV, which is in contradiction to larger studies that
demonstrate no difference between groups, or favor coiling for lower risk.165, 166, 169, 170
We are uncertain of why coiled patients in this study tended to have more CV, although
we cannot exclude that those patients less prone to CV were more often surgically
clipped.
Individuals with the Hp2-2 phenotype had increased mortality and poor aSAH
outcomes, as identified by both the GOSE and mRS scales on a continuous basis at
discharge, 6 weeks, and 1 year. No previous studies have correlated Hp phenotype and
38
long-term (1 year) functional outcomes after aSAH, or provided outcome data on a
continuum. Further, the observed differences in outcomes between the Hp phenotype
groups become more exaggerated with increased time post-bleed, suggesting that Hp
phenotype may be a valuable marker for predicting long-term neurologic disability. In a
recent study by Kantor et al., Hp2-2 genotype was associated with poor outcome
identified by dichotomized mRS at 3 months post-bleed; although, no significant
differences were seen with mortality or dichotomized GOS.145 In contrast, Ohnishi et al.
found no association between Hp phenotype and 3 month dichotomized mRS score.146
As they pointed out, the results of their study are not directly applicable to other
populations given the racial differences and marked variation in Hp genotype
frequencies depending on geographic location.
The combination of these previous studies and the current one suggests that
racial background is not an important confounding variable when evaluating Hp
phenotype and the risk for developing CV, though may be important when evaluating its
role in predicting outcomes. The former is likely indicative of the inherent biological roles
of Hb and Hp, where Hb is a primary instigator of CV and Hp is important in mediating
clearance of toxic free Hb from the body, which is Hp phenotype dependent. The latter
point may suggest that other genetic factors may also be responsible for poor outcomes
after aSAH, given that positive correlations between Hp phenotype and outcomes have
been shown in Western populations and no such association was found in an Asian
population. In support of this hypothesis, it was recently suggested in a cross-sectional
study of hospital discharges in the United States, that individuals of Asian/Pacific
Islander decent had worse outcomes after SAH when compared to other racial/ethnic
39
groups.171 Alternatively, different clinical management approaches could also explain
the conflicting results of these studies regarding the predictive potential of Hp
phenotype and poor aSAH outcomes.
A main limitation of this study is the retrospective nature, particularly with regard
to determining DCI. Accurate determination of DCI, even in the prospective setting,
remains difficult due to the complex clinical course of aSAH patients. It can be
challenging to discern CV-induced DCI from other causes of neurologic change
including metabolic derangements, fever, infection, hydrocephalus, seizure, and
respiratory complications. In this study, we performed a thorough chart review to look
for these other possible causes of clinical deterioration, and when possible, we
confirmed true CV-induced DCI by brain imaging demonstrating ischemia/infarction
and/or clinical improvement following CV treatment (balloon angioplasty, intra-arterial
infusion of verapamil). In contrast to DCI, the retrospective nature of this study does not
affect our evaluation of aSAH outcomes or CV since these determinations were
performed prospectively or by review of imaging collected as part of routine care,
respectively.
Other limitations of this study stem around our methodology used for evaluation
of CV. A single expert reviewed imaging and thus the inter-reader variability is unclear;
although, to reduce potential bias, the reader was blinded to Hp phenotype and aSAH
clinical course. In addition, there is no validated methodology for assessing CV or
standard way of reporting these data. Many groups dichotomize this outcome to “yes” or
“no” CV, which likely reduces the variability, but provides less information regarding the
location and severity. Other groups present the data with a grading of CV; although,
40
these methods also vary in terms of the grading system (number of groups) and criteria
for each grouping (% reduction in vessel diameter). Here, we have used two methods to
evaluate CV, an individual comparison of the number of vessels with a particular degree
of CV and an approach we termed global CV. While such techniques have not been
extensively validated, we have used these methods in order to obtain a comprehensive
view of CV. A study using similar methods for determining the degree of CV in each
vessel has shown good interobserver variability.158
41
Figure 2-1. Demonstration of Hp typing methods. Examples are shown for both the
aSAH patients and longitudinal controls of known Hp type that were incorporated in all Hp typing of patients. (A) Hp genotyping of control DNA samples. Lanes 1 and 12 show DNA ladders, lanes 3, 5, 7, 8, and 11 show the Hp1-1 genotype, lanes 2, 6, and 9 show the Hp2-1 genotype, and lanes 4 and 10 show the Hp2-2 genotype. The bands corresponding to Hp1 and Hp2 were subsequently confirmed to be specific by restriction enzyme analysis. (B) Hp phenotyping of serum samples from controls and aSAH patients. Lane 1 shows a molecular weight marker, lanes 3, 5, 6, and 8 show the Hp 1-1 phenotype, lanes 2 and 9 show Hp 2-1 individuals, and lanes 4 and 7 show the Hp2-2 phenotype. The controls in lanes 2-4 correspond to the same controls in lanes 2-4 in (A), demonstrating the matching Hp types between the two methods.
42
Figure 2-2. A prototypical example of a 34 year old female with aSAH. (A) Non-contrast
head CT showing diffuse Fisher 3+4 SAH and layering intraventricular hemorrhage. (B) Initial cerebral angiography of the right ICA demonstrates a right posterior communicating artery aneurysm. (C) Repeat cerebral angiography post-treatment by coiling. (D) Day 6 post-coiling the patient developed progressive confusion and stupor with a left hemiparesis. Cerebral angiography demonstrates severe right M1, moderate right M2, and moderate right A2 spasm. (E) Following intra-arterial infusion of verapamil, there is minimal change in the degree of vasospasm. (F) Angioplasty is performed. A balloon is inflated in the right M1. (G) There is significant improvement of the spasm in the right M1 following angioplasty. (H) Angiography of the left ICA shows moderate left A1 and A2, and moderate left M2 spasm, which was treated with intra-arterial verapamil infusion only.
43
Table 2-1. Demographics, patient characteristics, and subarachnoid hemorrhage severity stratified by Hp phenotype Variable Overall (n=74) Hp1-1 (n=11) Hp2-1 (n=39) Hp2-2 (n=24) Hp1-1/2-1 (n=50) P Value
*
Age, mean±SD 54.7±15.3 54.7±18.0 56.6±14.0 51.6±16.3 56.2±14.7 .254
Gender, n (%)
Female 54 (73.0) 9 (81.8) 28 (71.8) 17 (70.8) 37 (74.0) .785
Male 20 (27.0) 2 (18.2) 11 (28.2) 7 (29.2) 13 (26.0)
Race, n (%)
Black 14 (18.9) 3 (27.3) 9 (23.1) 2 (8.3) 12 (24.0)
.223 White 57 (77.0) 7 (63.6) 29 (74.4) 21 (87.5) 36 (72.0)
Hispanic 3 (4.1) 1 (9.1) 1 (2.6) 1 (4.2) 2 (4.0)
GCS, mean±SD 12.0±3.3 9.4±4.1 12.3±3.1 12.8±2.7 11.7±3.5 .169
WFNS, mean±SD 2.5±1.3 3.5±1.2 2.4±1.2 2.3±1.3 2.6±1.3 .363
Fisher Grade, n (%)
2 7 (9.5) 0 (0.0) 3 (7.7) 4 (16.7) 3 (6.0)
.256 3 24 (32.4) 4 (36.4) 14 (35.9) 6 (25.0) 18 (36.0)
3+4 43 (58.1) 7 (63.6) 22 (56.4) 14 (58.3) 29 (58.0)
Hunt-Hess Grade, mean±SD 2.8±0.9 3.5±0.9 2.7±0.9 2.6±0.9 2.9±1.0 .197
Aneurysm Size (mm), mean±SD 6.3±3.1 5.4±2.6 6.4±2.8 6.7±3.9 6.2±2.7 .903
Treatment, n (%)
Clipping 41 (55.4) 8 (72.7) 16 (41.0) 17 (70.8) 24 (48.0) .083
Coiling 33 (44.6) 3 (27.3) 23 (59.0) 7 (29.2) 26 (52.0) *For comparison between Hp2-2 and Hp1-1/2-1
44
Table 2-2. Multivariate analysis of radiographic vasospasm and delayed cerebral ischemia Outcome Overall (n=74) Hp1-1 (n=11) Hp2-1 (n=39) Hp2-2 (n=24) Hp1-1/2-1 (n=50) 95% CI
* P Value
*
No. Vessels with Mild CV, mean±SD 5.1±3.3 5.6±3.4 5.1±3.6 4.9±3.0 5.3±3.5 .536 - 1.38 .531
No. Vessels with Moderate CV, mean±SD 2.8±2.4 2.1±1.7 1.9±2.6 2.9±2.4 2.4±2.4 1.12 – 2.98 .014
No. Vessels with Severe CV, mean±SD 1.5±2.6 1.4±3.2 1.1±2.2 2.3±3.0 1.2±2.4 1.33 - 11.5 .008
Global CV, mean±SD 15.2±9.6 15.9±10.6 12.8±9.0 19.1±9.3 13.6±9.4 1.39 - 11.9 .014
CV-induced DCI, n (%) 22 (29.7) 3 (27.3) 9 (23.1) 10 (41.7) 12 (24.0) .706 - 6.00†
.180†
*For comparison between Hp2-2 and Hp1-1/2-1 controlling for age, GCS, WFNS, Fisher Grade, Hunt-Hess Grade, aneurysm size, and treatment type
†OR=2.1, for comparison between Hp2-2 and Hp1-1/2-1 controlling for age
45
Table 2-3. Covariate results for multivariate analysis of radiographic vasospasm and delayed cerebral ischemia
CV-induced DCI No. Vessels with Mild CV No. Vessels with Moderate CV No. Vessels with Severe CV Global CV
Outcome CI P Value CI P Value CI P Value CI P Value CI P Value
Hp Phenotype* .706 - 6.00 .180 .536 - 1.38 .531 1.12 - 2.98 .014 1.33 - 11.5 .008 1.39 - 11.9 .014
Age .941 - 1.01 .150 .992 - 1.02 .365 .962 - .992 .003 .887 - .954 <.0001 -.535 - -0.215 <.0001
GCS - - .940 - 1.13 .563 .849 - 1.07 .366 .629 - 1.17 .210 -1.73 - .682 .195
WFNS - - .667 - 1.20 .503 .751 - 1.51 .720 .462 - 2.03 .940 -2.10 - 4.85 .869
Hunt-Hess Grade - - .885 - 1.78 .245 .677 - 1.57 .881 .230 - 1.63 .210 -5.87 - 2.88 .442
Aneurysm Size - - .922 - 1.07 .850 .924 - 1.08 .967 .754 - .989 .062 -1.66 - .033 .043
Treatment Type - - .471 - 1.19 .222 .596 - 1.61 .938 2.13 - 18.0 .001 .953 - 11.3 .047 *For comparison between Hp2-2 and Hp1-1/2-1
46
Table 2-4. Multivariate analysis of functional outcomes and mortality Outcome Overall (n=74) Hp1-1 (n=11) Hp2-1 (n=39) Hp2-2 (n=24) Hp1-1/2-1 (n=50) 95% CI
* P Value
*
GOSE at discharge, mean±SD 3.1±1.5 3.3±1.3 3.4±1.5 2.7±1.5 3.4±1.5 -1.60 - .121 .091
GOSE at 6 week, mean±SD 3.9±2.0 4.5±1.9 3.9±1.8 3.6±2.2 4.1±1.8 -1.86 - .507 .256
GOSE at 12 month, mean±SD 5.2±2.5 5.4±2.7 5.5±2.3 4.6±2.8 5.5±2.3 -2.92 - .030 .055
mRS at discharge, mean±SD 3.9±1.3 4.1±1.2 3.7±1.3 4.0±1.4 3.8±1.3 -.141 - 1.25 .114
mRS at 6 week, mean±SD 3.1±1.7 3.4±1.8 2.8±1.6 3.5±1.8 2.9±1.7 -.090 - 1.78 .076
mRS at 12 month, mean±SD 2.5±2.0 2.5±2.3 2.2±1.7 2.9±2.3 2.2±1.9 -.006 - 2.38 .051
Mortality, n (%) 11 (14.9) 1 (9.1) 4 (10.3) 6 (25.0) 5 (10.0) .871 - 13.3† .079
†
*For comparison between Hp2-2 and Hp1-1/2-1 controlling for age, GCS, WFNS, Fisher Grade, Hunt-Hess Grade, aneurysm size, and treatment type
†OR=3.3, for comparison between Hp2-2 and Hp1-1/2-1 controlling for age
47
Table 2-5. Covariate results for multivariate analysis of functional outcomes and mortality GOSE at discharge GOSE at 6 week GOSE at 12 month mRS at discharge mRS at 6 week mRS at 12 month Mortality
Outcome CI P Value CI P Value CI P Value CI P Value CI P Value CI P Value CI P Value
Hp Phenotype* -1.60 - .121 .091 -1.86 - .507 .256 -2.92 - .030 .055 -.141 - 1.25 .114 -.090 - 1.78 .076 -.006 – 2.38 .051 .871 - 13.3 .079
Age -.039 - .020 .529 -.038 - .040 .969 -.061 - .039 .666 -.020 - .025 .900 -.027 - .034 .824 -.021 - .059 .344 .977 - 1.07 .381
GCS -.130 - .289 .448 -.193 - .373 .526 .033 - .746 .033 -.218 - .142 .604 -.413 - .071 .163 -.538 - .034 .082 - -
WFNS -.628 - .636 .990 -1.03 - .683 .684 -.337 - 1.81 .174 -.435 - .546 .971 -.681 - .662 .978 -1.14 - .651 .583 - -
Hunt-Hess Grade -.632 - .737 .879 -.670 - 1.18 .581 -1.37 - 1.03 .775 -.242 - .916 .228 -.835 - .682 .841 -1.12 - 1.06 .952 - -
Aneurysm Size -.251 - .036 .139 -.260 - .141 .554 -.195 - .303 .664 -.174 - .053 .294 -.189 - .124 .682 -.251 - .146 .599 - -
Treatment Type -.727 - 1.18 .633 -1.05 - 1.55 .702 -2.29 - 1.05 .457 -.231 - 1.28 .186 -.963 - 1.11 .888 -.702 - 1.96 .347 - - *For comparison between Hp2-2 and Hp1-1/Hp2-1
48
CHAPTER 3 INCREASED BRAIN HAPTOGLOBIN LEVELS IMPROVES OUTCOMES FOLLOWING
EXPERIMENTAL INTRACEREBRAL HEMORRHAGE
Introduction
Among the various stroke subtypes, intracerebral hemorrhage (ICH) is one of the
most disabling and has high mortality rates.16, 172 No therapies exist for the treatment of
ICH and the clinical management of patients is limited to supportive measures since
surgical and medical management approaches have failed to improve outcomes.61, 173-
175 Primary injury occurs early after the bleed from hematoma mass effects that cause
mechanical disruption of neurons and glia.58 Occurring later, secondary injury is largely
due to the presence of blood components and their breakdown products that cause
many parallel-operating neurotoxic processes leading to irreversible brain damage and
poor outcomes.58 These processes include oxidative stress, inflammation, blood-brain
barrier breakdown, edema, oligemia, mitochondrial dysfunction, excitotoxicity, spreading
depression, and cell death.12, 58, 60 Consequently, therapeutic paradigms aimed at
detoxifying and improving the clearance of blood products would represent a clinically
relevant treatment strategy for ICH.
Hemolysis within the hematoma releases large quantities of hemoglobin (Hb), the
main component of red blood cells. Extracorpuscular Hb is highly neurotoxic and
represents a key upstream precipitating factor for delayed secondary brain damage and
poor ICH outcomes.95, 176 Haptoglobin (Hp) is the primary defense mechanism in the
body against the toxicity of extracorpuscular Hb. Hp provides immediate, irreversible,
and direct protection from Hb through direct binding, and subsequently facilitates the
clearance and safe degradation of Hb and its toxic degradation products through the
CD163 scavenger receptor. Hp has also been shown to have potent angiogenic,
49
vasculogenic, anti-inflammatory, and wound healing effects, additional properties that
would further improve ICH outcomes.177, 178
Although some studies have shown that Hp is present endogenously in the
brain,179, 180 the levels are far too low to combat the Hb toxicity load seen after ICH. The
present study was designed to evaluate whether specific and local Hp overexpression
improves ICH outcomes, and if so, to investigate the potential in vivo mechanisms of
Hp-mediated neuroprotection.
Methods
Mice
All animal procedures were approved by the University of Florida Institutional
Animal Care and Use Committee and conducted in accordance with the National
Institutes of Health PHS policy on Humane Care and Use of Laboratory Animals.
C57BL/6N mice were bred and maintained in our animal facilities in a temperature-
controlled environment (23±2°C) on a 12h reverse dark/light cycle so behavioral testing
could be performed during the awaken phase. Mice were maintained on ad libitum food
and water, including pre- and post-surgical procedures, and all efforts were made to
minimize the possible suffering of the animals.
rAAV1 Construction and Preparation
Recombinant adeno-associated virus serotype 1 (rAAV1) vectors expressing
eGFP, mouse Hp (accession number BC138872), Hp-eGFP, and Hp-V5 under the
control of the cytomegalovirus enhancer/chicken β-actin promoter, woodchuck post-
transcriptional regulatory element, and bovine growth hormone poly(A) were generated
as described.181 Tags were fused to the C-terminus of the Hp gene. The V5 tag used
has the following amino acid sequence: GKPIPNPLLGLDST. The capsid serotype and
50
timing of injection (see below) was selected such that transgene expression would be
highest in regions surrounding the hematoma and disrupted the least by the ICH, thus
resulting in sustained high local Hp protein levels.181 This approach results in
predominately neuronal transduction, although some astrocytic transduction is also
seen.181
Prior to virus preparation, Hp and control plasmids were independently validated
by nucleotide sequencing performed at our institutional Interdisciplinary Center for
Biotechnology Research. Viruses were prepared by Polyethylenimine Linear (PEI,
Polysciences, Warrington, PA) co-transfection of the Hp or control plasmid and the AAV
helper plasmid pDP1rs (Plasmid Factory, Germany) into HEK293T cells. At 72h after
transfection, cells were harvested and lysed in the presence of 0.5% Sodium
Deoxycholate and 50U/mL Benzonase (Sigma, St. Louis, MO) by repeated rounds of
freeze/thaws at -80˚C and 50˚C. Viruses were isolated using a discontinuous Iodixanol
gradient and samples were buffer exchanged to PBS using an Amicon ultra filter
100,000 MWCO centrifugation device (Millipore, Billerica, MA).
The genomic titer of each virus was determined by quantitative PCR using a
CFX384 detection system (Bio-Rad, Hercules, CA). Briefly, viral DNA samples were
prepared by treating the isolated virus with DNaseI (Life Technologies, Carlsbad, CA),
heat inactivating the enzyme, digesting the protein coat with Proteinase K (Life
Technologies), followed by a second heat inactivation. A standard curve of supercoiled
plasmid diluted from 1x103 to 1x107 genomic equivalents/mL was used for comparison.
Freshly prepared rAAV1s were aliquoted and stored at -80˚C. When needed for
51
injection, viruses were diluted to the same injection titer of 1x1013 genome
equivalents/mL in sterile 1X DPBS (pH 7.2) and used immediately.
rAAV1 Injection
Neonatal rAAV1 injection procedures are adapted from a previous report.182 For
consistency in spatial transduction patterns, injections were performed within 12h of
birth.181 C57BL/6N mouse pups were cryoanesthetized by placing them within an
aluminum foil boat surrounded by ice for 3-4min to reduce body temperature to <10˚C,
at which point the skin color visually changes from pink to purple and the pups are
motionless so injections can easily and accurately be performed.181 Bilateral
intracerebroventricular injection of 2µL of rAAV1 was performed using a 10µL syringe
with a 33 gauge needle and 30˚ bevel (Hamilton Company, Reno, NV) at a 45˚ angle to
a depth of 1.5mm. Following injection, the needle was slowly retracted and the pups
were placed on a heating pad to fully recover from cryoanesthesia and then returned to
their mother and home cage. Mice were aged for 2.5-4.0mo at which point an ICH was
surgically induced or brains were collected from naïve littermates to assess the level
and localization of transgene-specific expression.
Randomization, Exclusion, Blinding
Randomization in this study occurred with rAAV1 injection at birth. First, the total
number of litters required for this study was calculated, assuming a 50:50 male:female
ratio, and a 20% addition was included to account for unforeseen loss (small litter size,
skewed male:female ratio, cannibalism, etc.). Randomization of the particular rAAV1 to
be injected (rAAV1-Hp, rAAV1-Hp-V5, rAAV1-Hp-eGFP, rAAV1-eGFP) or no rAAV1
injection was performed by a single person not otherwise involved in the study and who
had no knowledge of or contact, including no visual inspection, with the litters. Exclusion
52
criteria were defined a priori and included any mouse with apparent abnormal conditions
(skin, eye, abdominal, whisker, signs of infection, etc.), ear puncture during placement
in the stereotactic device, blood reflux at any point during the autologous blood
infusions, and bleeding on needle insertion (which precludes the ability to detect blood
reflex). No mice were excluded from this study. Mice in different experimental groups
were visibly indistinguishable (anatomically and behaviorally), thus blinding was
incorporated throughout the study. The surgeon and investigators performing
neurobehavioral testing had no knowledge of the experimental groups. Additionally, all
anatomical outcomes were quantified in a blinded manner. In all cases, a unique
numbering code was used with a linking list to the experimental treatment group and
individual animal.
Neuronal-Glial Mixed Primary Cultures and rAAV1 Transduction
To confirm the cellular versus secretory localization of our rAAV1-Hp, rAAV1-Hp-
V5, rAAV1-Hp-eGFP, and rAAV1-eGFP vectors, we utilized primary neuronal-glial
cultures prepared as described.183, 184 Briefly, cerebral cortices from P0 mouse brains
were dissected and dissociated in 2mg/mL papain (Worthington, Lakewood, NJ) and
50μg/mL DNAase I (Sigma) in sterile Hank’s Balanced Salt Solution (HBSS, Life
Technologies) at 37°C for 20min. To inactivate the papain, they were washed three
times in sterile HBSS and then switched to Neurobasal-A (Gibco, Waltham, MA) plating
media containing 1% fetal bovine serum (HyClone, Logan, UT), 0.5mM L-glutamine
(Gibco), 0.5mM GlutaMax (Life Technologies), 0.01% antibiotic-antimycotic (Gibco), and
0.02% SM1 supplement (Stemcell, Canada). The tissue mixture was then triturated
three times using a 5mL pipette followed by a Pasteur pipette and strained through a
70μm cell strainer. Following, centrifugation at 200xg for 3min, cells were resuspended
53
in fresh plating media and plated onto poly-D lysine 6-well plates at around 100,000-
200,000 cells/cm2. The following day, the media was replaced with maintenance media
consisting of plating media without fetal bovine serum. Cells were maintained for seven
days with maintenance media prior to transduction with rAAV1s.
After a fresh media change, 5μL of rAAV1-Hp, rAAV1-Hp-V5, rAAV1-Hp-eGFP,
or rAAV1-eGFP was added directly to the media. Wells with no rAAV1 transduction
were included as negative controls. The media was not changed after this point. Three
days later, images of the cultures were obtained using an EVOS FL cell imaging system
(ThermoFisher Scientific, Waltham, MA). The media was then removed, centrifuged to
remove any debris, and the supernatant, later referred to as media, was saved and
stored at -80˚C until later immunoblotting. After the initial plating and up until harvesting,
cells were kept at 37˚C in a humidified 5% CO2 chamber.
Collagenase ICH Model
ICH was induced as described with a few modifications.185, 186 Additional changes
were incorporated in order to avoid needle insertion through the motor cortex and thus
the possibility of confounding results on behavioral analyses, and to improve the
modeling of clinical deep basal ganglia hemorrhages, where concomitant
intraventricular hemorrhage is seen in 40% of nontraumatic ICH cases and is
associated with poor long-term prognosis.187, 188 Changes were accomplished by
modifying the site and angle of craniotomy/needle insertion and the site of injection
within the striatum. Briefly, stereotactic equipment was first manipulated so the injection
could be performed into the left hemisphere at a 40° angle from the vertical plane. Mice
were anesthetized using isoflurane (4% induction, 1.5-2% maintenance), and
immobilized on a stereotactic frame (Stoelting, Wood Dale, IL). A small left-sided
54
incision in the skin overlying the skull was made in a coronal plane mid-way between
the left eye and ear. A craniotomy was performed at an angle matching that of the
stereotactic angle at the following coordinates: 0.0 mm anteroposterior and 3.8 mm left,
relative to bregma. A syringe with a 26 gauge needle (Hamilton Co., Reno, NV) was
inserted 3.6 mm ventral from the skull surface and 0.04 units of collagenase type VII-S
(Sigma, St. Louis, MO) dissolved in 0.40 µL of sterile water was infused at 0.20µL/min
using an automated injector (Stoelting). The needle was left in place for 5min, and then
slowly removed over a 15min period. Rectal temperatures were maintained at
37.0±0.5°C throughout all surgical procedures and mice were allowed to fully recover in
temperature and humidity-controlled chambers post-operatively.
Autologous Whole Blood ICH Model
ICH was induced using the autologous whole blood double infusion model (30µL
total infusion).189 Mice were anesthetized with isoflurane (4% induction, 1.5-2%
maintenance) and immobilized on a stereotactic frame (Stoelting). After making a small
midline sagittal incision in the skin overlying the skull, a craniotomy was performed
0.5mm anterior and 2.4mm right relative to bregma. Autologous blood was collected
onto a sterile surface by needle prick of the tail artery after first cleaning the area with
70% ethanol and warming the tail gently for 2min with a heat lamp. Blood was
immediately drawn into PE-20 tubing (Instech, Plymouth Meeting, PA) connected on
one side to a 50µL syringe with a 26 gauge luer tip needle (Hamilton Company) located
within an automated injector, and the other side to a 26 gauge needle with the bevel
end inserted into the tubing. The blunt end of this needle was inserted 3.9mm ventral
from the skull surface, removed to 3.6mm, and left in place for 7min. Ten microliters of
blood was infused, followed by a 5min waiting period prior to the second infusion of
55
20µL. All injections were performed at 1.0µL/min using an automated injector
(Stoelting). The needle was left in place for 10min after the second infusion prior to slow
removal over a 25min period. Rectal temperatures were maintained at 37.0±0.5°C
throughout all surgical procedures and mice were allowed to fully recover in
temperature and humidity-controlled chambers post-operatively.
For the collagenase cohort, the control and experimental mice are rAAV1-eGFP
(n=9) and rAAV1-Hp (n=9), respectively. For the autologous whole blood cohort, the
control mice (total n=18) include rAAV1-eGFP (n=7) and no rAAV1 injection (n=11), and
the two groups were combined for statistical comparisons since no differences were
observed. The experimental groups (total n=21) consisting of rAAV1-Hp (n=10), rAAV1-
Hp-V5 (n=6), and rAAV1-Hp-eGFP (n=5) were similarly combined and herein referred to
as Hp mice.
Functional Outcomes
Functional outcomes were assessed daily post-ICH by neurological deficit
scoring (NDS), accelerating rotarod performance, and open field locomotor activity.
Testing was performed during the dark cycle (awaken phase) by investigators blinded to
genotype. Each test was performed at the same time of the day and mice were allowed
1h of rest in between tests. NDS: two blinded investigators independently assessed
mice for focal neurological deficits daily post-ICH by neurological deficit scoring (NDS)
as we have described.190, 191 Briefly, a score of 0 (no deficits) to 4 (severe deficits) was
assigned for six individual parameters including body symmetry, gait, circling behavior,
climbing, front limb symmetry, and compulsory circling. NDS is reported as the average
of the sum of the individual scores for the two investigators. Accelerating rotarod
performance: mice were evaluated for motor deficits and coordination, endurance, and
56
balance using an accelerating rotarod Rotamex-5 machine and software (Columbus,
OH). Rotational speed started at 4rpm and ended at 30rpm, and the latency to fall was
automatically collected by the software. On the three consecutive days prior to surgery,
mice were trained twice per day (morning and late afternoon) with three cycles per
training period. Average performance on the sixth training period served as baseline
functioning. Post-ICH testing consisted of one testing period per day with three cycles
and data is reported as the average latency to fall. Open field locomotor activity:
ambulatory distance and stereotypic time was measured using an automated open field
activity monitor and video tracking interface system (MED associates, St. Albans, VT).
In this context, stereotypic behavior represents fine motor ability defined as any
movement confined within a 4.8x4.8cm space relative to the mouse center point.
Baseline locomotor activity was assessed the day prior to surgery, before rotarod
training and pre-testing. For baseline and post-ICH testing, mice were placed
individually in 4 transparent acrylic cages and their locomotor activity was recorded over
a 30min period. The first 5min of recorded data was omitted to exclude for initial anxiety
responses.
Tissue and Biospecimen Harvesting
For those mice that underwent ICH, all collection procedures occurred at 72h
after surgery. Mice were transcardially perfused with PBS followed by 4%
paraformaldehyde. Brains were collected and kept in 4% paraformaldehyde for 24h
prior to cryopreservation in a 30% sucrose/PBS solution for subsequent histology.
Naïve littermates that received the same rAAV1 injections (and at the same time) as
those mice that underwent ICH were used for confirmation of transgene-specific
expression. Brain tissue was harvested after deep anesthetization with isoflurane and
57
PBS perfusion. The cerebellum and olfactory bulbs were removed and brains were snap
frozen in pre-cooled 2-methylbutane and stored at -80˚C for subsequent
homogenization.
Western Blotting
Western blotting of in vivo and in vitro samples was performed to characterize
and localize rAAV1-mediated Hp overexpression. Brain tissue was homogenized in
radioimmunoprecipitation assay buffer supplemented with Halt protease inhibitor
cocktail and protein content was subsequently estimated using the bicinchoninic assay
(ThermoFisher Scientific). Sample preparation for the in vitro experiments is described
above and a constant volume of media was loaded on the gel. Sodium dodecyl
polyacrylamide gel electrophoresis and Western blotting were performed according to
standard procedures under non-reducing and reducing conditions. Table 3-1 provides
details on the primary and secondary antibodies used. Chemiluminescence was
visualized using the SuperSignal West Pico substrate (ThermoFisher Scientific) and a
FluorChem E detection system (ProteinSimple, San Jose, CA). Near-infrared
fluorescence detection was performed using an Odyssey system (Li-Cor, Lincoln, NE).
Histology and Quantification
Histological staining and quantification procedures were performed by blinded
investigators as we have described.190, 191 Ten sets of sixteen sections equally
distributed throughout the entire hematoma and anteroposterior brain regions were
processed on a CM 1850 cryostat (Leica Biosystems, Buffalo Grove, IL) at 30µm and
stored at -80˚C for later histological procedures. In this way, for each animal, multiple
staining procedures can be performed and the staining pattern throughout the whole
brain can be analyzed. Cresyl violet staining was used to assess lesion volume,
58
perihematomal tissue injury, and hematoma volume. Perls’ iron staining was performed
to evaluate iron content. The antibodies used for immunohistochemistry to evaluate the
localization of rAAV1 expression, HO1 expression, lipid peroxidation, astrogliosis, and
microgliosis are provided in Table 3-1. All slides were scanned using an Aperio
ScanScope CS and analyzed with ImageScope software (Leica Biosystems).
Quantification was performed in a blinded manner and to reduce any potential
bias and interindividual variability, for a given histological stain, all slides simultaneously
underwent the staining protocol and a single investigator performed the quantification.
For quantification procedures in which total brain pathology was analyzed (lesion
volume, perihematomal tissue injury, hematoma volume, ferric iron content, and HO1
expression), all 16 sections were quantified for each animal. 4-HNE was evaluated on
the two sections for each animal representing maximal lesion area. To assess
astrogliosis and microgliosis, four sections for each animal representing maximal lesion
area were analyzed. Lesion volume: injured brain regions were outlined, areas
abstracted from the ImageScope software, and a volume was calculated using these
areas, known distance between each section, and section thickness. Injured brain areas
are defined as the hematoma and perihematomal tissue injury/cell death as shown in
Figure 3-1. For all other quantification procedures (hematoma volume, perihematomal
tissue injury, iron content and immunohistochemical stains), an ImageScope Positive
Pixel Count algorithm was used for quantification after the appropriate brain regions
were outlined (see below). Each algorithm was tuned for each of the individual stains
such that the appropriate signal and strength of signal were evaluated.190 Thresholds
were set intermediate between the signals seen in the two experimental groups on a
59
representative slide in each group such that the algorithm allowed for optimal detection
in either direction (i.e. more intense versus less intense). After running an algorithm, all
slides were checked for specificity and accuracy and to ensure minimal interference
from artifact before the signal data was abstracted from the ImageScope software.
Hematoma volume: the injured brain regions described above were outlined. A volume
was calculated in an identical manner to that described for lesion volume quantification.
Perihematomal tissue injury: Using the calculated lesion and hematoma volumes, the
amount of perihematomal tissue injury was calculated by subtracting the hematoma
volume from total lesion volume. HO1, iron, and 4-HNE: the injured brain regions and
surrounding areas were outlined. Microgliosis and astrogliosis: cortical gliosis was
analyzed by placing identically sized boxes of 1000x1000 pixels in the ipsilateral and
contralateral motor cortex. Striatal gliosis was analyzed by outlining of the ipsilateral and
contralateral striatum, excluding the lesion area, and these data are presented as the
signal per area quantified. Since gliosis differences were noticed in the contralateral
hemisphere between treatment groups, ipsilateral data was not normalized for
contralateral signal; instead, the data are presented separately for comparison. After all
analyses, the appropriate algorithm was run and signal data was abstracted from the
ImageScope software.
Statistics
Statistical analyses were performed using SAS-JMP (Cary, NC) by or in
consultation with a biostatistician. Mortality and NDS were analyzed using a χ2 test and
nonparametric Mann-Whitney U test, respectively. The remaining data sets were
checked for differences in variances between groups and normality and the appropriate
statistical test was used, either a Mann-Whitney U test or an unpaired two-tailed
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Student’s t test with or without Welch’s correction. Data are expressed as mean±SEM
with p<0.05 considered statistically significant.
Results
Characterization of rAAV1 Expression
The secretory nature of the rAAV1-Hp-(tag) protein products was validated by in
vitro transduction of mixed neuronal-glial cell cultures and Western blotting (Figure 3-2).
To demonstrate in vivo transgene spatial expression, immunohistochemistry for GFP
was performed using sections from rAAV1-eGFP mice. rAAV1 expression is primarily
neuronal and observed in cortical, striatal, thalamic, and hippocampal brain regions, as
well as in the corpus callosum, internal capsule, several other white matter tracts, and
periventricular areas (Figure 3-3). No GFP staining is seen in the negative control mice
that did not receive a rAAV1 injection (Figure 3-3). Western blotting was performed to
characterize the level of Hp protein in control and Hp mice. High levels of Hp are seen
in brain homogenates from naïve rAAV1-Hp-V5 mice (Figure 3-3).
ICH-Induced Brain Injury and Functional Outcomes
Following collagenase-induced ICH, Hp mice have significantly smaller lesion
volumes that are associated with less hematoma volume and perihematomal tissue
injury, and reduced ipsilateral hemispheric enlargement. Hp mice display 35.7±6.3%
smaller lesion volumes (Control: 12.1±1.3mm3, Hp: 7.7±0.8 mm3, p=0.0392; Figure 3-4),
35.0±7.8% smaller hematoma volumes (Control: 2.5±0.3mm3, Hp: 1.7±0.2mm3,
p=0.0175; Figure 3-4), 36.0±6.2% less perihematomal tissue injury (Control:
9.5±1.2mm3, Hp: 6.1±0.6mm3, p=0.0124; Figure 3-4), and 57.2±8.4% less ipsilateral
hemispheric enlargement (Control: 11.5±3.9%, Hp: 4.9±1.0%, p=0.0090; Figure 3-4).
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In accordance with reduced collagenase-induced ICH anatomical damage, Hp
mice demonstrate significantly reduced neurological deficits on NDS at 72h (Control:
10.5±0.8, Hp: 7.6±1.2, p=0.0487; Figure 3-5). Regression analyses show that Hp mice
have improved neurological recovery on NDS (p=0.0315), trends toward improved
recovery of stereotypic time (p=0.1093) and total resting time (p=0.1251), but no
significant difference in recovery of ambulatory distance (p=0.6122) or latency to fall on
an accelerating rotarod (p=0.5298) was seen (Figure 3-5).
Following autologous whole blood-induced ICH, Hp mice have smaller lesion
volumes that are associated with trends toward less hematoma volume and significantly
less perihematomal tissue injury. Hp mice display 63.0±11.0% smaller lesions (Control:
9.7±2.0mm3, Hp: 3.6±1.1mm3, p=0.0224; Figure 3-6), 63.9±8.7% less residual blood
(Control: 3.1±0.8mm3, Hp: 1.1±0.2mm3, p=0.1423; Figure 3-6), and 60.4±15.0% less
perihematomal tissue injury (Control: 6.6±1.3mm3, Hp: 2.5±0.8mm3, p=0.0138; Figure 3-
6). Consistent with reduced brain damage, Hp mice exhibit reduced neurological deficits
at all time points post-ICH (24h: p=0.0308, 48h: p=0.0685, 72h: p=0.0318; Figure 3-6).
Hemoglobin
To understand whether high local levels of Hp aid in the clearance/degradation of
Hb after ICH, immunohistochemical staining for Hb was performed. Hp mice tend to
have 35.0±8.6% less Hb (Control: 3.4±1.0x107A.U., Hp: 1.3±0.3x107A.U., p=0.0726;
Figure 3-7). After individually normalizing for lesion volume, the trend was maintained
(Control: 2.5±0.1x106A.U., Hp: 1.7±2.4x105A.U., p=0.2986; Figure 3-7).
Heme Oxygenase 1
To further understand the contribution of high local Hp levels to Hb
clearance/degradation after ICH, immunohistochemistry for HO1 was performed. HO1
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staining was observed in glial cells and the vasculature. Hp mice display significantly
reduced HO1 expression (Control: 7.0±2.4x106A.U., Hp: 4.2±1.6x105A.U., p=0.0083;
Figure 3-8). After individually normalizing for lesion volume, this difference was
maintained (Control: 7.6±1.9x105A.U., Hp: 1.5±0.3x105A.U., p=0.0092). A greater
proportion of control mice display HO1 expression in regions distant from the hematoma
(Control: 63.6%, Hp: 0.0%, p=0.0337).
Perls’ Iron
Iron accumulation (blue) was constrained to perihematomal regions for all mice in
the study. Following collagenase-induced ICH, Hp mice tend to have 30.8±14.9% more
iron (Control: 2.7±0.2x108A.U., Hp: 3.5±0.4x108A.U., p=0.1392; Figure 3-9). After
individually normalizing for lesion volume, this trend became significant, with Hp mice
showing 121.4±44.5% more iron (Control: 2.3±0.2x107A.U., Hp: 5.1±0.1x107A.U.,
p=0.0489; Figure 3-9).
Following autologous whole blood-induced ICH, no difference in iron content is
seen (Control: 2.0±0.2x106A.U., Hp: 2.0±0.5x106 A.U., p = 0.8680). However, after
individually normalizing for lesion volume, Hp mice demonstrate significantly more iron
(Control: 3.6±0.8x105A.U., Hp: 10.1±3.4x105A.U., p=0.0430, Figure 3-10).
Lipid Peroxidation
Additional histology was used to start identifying local mechanisms of Hp-
mediated neuroprotection after ICH. Immunohistochemical staining for 4-HNE was
conducted to assess lipid peroxidation. Staining is observed primarily in the
perihematomal region and quantification reveals that Hp mice have 64.2±5.7% less lipid
peroxidation (Control: 14.1±4.7x105A.U, Hp: 5.0±0.8x105A.U. p=0.0879; Figure 3-11).
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After individually normalizing for lesion volume, no significant difference is seen
between groups (Control: 2.2±0.9x105A.U., Hp: 1.4±0.3x105A.U., p=0.6753).
BBB Integrity
Immunohistochemistry for IgG was performed to assess BBB integrity.
Quantification demonstrates that Hp mice have 46.8±15.7% less BBB dysfunction
(Control: 4.2±0.4x107A.U., Hp: 2.2±0.7x107A.U., p=0.0686; Figure 3-12). After
individually normalizing for lesion volume, this difference was maintained (Control:
3.6±0.3x106A.U, Hp: 2.1±0.5x106, p=0.0569; Figure 3-12).
Angiogenesis/Neovascularization
Immunohistochemistry for PECAM-1 and VEGF was performed to assess
angiogenesis/neovascularization. Hp mice show 82.6±3.7% less neovascularization
(Control: 3.3±1.4x106A.U., Hp: 5.7±1.3x105A.U., p=0.0168; Figure 3-13). After
individually normalizing for lesion volume, this difference was maintained (Control:
2.9±1.5x105A.U., Hp: 7.5±1.6x104A.U., p=0.0581; Figure 3-13). Hp mice have
60.0±12.3% less VEGF immunoreactivity (Control: 13.8±4.7x106A.U., Hp:
5.5±1.7x106A.U., p=0.0455). After individually normalizing for lesion volume, no
significant difference is seen (Control: 10.3±3.0x105A.U., Hp: 6.8±1.9x105 A.U.,
p=0.3768).
Astrogliosis
GFAP immunohistochemical staining was performed to evaluate cortical and
striatal astrogliosis (Figure 3-15). No significant differences in ipsilateral or contralateral
cortical or striatal astrogliosis is seen between control and Hp mice, although Hp mice
tended to have overall reduced astrogliosis. Ipsilateral cortical astrogliosis was
significantly increased compared to the contralateral in both Hp (p=0.0103) and control
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(p=0.0151) mice. Ipsilateral striatal astrogliosis was also significantly increased
compared to the contralateral in both Hp (p=0.0152) and control (p=0.0003) mice.
Contralateral striatal astrogliosis was significantly increased compared to the cortex in
Hp mice (p=0.0229), but this difference was not observed in control mice (p=0.3727).
No difference in ipsilateral astrogliosis was observed between the striatum and cortex
for both control (p=0.3599) and Hp (p=0.5962) mice.
Microgliosis
Iba1 immunohistochemistry was performed to assess cortical and striatal
microgliosis. Overall, Hp mice have more microglial activation and morphological
changes compared to controls in both the ipsilateral and contralateral hemispheres
(Figures 3-16 and 3-17).
Following collagenase-induced ICH, Hp mice have 130.5±20.0% more ipsilateral
cortical (Control: 1.8±0.6x10-2A.U., Hp: 4.1±0.4x10-2A.U, p=0.0084; Figure 3-16) and
138.2±31.4% more ipsilateral striatal (Control: 1.8±0.6x10-2A.U, Hp: 4.4±0.6x10-2A.U,
p=0.0293; Figure 3-16) microgliosis. In the contralateral hemisphere, Hp mice also have
291.2±148.0% more cortical (Control: 3.7±1.8x10-3A.U., Hp: 1.5±0.6x10-2A.U, p=0.0911;
Figure 3-16) and 345.7±78.04% more striatal (Control: 4.5±1.7x10-3A.U, Hp:
2.0±0.4x10-2A.U, p=0.0082; Figure 3-16) microgliosis. Ipsilateral cortical microgliosis is
significantly greater compared to the contralateral for Hp (p=0.0151) and control
(p=0.0421) mice. Ipsilateral striatal microgliosis is also significantly increased compared
to the contralateral for Hp (p=0.0091) and control (0.0779) mice.
Following autologous whole blood-induced ICH, Hp mice display 229.8±19.9%
more ipsilateral cortical (Control: 1.2±0.2x10-2A.U., Hp: 3.8±2.3x10-2A.U., p=0.0431;
Figure 3-17) and 47.3±12.8% less contralateral cortical (Control: 0.75±0.1x10-2A.U., Hp:
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0.4±0.1x10-2A.U., p=0.0872; Figure 3-17) microgliosis. No significant ipsilateral or
contralateral difference is seen for striatal microgliosis (Figure 3-17). Ipsilateral cortical
microgliosis was significantly increased compared to the contralateral in Hp mice
(p=0.0012), and tends to be greater in control mice (p=0.1113). Control mice have
significantly increased ipsilateral striatal microgliosis compared to the contralateral
(p=0.0068). Additionally, Hp mice tend to have greater striatal microgliosis in the
ipsilateral hemisphere compared to the cortex (p=0.0775). Ipsilateral striatal microgliosis
is significantly increased in control mice compared to cortex (p=0.0021). This difference
was not seen in Hp mice (p=0.7791). Greater striatal microgliosis was seen in both
control (p=0.0749) and Hp (p=0.0328) mice in the contralateral hemisphere.
Discussion
The present study reveals that high local Hp levels are neuroprotective after ICH
in two separate complementary mouse models. We show that rAAV1-mediated Hp
overexpression locally within the brain results in significantly smaller lesion volumes
associated with reduced hematoma volumes, perihematomal tissue injury, and edema.
This attenuated anatomical damage is accompanied by improvements in neurologic
function. Hp-overexpressing mice have significantly less residual Hb, HO1 expression,
lipid peroxidation, BBB dysfunction, and angiogenesis/neovascularization, increased
levels of iron and microgliosis, and no change in astrogliosis.
The endogenous production of Hp in the brain is unclear. Some have shown that
Hp is not synthesized in the normal CNS,192 whereas others report it is present at very
low levels in the parenchyma or in the CSF at concentrations suggestive of leakage
across the BBB.107, 108, 124 It is difficult to assess the level of local Hp synthesis following
hemorrhagic stroke since Hp is present in abundant quantities in the blood and enters
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the brain as part of the bleed. One group has suggested that Hp is increased in the
hemorrhage-affected striatum, peaking around 72h post-ICH, although it is unclear
whether it represents peripheral or central-derived Hp.107, 108 Although, the same
investigators have suggested that oligodendrocytes are able to produce Hp.107, 108 Even
with the Hp that enters as part of the bleed and some degree of local Hp upregulation
following brain injury, the Hp levels would still be inadequate to handle the massive Hb
burden after ICH. Thus, the low Hb-binding capacity of the CNS leaves the brain
vulnerable to even small amounts of extravascular hemolysis. Furthermore, it is
expected that the severe hemolysis seen after ICH would rapidly deplete Hp since it is
not recycled following endocytosis of the Hp-Hb complex. For instance, in the periphery,
severe hemolysis results in undetectable Hp or hypohaptoglobinemia, and it takes
approximately a week for the Hp levels to return to the baseline. Indeed, serum Hp
levels are clinically used as diagnostics for peripheral hemolytic disorders. With
inadequate Hp levels, free Hb and its degradation products are free to impose their pro-
oxidative and pro-inflammatory properties, which ultimately leads to tissue injury and
neuronal and glial cell death.
Here, we use rAAV1 vectors to specifically and constitutively increase brain Hp
levels, which results in very high in vivo expression as assessed by Western blotting of
brain homogenates from non-ICH mice. In vivo localization by immunohistochemistry
showed that transgene expression is predominately neuronal-mediated and highest
surrounding the brain regions normally affected by an ICH. With in vitro transduction of
mixed neuronal-glial cultures and Western blotting, we confirmed the secretory nature of
our rAAV1-Hp-(tag) protein products. The in vivo secretory nature was confirmed by
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Western blotting of CSF from ICH mice (data not shown). In those experiments, anti-tag
antibodies were used to specifically assess transgene-derived CSF Hp and not
endogenous CSF Hp or Hp present from potential blood contamination, although the
CSF was visibly clear. Thus, the highly overexpressed and extracellular Hp is perfectly
positioned to bind and neutralize extracorpuscular Hb released by hemolysis occurring
within the hematoma, thereby potentially protecting surrounding viable brain tissue from
secondary Hb-mediated damage. Indeed, Hp mice have significantly smaller lesion
volumes with less perihematomal cell death and reduced neurological deficits,
collectively demonstrating improved recovery after ICH.
Additional studies are needed to clarify the relative peripheral versus central
clearance of the Hp-Hb complexes. The balance between these two clearance
pathways is of importance since too much central internalization of Hp-Hb complexes
could in theory lead to uncontrollably high intracellular heme and iron levels, increased
oxidative stress, and persistent inflammation, if other protective heme degradation and
iron regulatory pathways are not concomitantly and locally induced to a safe level.
Although, the Hp-CD163 scavenging pathway is part of a complex overall Hb
degradation system. Hp-Hb complex internalization by CD163 coordinately increases
CD163 expression and several other molecules involved in Hb degradation, such as
HO1 for heme catabolism and ferritin for iron storage, as well as anti-inflammatory IL-6
and IL-10 secretion.97, 126, 129 However, it is still possible that the CD163 receptor
becomes saturated, and if so, Hp-Hb complexes would accumulate and a steep
peripheral-central concentration gradient would be established that would facilitate
complex filtering to the periphery. As such, if peripheral clearance mechanisms were
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also operating, it would allow some of the Hb clearance to be taken care of in the large-
capacity systemic system where scavenging pathways are not overloaded. Thus, these
additional studies should evaluate the relative peripheral versus central clearance of
Hp-Hb complexes from a temporal standpoint in combination with the level of local
CD163 expression, a difficult task given the current lack of adequate anti-mouse CD163
antibodies.
In the present study, Hp mice have reduced Hb and HO1, but increased iron.
Less Hb is indicative of overall improved Hb clearance that could be either peripheral or
central mediated. Reduced HO1 expression suggests a higher degree of peripheral
clearance, whereas, increased local iron suggests the opposite, more central clearance.
The aforementioned work should clarify these apparent discrepancies. Here, iron was
identified by Perls’ staining, which is largely specific for ferric iron (Fe3+), and thus
hemosiderin.193, 194 Briefly, formation of the coarse orange pigment, hemosiderin, takes
several days and begins with Hb degradation and iron storage as Fe3+-ferritin.
Hemosiderin is a water insoluble degradation product of ferritin and consists of more
than 25% Fe3+. Iron in this form cannot participate in metabolic processes. Indeed,
hemosiderin is an inert molecule that is invariably present in those that survive and a
marker of previous hemorrhage in autopsied brains.93 Thus, in the case of ICH, the
incorporation of iron into holoferritin likely provides neuroprotection by the formation of
hemosiderin, which prevents iron-catalyzed lipid peroxidation.93 In fact, in the current
study, the Hp mice that have increased iron do have less lipid peroxidation. Therefore,
this increased iron should not necessarily be interpreted as a poor outcome.
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Angiogenesis is a key physiologic mechanism that facilitates tissue repair
following acute injury, but must be tightly regulated to prevent excessive activity and
deleterious consequences. After ICH, regulation of angiogenesis within the appropriate
range in injured brain regions would allow for delivery of glucose and oxygen to support
the energy-requiring reparative processes and facilitate the necessary entry of
peripheral cells involved. Conversely, excessive angiogenesis could exacerbate ICH
outcomes by causing a leaky BBB and allowing for too much peripheral cell infiltration,
which could collectively augment neuroinflammation. Here, Hp mice have improved
BBB integrity and reduced angiogenesis/neovascularization, consistent with an overall
improved recovery response after ICH.
Glial cell activation and neuroinflammation are intimately connected and
important dynamic processes following ICH that can be both neurotoxic and
neuroprotective.58, 60 At this early time point, the increased microglial activation and
reduced residual blood in Hp mice may imply augmented phagocytic responses and
improved hematoma resolution. Activated microglia are also reported to be less
susceptible to heme toxicity.195 It is also possible that these findings are in part
independent of the ICH and a result of high local Hp levels throughout late adolescent
and adult life (it is unlikely to affect early developmental processes since rAAV1 vector
expression is delayed post-P0 injection), something that is suggested by the
contralateral differences noted here; however, brain-wide changes have been observed
in various models of acute focal brain injury, including ICH.196-198 In either case, these
glial cell activation and morphological changes are accompanied by significantly less
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ICH-induced brain injury in Hp mice and thus likely represent a positive modulation of
neuroinflammatory processes.
Collectively, these results suggest a neuroprotective role for Hp after ICH and
establish the possibility of administering exogenous clinical grade Hp locally as a
therapeutic strategy for the treatment of ICH.
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Figure 3-1. Demonstration of lesion volume quantification methods. Representative
cresyl violet stained brain image (left) is shown where the box denotes the location of the high magnification image (right) that shows the lesion volume quantification methods used throughout this work. The black line denotes the boundary between healthy and damaged tissue. Damaged tissue is identified on the basis of the characteristic intensely/darkly stained pyknotic nuclei and overall surrounding hypointense staining (that changes from purple to bluish/grey) and decreased cellularity.199-203 In this quantification, the equivalent healthy contralateral areas are used for comparison.
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Figure 3-2. In vitro characterization of rAAV1 expression. (A) Brightfield, fluorescent (GFP/mCherry), and overlay images of mixed primary neuronal-glial cell cultures 72h after no rAAV1 transduction or transduction with rAAV1-mCherry, rAAV1-Hp-eGFP, rAAV1-Hp-V5, or rAAV1-Hp are shown. As expected, no fluorescence is seen for the no rAAV1 transduction negative control and the rAAV1-Hp-V5 and rAAV1-Hp transduced cultures. Transduction with rAAV1-mCherry resulted in intense intracellular fluorescence throughout neurons. Whereas, transduction with rAAV1-Hp-eGFP had a significantly different pattern, with fluorescence restricted to perinuclear areas of neurons, consistent with a secretory protein cellular localization (white arrows). Furthermore, the comparatively low fluorescent intensity is consistent with the expected low intracellular levels of transgene-derived Hp protein. (B) To verify the secretory nature of the rAAV1-Hp-(tag) vector protein products, Western blotting of mixed-neuronal glial culture media (6μL) was performed 72h after transduction. Reducing (R) and non-reducing (NR) conditions were used to confirm the presence of disulfide bonds in the expressed protein. Hp is identified as green. The media from rAAV1-Hp, rAAV1-Hp-V5, and rAAV1-Hp-eGFP transduction shows a substantial amount of Hp, Hp-V5, and Hp-eGFP protein, which directly confirms the secretory nature of these rAAV1 vector protein products. This finding is not as a result of potential cross-reactivity with Hp present in fetal bovine serum since cultures were maintained in serum-free maintenance media. Further confirmation of specificity is provided by the lack of immunoreactivity in the rAAV1-eGFP and no rAAV1 transduction lanes. The observed molecular weight for non-reduced and reduced Hp was 110kDa and 55kDa, respectively, confirming in vitro the appropriate Hp dimer formation of the secreted Hp protein.
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Figure 3-3. In vivo characterization of rAAV1 expression. (A) Representative images showing the reproducible spatial localization of GFP immunoreactivity in two separate rAAV1-eGFP-expressing mice. Numbered boxes on whole brain images denote the location of high magnification images in the quadrants, where the left and right quadrants correspond to the anterior and posterior coronal sections, respectively. (B) Representative images of a no rAAV1 injection negative control showing no GFP immunoreactivity. (C) Western blotting of brain homogenates (15µg) from naïve rAAV1-eGFP and rAAV1-Hp-V5 mice was performed to evaluate Hp protein levels. An anti-V5 antibody was used to specifically detect the transgene-derived Hp protein and not any potential endogenous Hp (and to avoid the non-specificity of anti-mouse Hp antibodies). Reducing and non-reducing conditions were used to confirm in vivo the presence of disulfide bonds in the expressed protein and to ensure minimal tag interference of post-translational modification. Media from mixed neuronal-glial cultures transduced with rAAV1-Hp-V5 was used as a positive control. The observed molecular weight for non-reduced and reduced Hp-V5 was 110kDa and 55kDa, respectively, confirming in vivo the appropriate Hp dimer formation.
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Figure 3-4. High local levels of haptoglobin reduces collagenase ICH-induced brain
injury. At 72h, brains were harvested and Cresyl violet staining of coronal sections was performed to evaluate brain injury. (A) Representative images of control (top panels) and Hp (bottom panels) mice are provided. Within experimental groups, images are from the same animal, where left-to-right corresponds to anterior-to-posterior. (B) Quantification of lesion volume reveals that Hp mice have significantly less ICH-induced brain injury. (C) Quantification of hematoma volume shows that Hp mice have less residual blood within injured brain areas. (D) Quantification of tissue injury shows that Hp mice have less perihematomal cell death. (E) Hp mice have less ICH-induced ipsilateral hemispheric enlargement, a measure of edema. All comparisons include n=6-9 mice per group, *p<0.05, **p<0.001.
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Figure 3-5. Haptoglobin therapy improves functional outcomes following collagenase-
induced ICH. (A) Hp mice display less neurological deficits at 72h post-hemorrhage. (B) Hp mice demonstrate a significantly faster rate of neurologic recovery compared to control mice. (C) No significant difference in the rate of recovery in latency to fall on an accelerating rotarod is seen between groups. (D) A trend towards improved rate of recovery of stereotypic behavior is seen. (E) Hp mice demonstrate a tendency toward improved rate of recovery of less resting time. (F) No significant difference in the rate of recovery of ambulatory ability is seen between groups. All comparisons include n=7-9 mice per group, *p<0.05.
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Figure 3-6. High local levels of haptoglobin reduces ICH-induced brain injury in the
autologous whole blood model. At 72h post-ICH, brains were harvested and Cresyl violet staining of coronal sections was performed to evaluate brain injury. (A) Representative whole brain images are shown for control (left) and Hp (right) mice. (B) Quantification reveals that Hp mice have significantly less ICH-induced brain injury and perihematomal tissue injury. Hp mice also tend to have less residual blood within injured brain areas and display significantly less neurological deficits following ICH. All comparisons include n=7-14 mice per group, *p<0.05.
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Figure 3-7. High local levels of haptoglobin reduces the amount of hemoglobin after
ICH. Representative low magnification images of coronal brain sections showing Hb immunoreactivity in injured brain regions of control (top) and Hp (bottom) mice are provided. Square selections denote the location of magnified regions. Hp mice have less Hb after ICH (left axis), trends which are stable following individual normalization for lesion volume (right axis). All comparisons include n=5-7 mice per group.
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Figure 3-8. Haptoglobin therapy decreases heme oxygenase 1 expression after ICH.
Representative low magnification center images of coronal brain sections showing HO1 immunoreactivity in injured brain regions of control (top) and Hp (bottom) mice are provided. The leftmost and rightmost square selections denote the location of the left and right high magnification images, respectively. HO1 expression is clearly evident in perihematomal microglia/macrophages as well as glia more distant from the lesion and endothelial cells. Hp mice have significantly reduced HO-1 expression. All comparisons include n=5-11 mice per group, **p<0.01.
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Figure 3-9. High local levels of haptoglobin increases Perls’ iron content after
collagenase-induced ICH. Perls’ staining was performed at 72h post-ICH to evaluate brain iron content. Representative high magnification images of coronal brain sections showing iron accumulation (blue) in perihematomal regions from control (top) and Hp (bottom) mice. Square selections denote location of magnified regions. Quantification reveals that Hp mice tend to have more iron (left axis). After normalizing for lesion volume, Hp mice have significantly more iron (right axis). All comparisons include n=6-9 mice per group, *p<0.05.
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Figure 3-10. High local levels of haptoglobin increases Perls’ iron content after ICH in
the autologous whole blood model. Perls’ staining was performed at 72h post-ICH to evaluate brain iron content. Representative low magnification images (top) showing iron accumulation (blue) in perihematomal regions of control (left) and Hp (right) mice. Square selections in the inserts denote the location of magnified regions. Quantification reveals that Hp mice have significantly increased iron, which appears to be more densely accumulated. All comparisons include n=6-12 mice per group, *p<0.05.
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Figure 3-11. Haptoglobin therapy reduces lipid peroxidation after ICH.
Immunohistochemistry for 4-HNE was performed at 72h post-ICH to evaluate lipid peroxidation. Representative low magnification images showing 4-HNE staining in the perihematomal regions. Square selections in the inserts denote the location of magnified regions. Quantification reveals that Hp mice strongly tend to have decreased lipid peroxidation. All comparisons include n=5-10 mice per group.
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Figure 3-12. High local levels of haptoglobin improves blood-brain barrier integrity after
ICH. Immunohistochemistry for IgG was performed at 72h post-ICH to evaluate blood-brain barrier integrity. Representative low magnification images of coronal brain sections showing IgG immunoreactivity in the ipsilateral hemisphere of control (top) and Hp (bottom) mice. Square selections denote the location of magnified regions. Quantification reveals that Hp mice strongly tend to have less BBB dysfunction (left axis). After normalizing for lesion volume, this trend is maintained (right axis). All comparisons include n=3-4 mice per group.
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Figure 3-13. Haptoglobin therapy reduces angiogenesis/neovascularization after ICH.
Immunohistochemistry for PECAM was performed at 72h post-ICH to evaluate angiogenesis/neovascularization. Representative low magnification images of coronal brain sections showing PECAM immunoreactivity in injured brain regions from control (top) and Hp (bottom) mice. Square selections denote the location of magnified regions. Quantification reveals that Hp mice have significantly less angiogenesis/neovascularization (left axis). After normalizing for lesion volume, this trend is maintained (right axis). All comparisons include n=6-9 mice per group, *p<0.05.
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Figure 3-14. High local levels of haptoglobin reduces VEGF expression following ICH.
Representative low magnification images of coronal brain sections showing VEGF immunoreactivity in injured brain regions from control (top) and Hp (bottom) mice. Square selections denote the location of magnified regions. Quantification reveals that Hp mice have significantly less perihematomal VEGF immunoreactivity (left axis). After normalizing for lesion volume, this trend is maintained (right axis). All comparisons include n=7-9 mice per group, *p<0.05.
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Figure 3-15. Effect of haptoglobin therapy on astrogliosis after ICH.
Immunohistochemistry for GFAP was performed at 72h post-ICH to evaluate astrogliosis. (A and B) Representative high magnification images showing GFAP immunoreactivity in the ipsilateral and contralateral (A) cortex and (B) striatum for control (left panels) and Hp (right panels) mice are provided. Square selections in the inserts denote the location of magnified regions. No significant differences in astrogliosis were observed between the groups in either the cortex or striatum (results above bars). All comparisons include n=7-10 mice per group, #p<0.05, ###p<0.001.
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Figure 3-16. Haptoglobin therapy increases microgliosis following collagenase-induced
ICH. Immunohistochemistry for Iba1 was performed at 72h post-ICH to evaluate microgliosis. (A and C) Representative high magnification images showing Iba1 immunoreactivity in the ipsilateral and contralateral (A) cortex and (C) striatum for control (left panels) and Hp (right panels) mice are provided. Square selections in the inserts denote the location of magnified regions. Quantification reveals an increase in (B) cortical and (D) striatal microgliosis for Hp mice in both the ipsilateral and contralateral hemispheres. All comparisons include n=7-9 mice per group and results above bars represent comparisons between control and Hp mice, *p<0.05, **p<0.01.
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Figure 3-17. Effect of haptoglobin therapy on microgliosis after ICH in the autologous
whole blood model. Immunohistochemistry for Iba1 was performed at 72h post-ICH to evaluate microgliosis. (A and B) Representative low magnification images showing Iba1 immunoreactivity in the ipsilateral and contralateral (A) cortex and (B) striatum for control (left panels) and Hp (right panels) mice are provided. Square selections in the inserts denote the location of magnified regions. Quantification reveals that Hp mice have significantly increased cortical ipsilateral microgliosis, whereas no difference is seen in the striatum (results above bars). All comparisons include n=7-10 mice per group, *p<0.05, ##p<0.01.
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Table 3-1. Details of the antibodies used for Western blotting and/or immunohistochemistry
Antibody Species 1°/2° Clonality Reactivity Use Dilution Vendor Catalog # Hp Rabbit 1° Polyclonal Mouse Western 1:2500 CalBiochem P101
GFP Rabbit 1° Polyclonal - IHC 1:1500 Invitrogen A11122
V5 Mouse 1° Monoclonal - Western 1:5000 Life Technologies 460705
HO1 Rabbit 1° Polyclonal Mouse IHC 1:3000 Enzo Life Sciences SPA-895
Hb Rabbit 1° Polyclonal Mouse IHC 1:1000 MP Biomedicals 0855447
4-HNE Rabbit 1° Polyclonal Mouse IHC 1:1000 Abcam Ab46545
GFAP Rabbit 1° Polyclonal Mouse IHC 1:1000 Dako Z033429
Iba1 Rabbit 1° Polyclonal Mouse IHC 1:1000 Wako 019-19741
VEGF Goat 1° Polyclonal Mouse IHC 1:500 Santa Cruz sc-1836
PECAM Rat 1° Monoclonal Mouse IHC 1:500 Santa Cruz sc-18916
IgG (800) Donkey 2° Polyclonal Mouse Western 1:20000 Rockland 610-745-124
IgG (HRP) Horse 2° Polyclonal Mouse Western 1:10000 Vector PI-2000
IgG (HRP) Goat 2° Polyclonal Rabbit Western 1:10000 Vector PI-1000
IgG (Biotin) Horse 2° Polyclonal Goat IHC 1:500 Vector BA-9500
IgG (Biotin) Horse 1° Polyclonal Mouse IHC 1:300 Vector BA-2000
IgG (Biotin) Rabbit 2° Polyclonal Rat IHC 1:500 Vector BA-4001
IgG (Biotin) Horse 2° Polyclonal Rabbit IHC 1:500 Vector BA-1100
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CHAPTER 4 CD163 HAS DISTINCT TEMPORAL INFLUENCES ON INTRACEREBRAL
HEMORRAHAGE OUTCOMES
Introduction
Hematoma clearance following intracerebral hemorrhage (ICH) is required for
recovery of local homeostasis and neurologic function.204 This process involves
hemolytic events that result in large quantities of hemoglobin (Hb).58 Extracorpuscular
Hb/heme/iron initiates a neurotoxic cascade of free radical-induced damage, oxidative
stress and inflammation.58, 93 While the inflammatory response and associated cellular
activation is initially helpful in the clean-up process after ICH, resolution of
neuroinflammation is necessary or additional secondary brain damage may occur.60, 204
CD163 is a scavenger receptor expressed on cells of the monocytic-lineage that
facilitates the safe clearance of Hb.109 CD163 is also widely used as a marker of
alternatively activated anti-inflammatory macrophages that are abundant during the
resolution phase of the inflammatory process.97 A few other functions have been
postulated for CD163 and include regulation of the pro-inflammatory cytokine TWEAK
and participation in angiogenic repair mechanisms.132 CD163-positive
microglia/macrophages accumulate in the brain following ICH,125, 149, 150 and thus it is of
interest no studies have evaluated the role of CD163 after ICH.
Here, we reveal that CD163 has distinct temporal influences on ICH outcomes.
We also investigated local mechanisms including Hb clearance, blood-brain barrier
(BBB) integrity, and angiogenesis.
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Methods
Mice
All experimental procedures were approved by our Institutional Animal Care and
Use Committee. Mice were bred and maintained in our temperature-controlled (23±2°C)
animal facilities on a reverse light cycle (12h light/dark) so neurobehavioral testing could
be conducted during the awaken phase. Two cohorts of C57BL/6N wildtype (WT) and
CD163 knockout (CD163-/-)109 male mice were used. The 72h study used n=20 WT
(4.1±0.3mo) and n=19 CD163-/- (4.7±0.4mo) mice. The 10d study used n=30 WT
(3.1±0.2mo) and n=23 CD163-/- (3.6±0.1mo) mice. Computer-generated random
numbers were used with a unique code linking to the individual animal. No mice were
excluded from this study. All surgical procedures and anatomical and functional
outcomes were performed and assessed in a blinded manner.
ICH Model
ICH was induced using our described model.191 Briefly, mice were anesthetized
with isoflurane and immobilized in a stereotactic frame. An injection of collagenase type
VII-S (0.04U) dissolved in 0.4µL of sterile water was performed at a 40° angle from the
vertical plane into the left hemisphere at 0.2µL/min using an automated injector. The
injection site was 3.6mm ventral from the skull surface at 0.0mm anterior and 3.8mm
left, relative to bregma. The needle was left in place for 5min and then slowly removed
over a 15min period to prevent backflow. Rectal temperature was maintained at
37.0±0.5°C during surgery. Mice were allowed ad libitum food and water before and
after surgery and allowed to fully recover in humidity-controlled chambers
postoperatively. For euthanasia, mice were transcardially perfused with ice-cold PBS
and 4% paraformaldehyde.
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Functional Outcomes
Functional outcomes were assessed by open field locomotor activity, Rotarod,
and neurological deficit scores (NDS) daily post-ICH.191 Behavioral tests were
performed in the same order and at the same time of day, with a 1h break between
tests.
Histology and Quantification
Histology and quantification were conducted as described.191 Ten sets of sixteen
sections equally distributed throughout the hematoma and anteroposterior brain regions
were processed. Cresyl violet staining was used to assess lesion and hematoma
volume, tissue injury, percent ipsilateral hemispheric enlargement, and hematoidin-
pigment (bilirubin). Perls’ iron staining was used to evaluate iron. Primary antibodies
used for immunohistochemistry include heme oxygenase 1 (HO1) (1:3000, Enzo Life
Sciences), Hb (1:500, MP Biomedicals), immunoglobulin G (IgG) (1:300, Vector
Laboratories), glial fibrillary acidic protein (GFAP), (1:1000, Dako), platelet endothelial
cell adhesion molecule 1 (PECAM) (1:400, Santa Cruz), and vascular endothelial
growth factor (VEGF) (1:500, Santa Cruz). For a given stain, slides for all animals were
simultaneously stained. All slides were scanned using an Aperio ScanScope CS and
analyzed with ImageScope software (Leica Biosystems).
For quantification of total brain pathology (lesion and hematoma volume, tissue
injury, ipsilateral hemispheric enlargement, Hb, iron, bilirubin, IgG and HO1), all
sections were quantified. For GFAP, five sections representing maximal lesion area
were analyzed. For 72h PECAM and VEGF, three sections representing maximal lesion
area were used. For 10d VEGF, the section representing maximal lesion area was
evaluated. Lesion Volume: injured brain areas were outlined. Using these areas, known
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distances between sections, and section thickness a total brain lesion volume was
calculated. Percent ipsilateral hemispheric enlargement: ipsilateral and contralateral
hemispheres were outlined, volumes were calculated similar to lesion volume, and the
following equation was used, 100*[(ipsilateral-contralateral)/contralateral]. A positive
pixel count algorithm was used for quantification of appropriately outlined brain regions
for hematoma volume, iron, bilirubin, and immunohistochemical stains. These
algorithms were tuned for each stain such that the appropriate signal level was
detected. Hematoma volume: number of blood-positive pixels were converted into a
volume using pixel size. Tissue injury: hematoma volume was subtracted from total
lesion volume. Hb: data are presented as ipsilateral hemisphere signal normalized for
contralateral. GFAP: cortical, striatal, and hemispheric astrogliosis was analyzed by
placing 1000x1000 pixel boxes in the motor cortex, circling the ipsilateral (excluding
lesion area) and contralateral striatum, and hemispheres, respectively. PECAM: cortical
and hematomal PECAM was analyzed by outlining the motor cortices and hematomal
region with a constant distance away from the damaged area, respectively. Ipsilateral
data are reported. VEGF: identical to PECAM, except data are presented as relative
ipsilateral to contralateral signal.
Statistics
Statistics were performed using SAS-JMP by or in consultation with a
biostatistician. Mortality was evaluated using a χ2 test and Cox proportion hazard
models. Neurobehavioral endpoint and anatomical data were analyzed by unpaired two-
tailed Student’s t-tests. Neurobehavioral regressions were analyzed by repeated
measures linear mixed modeling to account for identified baseline differences between
groups and allow estimations of mortality dropouts. All data sets were checked for
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normality and differences in variance, and data was transformed or a nonparametric test
was used as applicable. Data are expressed as mean±SEM with p<0.05 considered
statistically significant.
Results
Mortality
No difference in 10d overall mortality was observed between WT (43.3%, 13/30)
and CD163-/- (39.1%, 9/23) mice (p=0.7566). However, further inspection revealed a
temporal disproportion. Mortality on or before 4d and after 4d significantly differed
between groups (p=0.0389). WT mice accounted for the majority of deaths on or before
4d (76.9% vs. 23.1%), whereas CD163-/- mice accounted for the majority of deaths after
4d (33.3% vs. 66.7%). The hazard ratio of 0.37 for mortality on or before 4d trended
toward statistical significance (95%CI:0.08-1.20, p=0.1002), which can be interpreted as
CD163-/- mice having a 63% reduced risk of death during this timeframe.
ICH-induced Brain Damage
Correspondingly, CD163 deficiency has distinct temporal influences on ICH-
induced brain damage (Figure 4-1). At 72h, CD163-/- mice have 33.2±4.5% smaller
lesion volumes (13.9±1.0mm3 vs. 9.3±0.6mm3, p<0.0001), 43.4±5.0% reduced
hematoma volumes (3.2±1.2mm3 vs. 1.8±0.5mm3, p=0.0002), and 34.8±3.4% less brain
damage (10.6±3.9mm3 vs. 6.9±1.2mm3, p=0.0003). No significant difference in percent
ipsilateral hemispheric enlargement was seen (WT: 10.0±1.4%, CD163-/-: 7.6±1.4%,
p=0.2327). At 10d, CD163-/- mice show 49.2±15.0% larger lesion volumes (1.8±0.3mm3
vs. 2.7±0.3mm3, p=0.0385). Hematoidin-pigment (bilirubin) appears around 10d post-
ICH,93 and no difference was seen (WT: 15.7±7.0x104A.U., CD163-/-: 16.8±5.7x104A.U.,
p=0.9233).
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Functional Outcomes
In accordance with ICH-induced brain damage, CD163 deficiency has temporal
influences on functional outcomes (Figure 4-2). At 72h, CD163-/- mice display less
neurological deficits (11.8±0.7 vs. 9.5±0.6, p=0.0488) and improved latency to fall on
Rotarod (83.5±15.3s vs. 98.1±27.2s, p=0.0421). At 10d, CD163-/- mice show greater
neurological deficits (6.9±0.8 vs. 9.7±0.7, p=0.0387), reduced ambulatory distance
(15.9±1.8m vs. 9.5±1.4m, p=0.0129) and stereotypic time (146.8±17.5s vs.
101.0±14.1s, p=0.0497), and increased resting time (1341±18.4s vs. 1393±14.8s,
p=0.0378). Neurological deficit regression reveals that the WT mice recover faster while
CD163-/- mice remain stable, leading to a mortality- and anatomical-corroborating
inflection at approximately 4d (WT slope: -0.6±0.14, CD163-/- slope: -0.1±0.1,
p=0.0011). CD163 deficiency led to overall reduced baseline function on Rotarod and all
measures of open field activity (p<0.05), although these differences were statistically
accounted for.
Hemoglobin
To begin understanding the role of CD163 in Hb clearance/degradation after ICH,
immunohistochemical staining for Hb was performed at 72h (Figure 4-3). CD163-/- mice
display 30.0±7.8% less Hb (4.6±0.4A.U. vs. 3.2±0.4A.U., p=0.0167). When individually
normalized for lesion volume, this difference is no longer observed (p=0.8710).
Heme Oxygenase 1 and Iron
Furthermore, HO1 expression and iron content was evaluated by histology
(Figure 4-4). HO1 expression was higher and primarily constrained to perihematomal
areas at 72h as compared to 10d where expression was lower and more diffuse
throughout the lesion. No difference in HO1 expression was seen at 72h (WT:
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2.8±0.7x106A.U., CD163-/-: 2.7±0.9x106A.U., p=0.8384) or 10d (WT: 6.6±2.3x105A.U.,
CD163-/-: 8.2±1.6x105A.U., p=0.3408). When individually corrected for lesion volume,
still no difference is seen at 72h (WT: 1.7±0.4x105A.U., CD163-/-: 7.5±4.7x105A.U,
p=0.4727) or 10d (WT: 2.0±0.9x104A.U., CD163-/-: 1.2±0.3x104A.U, p=0.9530).
Iron was only observed in injured brain regions. At 10d, Perls’ staining was seen
in glia concentrated around vessels. CD163-/- mice exhibit 51.4±7.0% less iron at 72h
(14.1±1.5x105A.U. vs. 6.8±1.0x105A.U., p=0.0004) and 86.5±28.5% more iron at 10d
(2.8±0.8x108A.U. vs. 5.3±0.8x108A.U., p=0.0221). When individually corrected for lesion
volume, this difference is no longer observed at 72h (WT: 6.8±1.8x104A.U., CD163-/-:
5.1±1.3x104A.U, p=0.3632) and 10d (WT: 1.6±0.3x108A.U., CD163-/-: 2.0±0.2x108A.U,
p=0.2306), suggesting that the differences between groups are due to the underlying
lesion size differences.
Blood-Brain Barrier Integrity
Immunohistochemical staining for IgG was performed to assess BBB dysfunction
at 72h (Figure 4-3). CD163-/- mice have 49.7±7.4% improved BBB integrity
(4.5±0.5x108A.U. vs. 2.3±0.3x108A.U., p=0.0036). When individually normalized for
lesion volume, this difference is maintained (p=0.0526).
Astrogliosis
Immunohistochemical staining for GFAP was performed to evaluate astrogliosis
(Figure 4-5). At 72h, CD163-/- mice demonstrate 125.3±55.8% (0.7±0.3x10-2A.U. vs.
1.5±0.4x10-2A.U., p=0.0419), 52.1±25.2% (2.9±0.2x10-2A.U. vs. 4.3±0.7x10-2A.U.,
p=0.0348), and 47.5±25.8% (2.2±0.2x10-2A.U. vs. 3.3±0.6x10-2A.U., p=0.0348)
increased ipsilateral cortical, striatal, and hemispheric astrogliosis, respectively. No
significant difference in contralateral cortical (WT: 0.3±0.2x10-2A.U., CD163-/-:
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0.4±0.1x10-2A.U., p=0.2556), striatal (WT: 0.4±0.1x10-2A.U., CD163-/-: 0.8±0.3x10-2A.U.,
p=0.1439), and hemispheric (WT: 0.9±0.1x10-2A.U., CD163-/-: 1.3±0.3x10-2A.U.,
p=0.1667) astrogliosis is seen. For CD163-/- mice, ipsilateral cortical astrogliosis is
significantly increased compared to the contralateral (p=0.0054), but not for WT mice
(p=0.1745). Striatal astrogliosis is increased in the ipsilateral hemisphere compared to
the contralateral for WT (p<0.0001) and CD163-/- (p=0.0022) mice. Ipsilateral
hemispheric astrogliosis is also increased compared to the contralateral for WT
(p=0.0002) and CD163-/- (p=0.0215) mice. Ipsilateral striatal astrogliosis is significantly
increased compared to the cortex in WT (p=0.0122) and CD163-/- (p=0.0008) mice. No
difference in contralateral astrogliosis is seen between the striatum and cortex in WT
(p=0.2101) and CD163-/- (p=0.1599) mice.
At 10d, CD163-/- display 560.2±91.1% decreased ipsilateral cortical
astrogliosis (8.1±2.3x10-2A.U. vs. 2.3±0.9x10-2A.U., p=0.0528). No difference is seen in
contralateral cortical astrogliosis (WT: 2.5±1.6x10-2A.U., CD163-/-: 0.8±0.2x10-2A.U.,
p=0.4168), ipsilateral striatal astrogliosis (WT: 13.7±3.0x10-2A.U., CD163-/-:
10.7±2.3x10-2A.U., p=0.3662), or contralateral striatal astrogliosis (WT: 2.8±1.0x10-
2A.U., CD163-/-: 3.2±0.8x10-2A.U., p=0.7491 CD163-/- mice show 50.0±11.8% reduced
ipsilateral hemispheric astrogliosis (10.2±2.4x10-2A.U. vs. 5.1±1.2x10-2A.U., p=0.0528).
The contralateral hemisphere of CD163-/- mice also tends to have reduced astrogliosis
(4.3±1.4x10-2A.U. vs. 2.6±0.7x10-2A.U., p=0.0919). Ipsilateral cortical astrogliosis tends
to be greater than the contralateral for CD163-/- mice (p=0.0996), but not for WT mice
(p=0.1904). Ipsilateral striatal astrogliosis is increased compared to the contralateral for
both WT (p=0.0809) and CD163-/- (p=0.0082) mice. Ipsilateral hemispheric astrogliosis
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also tends to be increased compared to the contralateral for CD163-/- mice (p=0.0921),
but not for WT mice (p=0.3827). Striatal astrogliosis is increased compared to the cortex
for CD163-/- mice in the ipsilateral (p=0.0082) and contralateral (p=0.0082)
hemispheres. No difference in astrogliosis is seen between the striatum and cortex for
WT mice in either the ipsilateral (p=0.3827) or contralateral (p=1.0000) hemispheres.
Angiogenesis/Neovascularization
To investigate angiogenesis/neovascularization, immunohistochemical staining
for VEGF and PECAM was performed (Figure 4-6). At 72h, quantification of hematomal
regions reveals no difference in PECAM (WT: 5.7±0.7x105A.U., CD163-/-:
5.2±0.3x105A.U., p=0.5160). After individual correction for lesion volume, still no
difference is seen (WT: 5.0±0.5x104A.U, CD163-/-: 6.4±0.5x104A.U., p=0.1439). CD163-
/- mice exhibit increased neovascularization in the ipsilateral motor cortex (2.8±0.6x10-
3A.U. vs. 4.9±0.6x10-3A.U., p=0.0439).
At 72h, no difference in VEGF expression is seen in the ipsilateral motor cortex
(p=0.7983) or hematomal regions (p=0.4860). After individual correction for lesion
volume, still no difference is seen in hematomal VEGF expression (p=0.4853). At 10d,
no difference in cortical VEGF expression is seen (p=0.2046), but CD163-/- mice
demonstrate increased hematomal VEGF expression (0.8±0.3A.U. vs. 4.7±1.6A.U.,
p=0.0243). After individual correction for lesion volume, the trend is maintained
(0.4±0.1A.U. vs. 2.0±0.7A.U., p=0.1407).
Discussion
This study is the first to evaluate the contribution of CD163 to ICH outcomes.
Acutely, CD163-/- mice display significantly smaller lesions with reduced hematoma
volumes and tissue injury, whereas larger lesions are seen at 10d. Temporally, these
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differences in anatomical damage correlate with functional outcomes, where CD163-/-
mice have less neurological deficits and improved performance on an accelerating
rotarod acutely, but greater deficits and worse ambulatory ability at 10d. Regression
analyses identified the inflection point to be 4d post-ICH. At or before this time, CD163-/-
mice do well and have significantly less mortality, but after this point, they have worse
neurologic function and increased mortality. At 72h, CD163-/- mice have significantly
less Hb, iron, and BBB breakdown, increased cortical, striatal, and hemispheric
astrogliosis and cortical neovascularization, and no detectable change in HO1
expression. At 10d, CD163-/- mice have increased iron and hematomal VEGF
immunoreactivity, no change in HO1 expression, and decreased astrogliosis.
Hematoma absorption involves erythrophagocytosis and detoxification/clearance
of hemolysis products. Hemolysis begins ~24h post-ICH resulting in the accumulation of
cytotoxic extracorpuscular Hb.58 Hb is an important instigator of delayed poor outcomes
after ICH.93 Haptoglobin (Hp) is an endogenous plasma protein that tightly binds and
detoxifies Hb.176 The resting central nervous system (CNS) Hb-binding capacity is
estimated to be 50,000-fold lower than that of the large capacity systemic system.124
Although Hp enters the brain as part of the ICH, the collective levels are inadequate to
handle the massive hemolytic release of Hb. Additionally, compared to humans, mice
have around a five-fold lower plasma Hb-binding capacity.205, 206
CD163 is the scavenger receptor for Hp-Hb complexes and also clears
uncomplexed Hb under severe hemolytic conditions associated with Hp depletion,
thereby serving as its own fail-safe Hb scavenger receptor.97, 109 In mice, the estimated
Kd values for complexed and uncomplexed Hb are 18nM and 61nM, respectively.109 It
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should be noted that an apparent greater difference in kinetic parameters exists in
humans, with complexed and uncomplexed Hb having Kd values of 19nM and 198nM,
respectively.109 Basal CNS CD163 expression is restricted to perivascular
macrophages,125-129 thus, the capacity for Hb uptake immediately following the bleed
must be limited. However, during acute hemorrhagic or non-hemorrhagic CNS
pathology, CD163-positive macrophages/microglia accumulate within and surrounding
the lesions.125-129 Following ICH, CD163-positive macrophages/microglia are elevated at
24h (coinciding with start of hemolysis) and continue to increase, with significant
changes observed in and around the clot and also in remote areas.125, 149, 150 With this
distant accumulation/expression and lack of predominance in hemorrhagic brain
lesions, a few have speculated a primary anti-inflammatory role for CD163, particularly
in the resolution of inflammation.125, 127, 129 Although, a clear interaction with Hb
clearance exists, as Hp-Hb complex internalization coordinately increases CD163
expression and several other molecules involved in Hb degradation, as well as IL-6 and
IL-10 secretion.97, 126, 129 Therefore, CD163 has two anti-inflammatory consequences, 1)
removal of pro-inflammatory Hb, and 2) polarization of microglia/macrophages to an
anti-inflammatory phenotype with an altered cytokine profile.
The current study is consistent with previous speculation that CD163 has
primarily an anti-inflammatory role after acute brain injury. If Hb clearance was the main
operating mechanism, CD163 deficiency would result in increased Hb and worse
outcomes at 72h post-ICH, where hemolytic events are pronounced and CD163-positive
microglia/macrophages are elevated. However, CD163-/- mice have less Hb and smaller
lesion volumes associated with reduced tissue injury, implying that CD163-/- mice
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acutely have an augmented Hb clearance ability. It is possible these results are due to
underlying compensatory mechanisms, which may have a greater influence early when
CD163-positive macrophages/microglial are not in peak quantity. Although, the smaller
hematoma volumes combined with lower Hb levels could hint at enhanced
erythrophagocytosis in the absence of CD163, an intriguing paradoxical notion, since
M2-alternatively activated macrophages have high phagocytic capacity and participate
in wound healing by producing IL-10, chemotactic, angiogenic and extracellular matrix
components.207 It is possible that lifting the anti-inflammatory feedback loop that CD163
initiates polarizes their phenotype to a further enhanced phagocytic potential and/or
erythrophagocytosis represents a distinct under-characterized phagocytic category. IL-
4, a canonical M2 stimuli, induces tissue macrophage accumulation and
erythrophagocytosis, but paradoxically decreases CD163 expression,97, 208 indirectly
suggesting that CD163-negative M2 macrophages could have enhanced
erythrophagocytic potential. The temporal interacting complexity between CD163,
macrophage polarization, activation stimuli, and erythrophagocytosis is not clear and
warrants further investigation. Chronically, CD163-/- mice display increased brain
damage, an intuitive finding given the well-characterized role for CD163-positive
microglia/macrophages in the resolution of tissue inflammation, which is damaging if
chronic and untamed. Importantly, these temporally varied anatomical outcomes
correlate with the 4d inflection point identified on functional repeated measures
parameters, including mortality and neurobehavioral testing.
The other outcome measures used in this study aimed to further characterize Hb
clearance, inflammation, and the additional roles for CD163 suggested in other
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pathologies. No difference in HO1 expression (an inducible heme-metabolizing enzyme)
was seen between groups; however, iron levels followed the temporal nature of this
study with less and more iron seen at 72h and 10d, respectively. There are several
possible explanations for these findings. CD163 deficiency would not allow the
coordinately increased expression of Hb degradation molecules (e.g. HO1) upon Hp-Hb
or Hb internalization, although heme itself (among many other factors) is a HO1-
inducer. Thus, there are two main differential forces on HO1 induction between the
groups, where WT mice would have more HO1 induction due to the presence of CD163,
and CD163-/- mice would have more induction due to the hypothesized increased
erythrophagocytosis-derived intracellular heme. It is also possible that cell-type specific
changes are occurring and/or that the results are independent of CD163 and result from
the underlying lesion size differences at a given study endpoint. Iron
redistribution/clearance/storage mechanisms are also likely different between groups. At
72h, the reduced Hb, iron, and tissue injury correlate well with the improved BBB
integrity in CD163-/- mice. In other settings, CD163 has been associated with
angiogenesis, although these have been mostly associations linked through the known
angiogenic properties of alternatively activated macrophages, rather than causative.
Interestingly, here, CD163-/- mice show increased cortical neovascularization acutely
with trends toward reduced cortical VEGF at 10d, and increased hematomal VEGF
expression at 10d, which suggests a more direct angiogenic role for CD163. Last, glial
scar formation is an important consideration following ICH, and CD163-/- mice again
display this temporal switch in outcomes, in this case, astrocyte activation and
morphological changes.
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Figure 4-1. CD163 deficiency temporally influences ICH-induced brain damage.
Representative images for WT and CD163-/- mice at (A) 72h and (B) 10d. Within genotype and study endpoint, images are from the same animal, where left-to-right corresponds to anterior-to-posterior sections. (A) At 72h, CD163-/- mice have significantly less overall ICH-induced brain damage, residual blood volume, and tissue injury. No significant difference is seen in ipsilateral hemisphere enlargement. (B) At 10d, CD163-/- mice have significantly more ICH-induced brain damage. No difference is seen in the amount of hematoidin-pigment (bilirubin) content. (C) Representative high magnification images are shown for blood content at 72h and bilirubin content at 10d for WT and CD163-/- mice. At 72h, comparisons include n=20 WT and n=19 CD163-/- mice. At 10d, comparisons include n=11 WT and n=12 CD163-
/- mice. *p<0.05, ***p<0.001, ****p<0.0001.
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Figure 4-2. CD163 deficiency temporally influences functional outcomes after ICH. (A) Regression analysis shows that CD163-/- mice have stable neurologic deficits, while WT mice demonstrate improved recovery. Given the identified temporal inflection on multiple study measures, endpoint analyses reveal that CD163-/- mice have reduced neurological deficits at 72h, but greater deficits at 10d. (B) On measures of locomotor activity, regression analyses show no differences in the rate of recovery between groups. Endpoint analyses show no differences at 72h. At 10d, CD163-/- mice have reduced ambulatory distance and stereotypic time, and increased resting time, while WT mice demonstrate values approximating that of their baseline function. (C) On Rotarod performance, regression analysis shows no difference in the rate of recovery between groups. At 72h, CD163-/- mice exhibit improved latency to fall, whereas no differences are seen at 10d. The identified baseline differences in locomotor activity and Rotarod testing between groups were statistically accounted for. Comparisons include n=23 WT and n=17 CD163-/- mice. *p<0.05, **p<0.01.
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Figure 4-3. CD163 deficiency reduces BBB dysfunction and Hb content. Representative
images for WT and CD163-/- mice showing (A) IgG and (B) Hb immunohistochemistry at 72h. Square selections on low magnification images denote the location of magnified regions. (A) CD163-/- mice have significantly less BBB dysfunction. (B) CD163-/- mice display significantly less Hb. Comparisons include n=7 WT and n=14 CD163-/- mice for IgG and n=5 WT and n=7 CD163-/- for Hb. *p<0.05, **p<0.01.
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Figure 4-4. Effect of CD163 deficiency on HO1 and Perls’ iron. Representative images
showing HO1 expression (top panels) and iron accumulation (bottom panels) for WT and CD163-/- mice at (A) 72h and (B) 10d. Square selections on the low magnification images denote the location of magnified regions. (A and B) For both endpoints, no difference in HO1 expression is seen. (A) At 72h, CD163-/- mice have significantly less iron. (B) At 10d, CD163-/- mice have significantly more iron. At 72h, comparisons include n=14 WT and n =16 CD163-/- mice for Perls’ iron and n=10 WT and n=10 CD163-/- mice for HO1. At 10d, comparisons include n=9 WT and n=12 CD163-/- mice for Perls’ iron and n=7 WT and n=9 CD163-/- mice for HO1. *p<0.05, ***p<0.001.
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Figure 4-5. CD163 deficiency temporally influences astrogliosis. Representative images
showing ipsilateral and contralateral cortical, striatal, and hemispheric GFAP immunoreactivity for WT (top panels) and CD163-/- (bottom panels) mice at (A) 72h and (B) 10d. Square selections on the low magnification center whole-brain images denote the location of magnified regions. (A) At 72h, CD163-/- mice demonstrate significantly increased ipsilateral cortical, striatal, and hemispheric astrogliosis. The ipsilateral cortex, striatum, and hemisphere of WT and CD163-/- mice have more astrogliosis than the contralateral equivalents. (B) At 10d, CD163-/- mice show strong trends toward reduced astrogliosis in the ipsilateral cortex and hemisphere, but no difference is seen in the striatum. WT and CD163-/- mice have greater ipsilateral cortical, striatal, and hemispheric astrogliosis compared to the contralateral equivalents. At 72h, comparisons include n=5 WT and n=8 CD163-/- mice. At 10d, comparisons include n=4 WT and n=6 CD163-/- mice. *p<0.05, #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001.
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Figure 4-6. Effect of CD163 deficiency on angiogenesis/neovascularization.
Representative images showing ipsilateral cortical and hematomal VEGF and PECAM immunoreactivity for WT and CD163-/- mice at (A) 72h and (B) 10d. Square selections on the low magnification center hemi-brain images denote the location of magnified regions. (A) At 72h, CD163-/- mice have significantly increased neovascularization in the motor cortex. No difference in hematomal neovascularization or cortical and hematomal VEGF expression is seen. (B) At 10d, CD163-/- mice have increased hematomal VEGF expression, but no difference in cortical expression. At 72h, comparisons include n=5 WT and n=7 CD163-/- mice for PECAM and n=7 WT and n=7 CD163-/- mice for VEGF. At 10d, comparisons include n=6 WT and n=10 CD163-/- mice. *p<0.05.
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CHAPTER 5 SUMMARY AND CONCLUSIONS
Summary
The Hp-CD163 scavenging pathway is crucial for clearing toxic extracorpuscular
Hb under conditions of peripheral intravascular or extravascular hemolysis. However, a
paucity of literature exists regarding similar central paradigms such as that seen
following hemorrhagic stroke, a surprising notion given the putative therapeutic
implications of targeting this pathway. The present work aimed to characterize the
contribution of the Hp-CD163 scavenging system following hemorrhagic stroke in a
clinical SAH population and preclinical ICH models with genetic and viral approaches.
This work provides several new contributions to the field of hemorrhagic stroke. Here,
we reveal that i) Hp phenotype is an independent risk factor for the development of focal
and global CV and also predicts poor functional outcomes and mortality after SAH, ii)
high local levels of Hp improve anatomical and functional outcomes in two models of
experimental ICH, and iii) CD163, the Hp-Hb and Hb scavenger receptor, has distinct
temporal influences on ICH outcomes with acute deleterious effects but delayed
beneficial properties.
Discussion
Hemorrhagic stroke accounts for ~17% of stroke patients worldwide each year.88
Although it collectively represents the minority of stroke types, SAH and ICH have by far
higher disability and mortality rates when compared to the more common ischemic
stroke.4 Between 35-52% of ICH patients will not survive the first 30 days, and only 20%
regain functional independence at six months post-insult.61, 62 Between 50-75% of SAH
patients have poor outcomes, and combined with the earlier mean age of onset, SAH
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carries a high toll in terms of productive life-years lost, and thus also results in
disproportionately high costs.29, 31 Several medical management approaches, mostly
aimed at preventing secondary complications of brain hemorrhage have been tried, but
have resulted in no significant improvement in patient outcomes.61, 209 A thorough
understanding of the specific pathophysiology of these secondary complications is
crucial for designing more effective treatments in the future.
Given the massive release of Hb from erythrocytes with the hemolysis that
occurs in the subarachnoid space or brain parenchyma following SAH or ICH,
respectively, and highly toxic nature of Hb, previously underexplored therapeutic
regimens aimed at detoxifying Hb are of considerable interest. Indeed, several studies
have shown that Hb is a primary instigator of delayed secondary brain damage following
hemorrhagic stroke.8, 33-35, 76-78, 80 In addition to extracorpuscular Hb, many of its
breakdown products are also neurotoxic at supraphysiologic concentrations. Heme can
readily be released from extracorpuscular Hb if not in complex with Hp. Heme toxicity
further contributes to secondary injury following SAH and ICH by a multifactorial
mechanism, and consequently represents an additional preventable source of brain
damage.95, 176 Moreover, this step in the Hb degradation pathway represents a
deleterious amplification since the stoichiometry of heme released from Hb is 4:1. Even
further contributing to delayed secondary damage, the heme degradation products, iron,
bilirubin, and CO, have their own overlapping and distinct injurious properties following
hemorrhagic stroke.
In the periphery, the Hp-CD163 scavenging pathway is well characterized as the
primary defense mechanism against the harmful effects of extracorpuscular Hb
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following intravascular or extravascular hemolysis. Hp provides immediate, irreversible,
and direct protection from Hb through direct binding. Hp-Hb complex formation i)
prevents the peroxidation of Hb, which initiates a chain of free radical reactions, ii)
inhibits reactions that deplete protective nitric oxide, and iii) prevents the release of toxic
heme moieties. Collectively, these Hp-mediated effects minimize the harmful oxidation
of membrane lipids, lipoproteins and other biological macromolecules (DNA, proteins,
etc.), prevents the induction and propagation of inflammatory cascades, and thwarts the
adverse alteration of cellular metabolism, ultimately resulting in less cell death and
tissue injury.210 Furthermore, it facilitates the safe clearance, storage, and/or
redistribution of vital Hb degradation products, an important homeostatic process that is
tightly regulated in the periphery. Taking into account the severe consequences of
extracorpuscular Hb and the additional distinct damaging properties of Hb degradation
products, it is clear that Hb detoxification represents the most upstream therapeutic
target after pathologic intravascular or extravascular hemolysis. Thus, the current work
focuses on understanding the respective contribution of this pathway in the brain
following hemorrhagic stroke, a devastating and under-characterized CNS-localizing
form of extravascular hemolysis.
In contrast to the large capacity systemic circulation, the total Hb-binding
capacity of the CNS is estimated to be 50,000-fold lower.124 This substantial difference
is due to the fact that the brain has a very limited ability to synthesize Hp under normal
or stressed conditions such as hemorrhagic stroke. It is thus quite plausible that
upregulation or administration of Hp locally within the CNS could directly attenuate Hb-
related toxicities by the aforementioned pathways. Furthermore, this paradigm could
114
also improve the clearance of toxic free Hb following intracranial hemorrhage through
the CD163 scavenger receptor, since CD163-positive macrophages/microglia
accumulate lesional and perilesional following acute brain injury. However, it should be
noted that a converse scenario is also possible. High local Hp levels could exacerbate
hemorrhagic stroke outcomes by leading to excessive central Hb degradation and
accumulation of toxic byproducts if the proteins responsible for heme degradation,
byproduct storage, and/or redistribution are not concomitantly induced to a sufficient
level. Although, clearance of extracellular Hb via the Hp-CD163 scavenging pathway
has been shown to increase these key proteins. For instance, Hp-Hb complex
internalization coordinately increases CD163 expression and several other molecules,
such as HO1 and ferritin for heme degradation and iron storage, respectively, as well as
anti-inflammatory IL-6 and IL-10 secretion.97, 126, 129 Therefore, in addition to facilitating
the clearance of proinflammatory Hb and its breakdown products, the Hp-CD163
scavenging pathway also has another protective inflammation-resolving function of
polarizing microglia/macrophages to an anti-inflammatory phenotype with an altered
cytokine profile.
In the present work, high local levels of Hp were found to be protective against
ICH-induced brain injury and improve neurologic functional outcomes. Importantly,
these effects were reproduced in two complimentary models of ICH. An acute 72h study
endpoint was selected, as this is the peak of the hemolytic cascade in mice, and thus
the optimal time to assess Hb-driven delayed secondary brain damage and the
associated putative therapeutic potential of Hp. Increased brain Hp was achieved by
rAAV1-mediated transgene delivery, which results in very high protein levels of
115
specifically the delivered gene of interest. This specificity is an important distinction
regarding previous studies assessing the role of Hp after ICH that used relatively non-
specific methods.107, 108 Additionally, in this particular paradigm, transgene expression is
highest surrounding the brain regions normally affected by an ICH. Thus, the highly
overexpressed and secretory Hp is perfectly positioned to bind and neutralize
extracorpuscular Hb released by hemolysis occurring within the hematoma, thereby
potentially protecting surrounding viable brain tissue from Hb-mediated secondary
damage. However, it should be noted that these experimental paradigms represent
proof-of-concept studies, in that rAAV1-mediated gene delivery of Hp for hemorrhagic
stroke therapy is not directly clinically translatable. The reason being the length of time
required for transgene expression to achieve levels adequate enough to combat
extracorpuscular Hb does not fit the hemolytic course of these acute disorders.
Additional studies are needed to assess the following intertwined questions: i) the
effects of high local Hp levels on long-term anatomical and functional outcomes after
ICH, ii) the relative central versus peripheral clearance of Hp-Hb complexes (in theory, it
is possible that some escape the CNS for peripheral degradation), iii) the downstream
byproduct homeostatic redistribution mechanisms, including iron handling capability,
and iv) evaluate the therapeutic potential of delivering exogenous purified Hp protein
locally, which is clinically relevant.
Clinical translation of preclinical Hp studies is complicated by the inherent genetic
differences between humans and other vertebrates. Humans are the only vertebrates to
possess a genetic polymorphism that results in distinct phenotypic forms of Hp.131 Two
human Hp alleles exist, Hp1 and Hp2, which allow for three possible Hp genotypes:
116
Hp1-1, Hp2-1, and Hp2-2. In the United States, the distribution is around 14.4% Hp1-1,
48.2% Hp2-1, and 37.4% Hp2-2.153 The importance of this polymorphism has been
demonstrated clinically in multiple disease states, as described elsewhere herein. In
general, Hp2-2 has been implicated as the “poor phenotype,” with these individuals
displaying worse outcomes as compared to Hp1-1 and Hp2-1 persons. Mechanistically,
the Hp2-2 protein has a reduced ability to bind and detoxify free Hb, and impairs the
safe clearance of the Hp-Hb complex.153 In contrast, mice are monoallelic possessing
the equivalent of the human Hp1 allele. The overall homology between mouse Hp and
human Hp1 is approximately 80%, where key motifs have an even higher percentage.
Thus, it is expected that the results observed here are translatable to the human Hp1-1
equivalent. While the translational implications on Hp2-1 and Hp2-2 persons are less
clear, all human Hp phenotypes are protective, although it is possible that the
therapeutic effect size would vary (larger or smaller) between the sub-stratified patient
populations. Additionally, delivery of exogenous purified Hp may require type-matching,
analogous to a blood transfusion or organ transplant, to prevent conformational
antibody production and clearance of the therapeutic Hp. In this setting, Hp2-1
individuals could receive Hp2-1 or Hp1-1 since some of the Hp protein in Hp2-1 persons
is in the phenotypic form of Hp1-1. Whereas, Hp2-2 and Hp1-1 individuals would have
to receive protein of their own Hp phenotype. However, in the case of hemorrhagic
stroke, given the acute therapeutic regimen, it may also not be necessary to type-match
and all individuals could receive the “more protective” Hp1-1. Moreover, it is unclear
whether administration of the different non-type-matched Hp phenotypes would actually
elicit a humoral immune response, but if so, it would have to be conformational
117
antibody-based since the Hp2 allele originates from an intragenic duplication of a
portion of the Hp1 allele.211
Given the complicated nature of preclinical to clinical translation in the Hp field,
and to further characterize the role and therapeutic potential of Hp in hemorrhagic
stroke, a clinical study in a SAH population was performed to evaluate the Hp
phenotypic influence on hemorrhagic stroke outcomes. Regarding SAH, Hb has been
implicated as a primary instigator of cerebral vasospasm (CV), a sustained
vasoconstriction of large arteries supplying the brain, which can cause secondary
ischemia and/or infarction. CV is a known delayed complication that is a key contributor
to poor outcomes after SAH. The findings of this work reveal that Hp2-2 phenotype is an
independent risk factor for the development of focal and global CV and predicts
mortality and poor outcomes after SAH. The availability of such a genetic marker to
predict CV, delayed cerebral ischemia, mortality and poor outcomes would aid in the
critical care management of SAH patients, which continues to pose a considerable
challenge to clinicians. Genotyping for the various Hp polymorphisms can easily be
performed upon admission and has the potential for use in risk stratification by allowing
for the identification of those patients requiring increased vigilance due to their inherent
genetic risk for developing CV, delayed cerebral ischemia, and poor outcomes. This
study needs to be replicated in a larger prospective cohort. We are actively
collaborating with several groups to increase enrollment, and confirm and extend the
results presented here in a multi-institutional setting since some of these statistical
analyses were limited by sample size. Furthermore, given the major role of Hp in
detoxifying free Hb by direct binding, it is critical to understand the dynamics of both Hp
118
and Hb levels in both the CSF and serum following SAH, studies which are currently
underway on the same individuals included here. This information in combination with
the Hp phenotyping will yield an additional understanding of the protective potential of
Hp in mitigating Hb-mediated damage after SAH, and is necessary for the design of
therapeutic regimens aimed at delivering exogenous purified Hp. Notably, direct access
to the CSF compartment is available in the large majority of SAH patients (~90-95%)
since external ventricular drains are commonly placed to control intracranial pressures.
These drains can directly be used to supply a Hp infusion into this space where
hemolytic cascades are occurring, and thus serve as the optimal therapeutic avenue.
To therapeutically target the Hp-CD163 scavenging system, in addition to Hp, it
is important to understand the role of the Hp-Hb and Hb scavenger receptor, CD163.
The present work represents the first investigation of the contribution of the CD163
receptor following hemorrhagic stroke, where a genetic approach was utilized in an
experimental model. Here, we reveal that CD163 deficiency has distinct temporal
influences on ICH outcomes, with early beneficial properties but delayed injurious
effects. Temporally, differences in anatomical damage correlate with functional
outcomes on multiple neurobehavioral measures. Statistical analyses identified the
inflection point at four days post-bleed. Before day four, CD163-/- mice have significantly
less mortality and improved neurologic function, but after four days, CD163-/- mice
perform worse and have increased mortality. While it is unclear why CD163 deficiency
is initially beneficial, the late injurious effects of absent CD163 are consistent with the
key anti-inflammatory role of the receptor in the recovery phase of tissue damage.
Although some questions remain unanswered, we believe the findings of this study
119
open the door for future studies that should focus on further exploring the refined
mechanisms and therapeutic potential of targeting the CD163 receptor. Last, it should
be noted that although a soluble form of CD163 exists, it is likely not contributing here
since mice do not have the consensus sequence for ectodomain cleavage.212 However,
future studies should explore the function and role of soluble CD163 following
hemorrhagic stroke since ectodomain shedding of CD163 increases under inflammatory
conditions.
In conclusion, the Hp-CD163 scavenging system appears to be a candidate
therapeutic target for SAH and ICH, neurological disorders with currently no effective
treatments. Additional preclinical and clinical studies are needed to further characterize
this pathway following hemorrhagic stroke, although the results presented here are
promising. Furthermore, we expect that the therapeutic value of targeting the Hp-CD163
scavenging pathway will extend beyond hemorrhagic stroke, applying to the various
other conditions in which blood is released within the brain, which affect millions of
people worldwide each year, such as traumatic brain injury.
120
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BIOGRAPHICAL SKETCH
Jenna Lizabeth Leclerc was born in Berlin, Vermont, USA in 1987. In 2005, she
graduated from Venice High School (Venice, Florida, USA) and began her
undergraduate studies at the University of Florida (Gainesville, Florida, USA) where she
earned summa cum laude highest honors dual Bachelor of Science degrees in
Chemical Engineering and Microbiology and Cell Science in 2010. She then proceeded
to an industry position in Product Development/Research and Development at a
biotechnology company, Banyan Biomarkers (Alachua, Florida, USA), where she
developed biomarkers and in vitro diagnostics for Traumatic Brain Injury on various
platforms, including point-of-care diagnostics. She left Banyan Biomarkers as a Senior
Scientist to enter the MD program at the University of Florida College of Medicine,
where she confirmed her passion for research, soon after, merging into the MD-PhD
combined program. She completed the first two years of medical school and then began
her graduate PhD work described herein. She graduated with a PhD in neuroscience in
2016 and is currently finishing her clinical MD studies.