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INVESTIGATING THE RO LE OF HAPTOGLOBIN IN EPICATECHIN MEDIATED PROTECTION AGAINST I NTRACEREBRAL HEMORRH AGE By SEAN ROBBINS A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2013
2 2013 Sean Robbins
3 ACKNOWLEDGMENTS I would like to thank Dr. Sylvain Dor professor of Anesthesiology, Neurology, Psychiatry, Neuroscience, College of Me dicine for allowing me the opportunity to work in his laboratory, where I have gained an enormous amount of knowledge. I would like to thank all my fellow laboratory colleagues for the advice, support and wisdom they have been kind enough to bestow upon me while I worked on my thesis. In addition to Dr. Dor, I would like to thank Drs. Eduardo Candelario Jalil and Hendrik Luesch for serving on my supervisory committee and for the advice during the completion of this thesis.
4 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 3 LIST OF ABBREVIATIONS ................................ ................................ ............................. 6 LIST OF FIGURES ................................ ................................ ................................ .......... 8 ABSTRACT ................................ ................................ ................................ ..................... 9 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 11 Intracerebral Hemorrhage (ICH) ................................ ................................ ............. 11 Role of Reactive Oxygen Species (ROS) in Brain Injury ................................ .. 13 Animal Models of ICH ................................ ................................ ....................... 14 Haptoglobin (Hp) ................................ ................................ ................................ ..... 15 Structure ................................ ................................ ................................ ........... 15 Metabolism ................................ ................................ ................................ ....... 16 Role of Haptoglobin in ICH ................................ ................................ ............... 17 Transcriptional Factor Nrf2 ................................ ................................ ..................... 18 Structure and Role ................................ ................................ ........................... 18 Adeno associated Virus (AAV) ................................ ................................ ............... 21 Rationale of Thesis ................................ ................................ ................................ 23 2 MATERIALS AND METHODS ................................ ................................ ................ 30 Animals ................................ ................................ ................................ ................... 30 Dose Response to Establish Minimum Effective Dose ................................ ........... 30 Tissue Collection and Processing ................................ ................................ ........... 31 Western Blot Analysis of Hp Expression ................................ ................................ 32 Autologous Blood Injection Model ................................ ................................ ........... 33 ICH in Epicatechin Dosed Animals ................................ ................................ ......... 34 ICH in Neonatally In jected Animals ................................ ................................ ......... 34 Behavioral Testing ................................ ................................ ................................ .. 35 Tissue Collection ................................ ................................ ................................ .... 35 Cresyl Violet Staining ................................ ................................ .............................. 36 Analysis of Lesion Volume ................................ ................................ ...................... 36 Virus Production ................................ ................................ ................................ ..... 37 Neonatal Mouse Injections ................................ ................................ ...................... 38 Statistical Analysis ................................ ................................ ................................ .. 38
5 3 EPICATECHIN DOSE RESPONSE AND EFFECTS OF MINIMUM EFFECTIVE DOSE AFTER I CH ................................ ................................ ................................ .. 39 Results ................................ ................................ ................................ .................... 39 Western Analysis of Hp levels ................................ ................................ .......... 39 qRT PCR Analysis of endogenous Hp, CD163, and Biliverdin reductase B (Blvrb) expression ................................ ................................ ......................... 40 Pretreatment with Epicatechin Lowest Effective Dose to Induce Hp Prior to ICH ................................ ................................ ................................ ................ 41 Epicatechin Effects on Histological Outcomes ................................ ................. 42 Western Analysis of Hp Post ICH ................................ ................................ ..... 42 Functional Outcomes ................................ ................................ ....................... 42 Discussion ................................ ................................ ................................ .............. 43 4 AAV INDUCED HP OVEREXPRESSION IN WILDTYPE MOUSE BRAINS ........... 56 Results ................................ ................................ ................................ .................... 56 Hp Vector Plasmids Transfect HEK 293 Cells in vitro and Induce Hp Expression ................................ ................................ ................................ .... 56 AAV Induced Overexpression of H p in Animals Subjected to ICH ................... 56 Functional Outcomes ................................ ................................ ....................... 57 Discussion ................................ ................................ ................................ .............. 57 5 CONCLUSIONS ................................ ................................ ................................ ..... 68 LIST OF REFERENCES ................................ ................................ ............................... 71 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 79
6 LIST OF ABBREVIATIONS / Kn ockout AAV Adeno associated virus ARE Antioxidant response element AVM A rteriovenous malformations BCA Bicinchoninic acid assay Blvrb Biliverdin reductase B CD163 Cluster of d ifferentiation 163 CO Carbon monoxide DNA Deoxyribonucleic acid EC Epicatechin eG FP Enhanced green fluorescent protein Fe 2+ Ferrous iron Fe 3+ Ferric iron Hb Hemoglobin Hp Haptoglobin ICH Intracerebral hemorrhage ICV Intracerebroventricular Keap1 Kelch like ECH associated protein 1 Nrf2 Nrf2 transcription factor PBS Phosphate buffered saline PFA Paraformaldehyde qRT PCR R eal time reverse transcription quantitative polymerase chain reaction RIPA Radioimmunoprecipitation assay buffer RNA Ribonucleic acid
7 ROS Reactive oxygen species TBP TATA binding protein TBS Tris buffered saline WT Wild type
8 LIST OF FIGURES Figure page 1 1 Structure of Hp1 and Hp2 alleles ................................ ................................ ........ 25 1 2 Hp phenotype and polymeric forms as a result of Hp2 allele and duplication chain ................................ ................................ ................................ ............ 25 1 3 Metabolism of Hp Hb complex via CD163 ................................ .......................... 26 1 4 Translocation of transcriptional factor Nrf2 under oxidative stress and degradation under normal basal state ................................ ................................ 27 1 5 List of proteins upregulated via Nrf2 activ ation ................................ ................... 28 1 6 Structure of ( ) epicatechin ................................ ................................ ................ 29 1 7 Role of epicatechin in Nrf2 activation ................................ ................................ 29 3 1 Epicatechin upregulates Hp in a dose dependent manner ................................ 49 3 2 Real time reverse transcription quantitative PCR analysis of brain mRNA expression levels of Hp, CD163, and Biliverdin reductase B .............................. 50 3 3 Epicatechin treatment reduces lesion volume ................................ .................... 51 3 4 Neurological deficit score after ICH ................................ ................................ .... 52 3 5 Adhesive removal time after ICH in treated animals ................................ ........... 53 3 6 Grip strength after ICH in epicatechin treated animals ................................ ....... 54 3 7 Epicatechin significantly upregulates Hp post ICH in WT animals but not Nrf2 / animals ................................ ................................ ................................ .... 55 4 1 Hp plasmid constructs are able to transfect HEK cells and induce expression of transgene ................................ ................................ ................................ ........ 62 4 2 AAV1 is able to transduce neurons after neonatal injection and induce expression of transgene out to 3 months ................................ ............................ 63 4 3 Overexpression of Hp is able to reduce lesion volume ................................ ....... 64 4 4 Neurological defi cit score after ICH in Hp overexpressing mice ......................... 65 4 5 Grip strength after ICH i n Hp overexpressing mice ................................ ............ 66 4 6 Adhesive removal times after ICH in Hp overexpressing mice ........................... 67
9 A bstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science INVESTIGATING THE RO LE OF HAPTOGLOBIN IN EPICATECHIN MEDIATED PROTECTION AGAINST I NTRACEREBRAL H EMORRHAGE By Sean Robbins August 2013 Chair: Sylvain Dor Major : Medical Sciences Intracerebral hemorrhage (ICH) represents approximately 10 15% of all strokes per year yet represents a majority of the mortality within 30 days of ictus. ICH is characte rized by extravasation of the blood into the brain parenchyma and formation of a hematoma. Within hours, the red blood cells present in the hematoma begin to lyse and release hemoglobin. Free hemoglobin is a strong pro oxidant that is associated with c ytot oxicity, blood brain barrier breakdown, free radical formation, inflammation and apoptosis. The primary mechanism for the removal of free hemoglobin is the glyc oprotein haptoglobin (Hp) The binding of Hp to hemoglobin mitigates the toxicity of hemoglobin and facilitates the clearanc e of free hemoglobin via the CD 163 receptor. H a p toglobin can be upregulated via the activation of the Nrf2 transcription factor This thesis focuses on the role of over express ed of h aptoglobin ( Hp ) as a method to improve outcomes post ICH. We tested the hypothesis that epicatechin mediated neuroprotection in ICH is regulated through Nrf2 and specifically that the incre ased expression of Hp will lead to improved outcomes post ICH. We found that epicatechin was able to upregulate Hp in a
10 time and dose dependent manner in sham operated mice. We found that the lo west effective dose of epicatechin was able to reduce ICH lesion volume in wildtype mice however; this effect was abolished in Nrf2 / mice These findings suggest that epicatechin confers protection through the Nrf2 transcriptional factor and that the upregulation of Hp is key to reducing injury in ICH. The second project focused on determining the effect of a deno associated virus ( AAV ) induced overexpression of Hp in the brain on outcomes following ICH. We hypo thesized that overexpression of Hp within the brain using AAV serotype 1 ( AAV1 ) will confer neuroprotection and better functional outcomes following experimentally induced ICH. AAV 1 expressing Hp was injected intracerebroventricularly in day 0 neonates C5 7BL/6 wildtype pups We found that overexpression of Hp within the brain lead s to reduced lesion volumes and to improved functional outcomes post ICH. This suggests the over expression of Hp with AAV represents a promising therapeutic for hemorrhagic stroke.
11 CHAPTER 1 INTRODUCTION Intracerebral Hemorrhage (ICH) Intracerebral hemorrhage ( ICH) represents a major public health problem and accounts for 2 million of about 15 million stroke patients worldwide each year. Someone in the world dies from stroke every 6 seconds, and someone has a stroke every 40 seconds in the Unite d States. The direct cost of ICH in the US is approximately $ 12.7 billion an nually. 2 Despite active research, it is still the least treatable type of stroke and a leading cause of morbidity, disability and death worldwide 3 While ICH rep resents a small percentage of strokes, it has a mortality rate of 30 40% with half of the mortalities occurring within 48 hours of ictus. Only 20% of peoples who survive achieve functional independence at 6 months post ICH. 4 Thus, a search for a better understanding of the etiopathology is cruc ial for the design of better and more effective treatments. There are multiple risk factors and causes of ICH. The most common cause s of ICH are vascular abnormalities and trauma. The two most common abnormalities that can lead to ICH are aneurysms and ar teriovenous malformations (AVM s ) 3 4 Aneurysms typically occur in the deeper vessels of the cerebrum and tend to de velop in individuals over 40, ballooning out over a prolonged period. AVMs are congenital, tangled arteries that shunt arterial blood directly into the venous circulation bypassing the capillaries of the brain. Over time, these vessels weaken and rupture, most often due to h ypertension. In addition to hypertension major risk factors include h yper cholesterolemia, diabetes, and smoking. ICH is characterized by extravasation of the blood into the brain parenchyma and formation of a hematoma causing both p r imary and secondary damage. P rimary damage occurs concurrently with the ictus and this damage is extremely resistant to
12 treatment Primary damage is characterized by mass effects of the expanding hematoma, mechanical disruption of the neurons and glia, f ollowed by mechanical deformation leading to localized ischemia and the resulting loss of energy sources leading to mitochondrial dysfunction, membrane depolarization, edema and necrosis It is important to note that the primary damage is effectively impos sible to prevent and treat following ICH 3 5 8 The hematoma continues to enlarge for the next 3 24 hours depending on the extent of rebleeding events It is important to note that oral anticoagulants significantly increase the risk of rebleeding events 3 9 10 The ischemic mass effects in combination with increased intracranial pressure can quickly lead to si gnificant levels of ischemia in brain regions distal to the hematoma. Secondary damage occurs in the hours and days following the initial ictus and is characterized by a complex biochemical cascade. Secondary damage is precipitated by the release of coagul ation factor by products and hemoglobin released from damaged erythrocytes from the hematoma 11 Secondary damage is characterized by the breakdown of the blood br ain barrier, free radical formation, inflammation edema and apoptosis in neurons and glia Unlike primary damage, secondary damage is largely preventable and treatable 3 Secondary damage is the primary contributor of ICH damage Hemoglobin (Hb) is thought to be the primary contributor to secondary damage 4 12 H emoglo b in is a strong cytotoxic, pro oxidative and pro inflammatory protein when it is released extracellularly It initiates deleterious reaction s including oxidative damage to lipid s DNA, and, proteins which can lead to apoptosis and neuronal damage 12 13 Hemoglobin is an iron containing metalloprotein responsible for o xygen transport and it is one of the most abundant proteins in the body. H emoglobin is characterized as a globular protein with several different isoforms T he most
13 predominant isoform is h emoglobin A. It is found as a tetramer o f two and two chains, the four chains fold in such a way as to form a pocket that binds to a heme prosthetic group. The heme group coordinates with a single ferrous iron atom which serves as the binding site for oxygen used in transport Under homeostatic conditions and in the absence of pathology, the hemoglobin and its contained heme groups are safely contained within erythrocytes. However, under hemolytic and other conditions Hb and heme are released from erythrocytes directly into circulation. The free Hb is a potent pro oxi dant owing to the presence of the heme group and its coordination wit h ferric iron. The free Hb can participate in the Fenton reaction, producing reactive oxygen species that produce cellular damage and death by reacting with various macromolecules 14 15 In addition, t hese ROS can in turn induce the activati on of caspases that produce apoptosis 16 T herefore, scavenging free Hb and facilitation the fast metabolism and clearance of free Hb serve s as a potential therape utic target for ICH. Role of Reactive Oxygen Species (ROS) in B rain I njury Following ICH the complex cytotoxic cascade of secondary injury is initiated by the release of free Hb, and other cellular contents due to necrosis and blood breakdown products. T h e release of free Hb is one of the principle mechanisms of pathology following ICH. 7 17 T he formation of reactive oxygen species mediated by the pro oxid ant properties of the free Hb is important in this cytotoxic cascade. Under non pathological conditions, the production ROS is inevitable due to oxidative metabolism but the process occur s at low enough levels that t he ROS are quickly quenched via endogenous antioxidant molecules and proteins prior to causing extensive damage. However, following ICH the levels of ROS quickly rise to higher than normal levels due
14 to the release of free Hb, infiltration of neutrophils a nd necrotic cells. 18 19 Within approximately 24 hours post ictus erythrocytes begin to lyse and the subsequen t release of free Hb quickly overloads the endogenous anti oxidant pathways leading to the potentiation of oxidative stress. The dramatic increase in oxidative stress post ICH leads to large amounts of neuronal and glial cell death. Animal M odels of ICH M ultiple models of experimental ICH in animals are designed to replicate specific aspects of clinical ICH. The two most common models of ICH are striatal collagenase injection or autologous blood injection. The collagenase model involves the injection of ba cterial collagenase into the striatum. The collagenase then degrades the basal lamina around vessels, ultimately leading to vessel rupture and blood extravasation 20 The bleeding profile of the collagenase model replicates the rebleeding and hematoma evolution observed in the clinic however, the injection of an exogenous enzyme and the bleeding from multiple factors makes physiological analysis of mechanisms difficul t 21 23 The autologous model is one of the earliest models developed to replicate ICH. The model involves the injection of blood collected from a superficial arter y into the cerebrum. Specifically, t he most common target is the striatum 24 The injection of autologous blood more accurately mimics the inflammation and toxicity of the clinical presentation of I CH 25 One of the challenges of the autologous blood model is the volum e and rate of blood injection; larger volumes or fast injec tion rates tend to precipitate increased injury or intraventricular hemorrhage 26 Additionally the injection of blood into the brain parenchyma can reflux along the needle track or flow along the white matter.
15 T his can be avoid ed by employing the double injection model, in which an initial small volume of blood is allowed to clot around the needle and plug the tract before the remainder of blood is injected 27 Haptoglobin (Hp) Haptoglobin ( Hp) is an endogenous glycoprotein and is the primary hemoglobin binding protein present in blood plasma Haptoglobin helps in removal o f free Hb under normal hemolysis and hemorrhagic events In normal conditions, Hp binds to free Hb to form the less toxic Hb Hp complex which can then be endocytosed by microglia or macrophage through the CD163 scavenger receptor. 18 28 31 Structure and two chains with disulfide bridges connecting all the chains together. In humans, the Hp gene i s present in two allelic forms which are designated as Hp1 and Hp2 Therefore, there are three different genotypes possible, Hp1 1, Hp 2 1 and Hp2 2 (Fig ure 1 1). 14 The Hp1 allele only forms homodimers and 1 1 14 chain, designated 2 The additional alpha chain causes individuals with Hp 2 1 and Hp2 2 to form cyclic oligomers in contrast to the linear nature of Hp1 1. 7 1 4 The formation of the oligomers is a result of the duplication of the cysteine residues involved in the formation of the disulfide bridges between Hp chains (Figure 1 2) 19 28 The size of the Hp2 2 and Hp2 1 oligomer negatively affects movement across t he blood brain barrier. 6 32 The Hp1 1 isoform is associated with faster clearance of the Hp Hb complex. The Hp2 2 genotype is associated with lower hemoglobin binding affinity and an increased risk of vasospasm following subarachnoid hemorrhage and diabetes H owever, the higher order oligomers
16 are able to bind more Hb per molecule of Hp 32 Additionally the Hp2 gene is associated with increased risk of cardiovascular disease in diabetic individuals 14 33 Metabolism Haptoglobin is synthesized mainly in the liver by hepatocytes and is circulate d via the serum. The uncleaved form of Hp known as zonulin functions in tight junctions in the lining of the intestines, and possibly is involved in proper B cell maturation and differentiation 8 3 4 Hp is an acute phase protein that is rapidly and readily upregulated in response to inflammation and injury 19 Hp possesses an antioxidant response element (ARE) within the promoter. During inflammation and injury, the transcription factor Nrf2 binds to the ARE and induces expression. Upon hemolysis, a single Hp homodimer binds a single Hb tetramer by each chain binding a single Hb dimer 19 35 The binding of t he Hp Hb complex binds the CD163 receptor on monoc ytes, macrophages and microglia causes the entire complex to be internalized and trafficked to the lysosome where both Hp and Hb are degraded 29 36 The kupffer cells in the liver are the primary site of metabolism of circulating Hp Hb complexes. 29 Hp2 1 and Hp2 2 b ind to the CD163 receptor with greater affinity than the Hp1 1 isoform 28 Because Hp is not recycled by macrophages, it may take 5 7 d ays for the Hp level to r ecover after massive hemolysis. Therefore, m assive hemolysis like in ICH may lead to rapid and persistent hypohaptoglobinemia and extensive brain damage 7 The express ion and level of Hp is regulated by transcription factor Nrf2. Along with this, Nrf2 helps in regulating redox balance and stress response The expression of Hp has been shown to be inducible post ICH with the natural small molecule sulforaphane an activa tor of Nrf2 7
17 Role of Haptoglobin in ICH Haptoglobin is found in extremely low levels in the brain and under normal conditions is not synthesized in the brain at app reciable levels. I nstead it is suggested that Hp is able to cross the blood brain barrier at low levels 6 scavenge free Hb via Hp CD163 is som e 50,000 fold lower than the circulation 6 Under non pathological conditions normal serum levels of Hp varies over a range of 0.3 3mg/mL, with evidence that circulati genetics 19 30 The binding of Hp to Hb is an important mechanism for t he removal of free Hb and the mitigation of toxicity. The mechanism of alleviation of toxicity is a subject of debate with two predominate theories i ) the binding of Hb to Hp increases clearance of Hb or ii .) a reduction in the native pseudoperoxidase pro perties of Hb. 37 The binding of Hb to Hp is one of the strongest non covalent bonds in nature ( K D =10 12 M). T he half life of this complex is comparatively long at approximately 12h 38 39 40 42 It is the ferryl iron that is the primary contributor of ROS in free Hb, even more so under acidic conditions. 41 The reduction in pero xidase activity of Hb following Hp binding has been suggested to be due to the location of the binding site of Hp to Hb functions to shield and stabilize the ferryl intermediate in the heme. This stabilization is most profound in conditions of low pH, whic h are found in the ischemic areas of the hematoma. 31 Whether the mechanism through facilitated clearance or mitigation of Hb oxidative activity Hp is essential for the removal of Hb induced toxicity following ICH Therefore, Hp serve s as a potent therapeutic target and any agent that is able to increase the reservoir of Hp could be a powerful therapeutic for combating hypohaptoglobinemia
18 Transcriptiona l F actor Nrf2 Nuclear factor (erythroid derived 2) like 2 or Nrf2 is a transcripti on factor important in the regulation of cytoprotective genes with an antioxidant response element (ARE) in the promoter. Nrf2 is key to cellular responses to oxidative stress, inflammation and injury. These conditions as well as some pharmacological agent s are potent activators of Nrf2 driven expression of cytoprotective genes. Structure and Role factor 43 44 Under normal conditions Nrf2 is bound to Kelch like ECH associated protein 1 (Keap1) in the cytoplasm. Under the homeostatic conditions, Keap1 facilitates the ubiquitylation and subsequent proteosomal degradation of Nrf2 O verexpression of Keap1 results in the increased ubiquitylation and degradation of Nrf2 suggesting that Keap1 is integral to Nrf2 degradation. 45 In contrast, Keap1 / mice demonstrate postnatal lethality due to skin and esophageal abnormalities further suggesting the importance of Nrf2 regulation via Keap1. 46 Under non oxidative conditions when Nrf2 dissociates from Keap1 it has a relatively short half life of approximately 15 minutes before it is degraded via the proteasome pathway. H owever oxidative stress and electrophilic compounds are able to stabilize Nrf2 and prolong its half life. 45 47 48 When the cell is exposed to conditions that result in an increase in oxidative stress Nrf2 dissociates from Keap1 and translocates to the nucleus There Nrf2 binds to AREs thereby inducing transcription of cytoprotective genes such as s uper oxide dismutase, NAD(P)H dehydrogenase quinone 1 heme oxygenase 1 and haptoglobin. 49 50 If oxidat ive stress is allowed to continue, t he constitutively expressed antioxidants in the cell are rapidly depleted, which leads to a buildup o f reactive oxygen species (ROS). N otably the participation of iron from free Hb
19 results in elevated levels of the extr emely reactive hydroxyl radical via the Fenton reaction. This buildup of ROS results in damage to DNA, proteins and lipids essential for cellular function and if left unchecked will result in apoptosis of the e ffected cell. 51 The process of dissociation from Keap1 is thought to involve the reduction of cysteine residues via oxidative species or electrophilic compounds, such as epicatechin or its related flav anols. Some molecules might promote a conformational change in Keap1 thereby releasing Nrf2 from the ubiqutylation pathway. 52 Alternatively, it has been sugges ted that Nrf2 is constitutively located in the nucleus and Keap1 functions to shuttle it out of the nucleus for degradation. 53 54 Nrf2 translocated to the nucleus binds to AREs and induces transcription of cytoprotective genes to combat oxidative stress and inflammation. 55 Antioxidant response elements are cis acting enhancers that regulate the expression of cytoprotective genes in response to oxidative stress 55 ROS or antioxidant phenolic compounds can activate AREs and induce the cytoprotective genes. It has previously been shown that transcriptio nal factor Nrf2 is able to confer protection in both ischemic and hemorrhagic stroke 56 In addition, recent evidence supports the role of Nrf2 in mediating the prote ctive effects of epicatechin in ischemic stroke models. 57 To date, no study has been conducted to investigate the mechanism of action of epicatechin in ICH. ( ) Epic atechin ( ) Epicatechin belongs to the subclass of polyphenols known as flavanols and more specifically the flavan 3 ols. Epicatechin is a diastereoisomer of catechin. Polyphenols are a broad class of primarily natural compounds that consist of molecules m ade up of multiple phenol groups. The flavonoids class of polyphenols has been the most extensively studied, as it is one of the more common class es of polyphenols that
20 are regularly ingested. 58 The flavonoids are a diverse group of molecu les that include the flavanols, flavanones, flavones, anthocyanidins and isoflavones. Tea, cocoa, berries, grapes, and citrus all contain high levels of flavonoids. The composi tion and amount of flavonoids is unique to each food source. 59 For instance, in green tea the flavanol catechins make up almost 70% of the total polyphenolic content. 59 60 The beneficial effects of fruits, vegetables, green tea and cocoa have been attributed to these polyphenols. 61 Flavanols are most often encount ered as secondary metabolites in plants such as in green tea. Flavanol studies have indicated that these molecules possess strong antioxidant properties and that ingestion of large amounts of flavanol containing foods is associated with reduced risks of ca rdiovascular pathologies and stoke, both ischemic and hemorrhagic. 62 65 Studies have suggested that these protective effects are due to the direct anti oxidant properties of flavanols 66 However, the bioavailabi lity of polyphenols and specifically, epicatechin and other flavanols suggests that the mechanism of action lies elsewhere and it is more likely that these molecules act to induce cell signaling pathways, such as the activation of transcription factor Nrf2 57 67 68 Flavanols ingested orally are subject to extensive f irst pass metabolism in the liver, and are subject to extensive metabolism in the gut by intestinal enzymes or bacterial activity. 69 In addition, many flavanols a re readily absorbed by the intestines because of the large size of some flavanol polymers or the functional groups, smaller catechins tend to be more easily absorbed. 69 70 The small size of epicatechin makes it an ideal molecule for study using oral administration regimes. However, epicatechin, along with other catechin flavanols, still possess relatively low bio availability. Plasma levels of catechin flavanols peak within 2 4 hours after ingestion. 70 In the liver, flavanols undergo extensive enzymatic metabolism via sulfation,
21 methylation, and glucuronidation with evidence that th e majority of flavanols that are absorbed are metabolized within 20 minutes. 71 The extensive metabolism along with the decreased bioavailability of some flavano ls suggests that the stoichiometry would be too low to provide a direct anti oxidant mechanism of protection. This raises the question about the amount of native flavanol present in circulation and the ability of the metabolites to readily cross the blood brain barrier and induce neuroprotection in stroke. It is well documented that populations of individuals with high intakes of polyphenol containing foods have reduced incidences of cardiovascular disease and associated pathologies notably stroke. 58 Previous studies have shown that administration of flavanols either acutely or chronically prior to experimental induction of stroke in animal models has shown that polyphenols have the ability to mitigate stroke related damage 57 Importantly administration of flavanols has been shown to reduce oxidative stress and lesion volu me following ischemic stroke and reperfusion. Most studies have focused on the property of administered flavonols to reduce oxidative stress via lipid peroxidation and protein carbonylation, effectively assuming the effects were due to the direct anti oxid ant properties of flavanols. However, in studies in Nrf2 / mice receiving ischemic stroke and epicatechin pretreatment the protective effects of epicatechin where ablated 57 This suggests that the mechanism of action of flavanols and more specifically, epicatechin, is mediated through the activation of Nrf2, and the resulting cytoprotective protein cascade. A deno a ssociated V irus (AAV) Adeno associated virus (AAV) i s a class of small viruses that infect mammalian cells. AAVs are Dependovirus members of the Parvovirus family. 72 Native AAV is a non enveloped single stranded DNA virus that upon infection inserts its genetic material into
22 a specific site within human chromosome 19. AAVs are well suited for use as viral vectors for gene therapy because they possess low immunogenicity, can infect both dividing and non dividing cells, and the insertion of the transgene into the host genome allow s for stable transgene expression. 73 In addition, transduction is stable over an extended period. 73 Currently 12 serotypes of AAV are known (AAV 1 12) T he difference between the serotypes arises because of substitutions and changes to the viral capsid protein s. 74 These viral capsid proteins determine the c ells that each serotype infects 73 76 Currently gene therapy i s used to correct genetic disorders. Preclinical data for the treatment of muscular dystrophy has had great success restoring muscle function. 77 The use of AAV for gene therapy in animal models of muscular dystrophies, such as the mdx deficient mouse models has been studied and the results look promising for gene therapy in humans. 78 Recombinant AAV2 based gene delivery has demonstrated efficient transduction of target tissues and delivery of secreted proteins, such as factor IX in the treatment of hemophilia. 79 The ventricle system is a continuous series of large fluid filled spaces in the brain and spinal cord through which cerebrospinal fluid flows. Intracerbroventricular injection (ICV) targets the lateral ventricle in either hemisphere of the brain. Access to it would require a craniotomy and stereotaxic neurosurgery. ICV administration of recombinant VEGF has been shown to delay onset and increase survival in ALS mice. ICV a dministra tion of A AV1 or AAV2 expressing glucuronidase to neonate mouse brains showed very different transduction patterns between the two serotypes with respect to the regions that were transduced. 80 Importantly, the e nzyme spread was far greater throughout the AAV1 injected brain s In addition, the transduction and
23 expression was found to be stable to one year post injection. 80 ICV delivery of AAV1 in neonatal mice allows widespread gene expression throughout the brain. 81 ICV may be a useful delivery method for both secreted and intracellular molecules because of its wide transgene expression distribution. Finally, these injections were conducted in neonate mice where the ventricular brain barrier is not established The lack of this barrier is pivotal to infection of the majority of the neurons in order to have an in creased reservoir of cells over expressing Hp during and after ICH. 75 Rationale of T hesis ICH represents a serious public health problem with a high rate of mortality. Cur rently treatment options are limited to supportive therapy, hematoma removal, and/or rehabilitation; however, 6 month mortality remains high. The majority of damage occurs as a result of the release of free Hb and the subsequent oxidative stress and toxici ty. Targeting this free Hb and mitigating its toxicity is a promising therapeutic tool. Given the promise that targeting this pathway holds, the focus of this thesis is investigating the effect of upregulation or over expression of haptoglobin on outcomes following ICH. This thesis is divided into two major in vivo projects aimed at investigating the role of Hp in ICH. The first project investigated upregulating Hp prior to ICH with the natural compound epicatechin and the pathway involved in conferring n europrotection and up regulating Hp. The study was conducted in C57BL /6 WT mice and transcriptional factor Nrf2 / mice to determine the minimum effective dose of EC needed to induce Hp upregulation and confer neuroprotection. The establishment of a minimu m effective dose is important to mitigate potential toxicity of epicatechin and reduce any direct antioxidant confounds. The use of Nrf2 / allows for the elucidation of the
24 mechanism of action of epicatechin neuroprotection in ICH and Hp upregulation. The purpose of establishing a minimum effective dose to upregulate Hp is important to creating an enlarged reservoir of Hp that is able to bind free Hb during hemolytic events such as ICH and mitigate the toxicity of free hemoglobin. The ability of epicatechi n to upregulate Hp provides evidence that it can serve as a readily available therapeutic for those at risk for ICH. The second project in this thesis involves the investigation of the overexpression of Hp within the brain of WT mice receiving ICH. In thi s study, neonatal mice were intracerebroventricularly injected with AAV1 expressing Hp, Hp eGFP, and Hp V5 or eGFP and then survived out to three months before receiving ICH. The use of eGFP as a fluorescent label allowed for differentiation of intrathecal ly AAV induced synthesized Hp and Hp produced peripherally. The purpose of this study was to investigate the effects of Hp overexpression on outcomes following ICH. The importance being that ICV injection of AAV expressing Hp would dramatically increase t he hemoglobin binding capacity of the brain and serve a potentially powerful preventative therapeutic for ICH and other similar hemoly tic events.
25 1 1 1 1 1 1 2 1 1 2 2 2 2 2 2 2 2 2 Figure 1 1. Structure of Hp1 and Hp2 alleles. Brackets denote duplicated sequences. Figure 1 2 Hp phenotype and polymeric forms as a result of Hp2 allele and duplication of chain.
26 Figure 1 3. Metabolism of Hp Hb complex via CD163.
27 Keap1 Nrf2 Nrf2 Nrf2 ARE Gene transcription Keap1 Nrf2 Nrf2 Ub Ub Ub Ub Proteasome degradation Inducers, such as epicatechin or oxidative stress Basal State Figure 1 4. Translocation of transcriptional factor Nrf2 under oxidative stress and degradation under normal basal state.
28 Figure 1 5. List of proteins upregulated via Nrf2 activation 1
29 Figure 1 6. Structure of ( ) epicatechin. Figure 1 7. Role of epicatechin in Nr f2 activation. 1
30 CHAPTER 2 MATERIALS AND METHODS Animals C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME) were housed and bred in under approved protocols by Universi ty of Florida Institutional Animal Care and Use Committee (IACUC) in accordance with NIH guidelines. All animals were housed three to five to a cage and maintained on ad libitum food and water with a 12 h light/dark cycle. Dose R esponse to E stablish Minimu m E ffective D ose Three month old male wildtype C57BL/6 mice were weighed prior to oral administration of 5, 15, or 30mg/kg epicatechin dissolved in 0.2mL of 1.0% methylcellulose or given isovolume of 1.0% methylcellulose 90 minutes prior to craniotomy. Mi ce were anesthetized using h alothane (5% for induction and 1 2% for maintenance). The tops of the heads were shaved and the animals placed in a nose cone to maintain halothane anesthesia for the duration of the craniotomy procedure. The skin was cleaned wi th chlorohexidine gluconate then 0.9% saline. A single anterior posterior incision was made from behind the eyes to the top of the skull (approximately 1 2cm). The skin was retracted and the skull cleaned with sterile cotton tipped applicators soaked in sa line. The craniotomy (1 1.5mm) was drilled with a rotary burr drill bit, until just through the skull taking care not to pierce the dura. The dura was then raised and then pierced. Care was taken to prevent rupture of superficial arteries and in the event of a bleed, the bleeding was stopped and any superficial blood was wiped away with a sterile saline soaked cotton tip applicator. The incision was then sutured closed. Mice were given a 500L subcutaneous saline injection post surgery for
31 rehydration. Body temperature was maintained at 37 .0 0.5C with the use of a rectal probe and heating pad throughout surgery. The mice were allowed to recover for approximately 60 minutes in a 34C humidity controlled recovery chamber. After mice were able to ambulate th ey were placed back in their respective home cages containing moistened mouse chow. Tissue Collection and Pr ocessing Mice were divided into two survival time points post surgery 24 and 48 hours with equal numbers in each time point and dose. Mice were dee ply anesthetized with halothane. Blood was collected via cardiac puncture and placed in sterile 1.5mL tubes and allowed to clot for 30 minutes at room temperature. Mice were then transcardially perfused with ice cold 0.1M phosphate buffered saline for appr oximately 5 minutes to insure maximal removal of blood and Hp from vessel to prevent blood contamination of brain and liver. The liver was quickly collected and placed in 2mL of 2x RIPA buffer containing protease and phosphatase inhibitors and stored on ic e. The brain was removed and washed with ice cold PBS. The brains were then divided in half a long the medial sulcus with a sterile razor blade T he right half was then immediately flash frozen in 2 methyl butane on dry ice for RNA extraction later. The lef t half of the brain was placed in 2mL of 2x RIPA buffer containing protease and phosphatase and stored on ice. Clotted blood was centrifuged at 4C at 3000xg for 15 min ute s until red cells were pelleted. Serum was collected and both serum and red cells st ored at 80C. Left hemispheres and livers were homogenized with a sterile rotor stator homogenizer on high for 60 seconds on ice then allowed to lyse for 45 minutes on ice prior to
32 centrifugation at 14000xg for 20 minutes at 4C; supernatant was drawn off aliquoted and stored at 80C. Right hemispheres were processed for total RNA extraction. All equipment, materials and surfaces were sterilized and sprayed down with RNase AWAY (Life Technologies, Grand Island, NY ) to neutralize ribonucleases. Brains we re kept at 80C until immediately ready for processing to limit RNA degradation. Frozen brains were homogenized in 1mL of ice cold TRIzol reagent (Life Technologies, Grand Island, NY ) using a sterile rotor stator homogenizer on high for 60 seconds. Subseq uent separation and purification steps carried out according to manufacturer instructions using the combined TRIzol PureLink RNA minikit (Life Technologies, Grand Island, NY ) Total RNA concentration was d etermined with a NanoDrop 2000 (ThermoFisher Scient ific, Waltham, MA) Total protein concentration for liver, serum, and brain lysates was determined using bicincho ninic acid total protein assay (Bio Rad, Hercules, CA). Western B lot A nalysis of Hp E xpression Brain and liver lysates were mixed with lamelli l oading buffer contain ing 5% 2 mercaptoethanol and incubated for 5 minutes in a 95C heating block. The samples were then loading onto a 12% SDS polyacrylamide gel, 40g of total protein for brains and 10g total protein for livers and serum. Proteins were separated at 150 volts for 45 min ute Proteins were then transferred from the gel to a p olyvinylidene fluoride membrane in a semi dry system for 38 minutes at 10 volts The membrane was blocked in 0.5% casein in tris buffered saline ( TBS ) with agitation fo r 45 minutes at room temperature. Membranes were washed once with 1x TBS/0.01% Tween 20 for 1 minute. Primary rabbit anti mouse Hp (CalBioreagents, San Mateo, CA) was diluted 1:2500 in blocking buffer containing 0.2% Tween 20. The membrane was incubated
33 wi th the primary antibody overnight at 4C with rocking. The membrane was then washed with 1x TBS/0.01% Tween 20 and then incubated with anti rabbit conjugated with horse radish peroxidase (Vector Laboratories, Burlingame, CA) for 1 hour at room temperature with rocking. The secondary was diluted 1:1000 in blocking buffer containing 0.2% Tween 20 and 0.01% sodium dodecyl sulfate ( SDS ) The proteins were visualized by chemiluminescence. Membranes were then stripped for 30 min at room temperature with vigorous rocking. Membranes were washed twice with dH 2 O and then 1x TBS/0.01% Tween 20. The membrane was reblocked in 0.5% casein in TBS with agitation for 45 minutes at room temperature, and then washed once with 1x TBS/0.01% Tween 20. The membrane was then washed and incubated with anti beta actin diluted 1:3000 in blocking buffer containing 0.2% Tween 20 overnight with rocking at 4C. then the membrane was washed and incubated for 1 hour at room temperature with rocking with anti mouse conjugated with horse radis h peroxidase diluted 1:1000 in blocking buffer containing 0.2% Tween 20 and 0.01%SDS. The proteins were visualized by chemiluminescence. The images were analyzed in alpha view and levels of protein were normalized to its respective b actin The levels of e xpression were analyzed as relative densities. Autologous B lood I njection M odel The autologous blood double injection model was modified from previous studies 27 Mi ce were anesthetized with h alothane 5% for induction and 1 2% for maintenance. Mice skulls were immobilized in a Stoelting Co. Just for mouse stereotaxic (Stoelting Co., Wood Dale, IL) Blood was collected from the tail artery and drawn up into heparinized tubing connected to a 25g blunt needle. All animals received a total of 30L of autologous blood divided into two infusions of 10L and 20L. The
34 infusions were delivered at 1.5L/min ute with an autoinjector (Stoelting Co., Wood Dale, IL) Infusions were delivered at the following coordinates : 0.5 anterior, 2.4mm lateral and 3.4mm ventral to bregma This was done using a 25g blunt ended needle. The second infusion was administered 4 minutes after completion of the first. Ten minutes after completion of th e second infusion the needle was withdrawn at 0.1mm/min ute until reaching 2.0mm ventral, at which point the needle removal was stopped for five minutes to ensure complete coagulation of injected blood. Needle was then withdrawn at 0.1mm/min ute until reach ing the surface of the skull. The incision was then closed with tissue adhesive (3M St. Paul, MN ), mice given 1mL of 0.9% saline. Body temperature was maintained at 37 .0 0.5C with the use of a rectal probe and heating pad throughout surgery. The mice we re allowed to recover for approximately 60 minutes in a 34C humidity controlled recovery chamber. After mice were able to ambulate they were placed back in their respective home cages containing moistened mouse chow, and placed back in mouse room within 2 4 hours of surgery. ICH i n Epicatechin D osed A nimals Three month old male wildtype C57BL/6 mice or Nrf2 / were weighed prior to oral administration of 15mg/kg epicatechin (Sigma Aldrich, St. Louis, MO) dissolved in 0.2mL of 1.0% methylcellulose or given isovolume of 1.0% methylcellulose 24h prior to experimentally induced ICH. ICH in N eonatally Injected A nimals Neonatally injected mice were survived to three months and then autologous blood ICH was induced as above.
35 Behavioral T esting All mice were eva luated for sensory and motor deficits every day out to 72 hours post surgery. All mice were evaluated using neurological deficit scoring, grip strength and adhesive removal in that particular order to minimize confounding effects of prolonged stimulation a nd activity on activity. All behavioral testing were conducted by two trained experimenters blinded to genotype or treatment group NDS was modified from Clark et al. and scored animals on a 0 4 point scale in each of the following six categories open field gait, body asymmetry, climbing, circling, front limb asymmetry and compulsory circling for a maximum deficit score of 24 82 Grip strength was utilized to isolate motor deficits and consisted of a trained blinded experimenter obtaining forelimb grip strength using a force meter; baseline grip strength for each anima l was obtained 24 h ours prior to ICH. Adhesive removal was used to evaluate sensorimotor deficits in both forelimbs. The test consisted of a trained blinded experimenter training animals 72 and 48 hours prior to ICH with a baseline pretest taken at 24 h our s prior to induction of ICH. Tissue C ollection Mice were deeply anesthetized with halothane at 72 hours after ICH and after receiving the final behavior tests. CSF was collected from the cisterna magna, placed in sterile 200L tubes then immediately froze n on dry ice. Blood was collected via cardiac puncture and placed in sterile 1.5mL tubes and allowed to clot for 30 minutes at room temperature. Mice were then transcardially perfused with ice cold 0.1M phosphate buffered saline for approximately 5 minutes to insure maximal removal of blood and Hp from vessels to prevent blood contamination of brain and liver. The liver was quickly collected and flash frozen on dry ice. Mice were then perfused with ice cold 4% PFA in
36 0.1M PBS. The brain was removed and wash ed with ice cold PBS. The removed brains were placed in 15mL conical tubes and post fixed in 4% PFA in 0.1M PBS at room temperature for 24 hours. After post fixing, brains were placed in 30% sucrose in 0.1M PBS and stored at 4C until the brains became sat urated with sucrose, indicated by sinking to the bottom of the tube. Brains were then cryosectioned at 30m starting at the beginning of the caudate, collecting every section for 6mm or until hematoma was no longer visible Sections were stored at 80C un til staining and immunohistochemistry could be carried out. Cresyl V iolet S taining Sections were thawed and left covered at room temperature for 24 hours prior to staining and then dried at 40C for 45 minutes prior to staining. Tissues were rehydrated an d washed in dH 2 O for 5 minutes before being placed in 0.5% Cresyl violet acetate (Sigma Aldrich, St. Louis, MO) solution for 1 minute. Tissues were washed with dH 2 O to removed excess Cresyl violet and then dehydrated through increasing ethanol concentratio ns (50/70/95/100%). Finally, tissues were cleared in Histo clear (National Diagnostics, Atlanta, GA) and cover slipped with Permount (ThermoFisher Scientific, Waltham, MA) Slides were scanned using the Aperio scanscope XT system (Aperio Vista, CA ) Analy sis of Lesion V olume Scanned Cresyl violet stained sections were analyzed for lesion volume using ImageScope (Aperio Vista, CA ) The lesion border was defined as the line dividing healthy stained cells from areas devoid of cells, containing erythrocytes a nd/or pyknotic cells.
37 Virus P roduction AAV was prepared by methods previously described by Zolotukhin et al 83 AAV vectors expressing Hp, Hp eGFP, HP V5 and e GFP were under the control of the cytomegalovirus enhancer/chicken beta actin (CBA) promoter, Virus was g enerated by Polyethylenimine Linear ( Polysciences Warrington, PA ) transfection into a human kidney epithelial 293 ( HEK 293 ) cell line. C ells were cult Eagle medium (DMEM) supplemented with 10% fetal bovine serum Cells were co transfected with the AAV helper plasmid pXYZ1. The helper plasmid pXYZ1 encodes the AAV and Ad gene s necessary for creating the serotype At 72 hours af ter transfection, cells were harvested and lyse d in the presence of 0.5% s odium d eoxycholate and 50U/ml b enzonase (Sigma Aldrich, St. Louis, MO) by repeated rounds of freeze/thaws at 80 C and 50 C. The virus was isolated using a discontinuous Iodixanol gr adient. 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 Bio Rad CFX384 (Bio Rad, Hercules, CA) The viral DNA samples were prepared by treating the virus with DNase I (Life Technologies, Grand Island, NY ) heat inactivating the enzyme, then digesting the protein coat with Proteinase K (Life Technologies, Grand Island, NY ) followed by a second heat inact ivation. Samples were compared against a standard curve of plasmid diluted to 10 3 to 1 0 7 copies per ml. Viruses were aliquoted and stored at 80 C till further use. Viruses were diluted in sterile 1X PBS, pH 7.2, if needed and used immediately.
38 Neonatal Mouse I njections Wildtype C57BL/6 post natal day 0 pups were injected within six hours of birth The nave pups were covered in aluminum foil and completely surrounded in ice for 3 4 min utes to insure sufficient cryoanesthetization. Complete cryoanestheti zation was determined when all movement stopped and the skin color change d from pink to purple. Cryoanesthetized n eonates were injected using 10uL Hamilton syringes with 30g, 30 bevel (Hamilton Company, Reno, NV) at an angle of 45 to a depth of 1.5mm. 2 L of virus (titer 2.0x10 10 ) or 1x PBS was slowly injected into each ventricle and needle retracted slowly. After the injection pup s w ere immediately placed on a warming bl anket for recovery until all were recovered completely T hey were then returned to the home cage. Statistical Analysis One was used to compare control and treated groups, with P <0.05 considered statistically significant. For tests involving two groups, statistical significanc e between groups was t test, with P <0.05 considered statistically significant.
39 CHAPTER 3 EPICATECHIN DOSE RESPONSE AND EFFECTS OF MINIMUM EFFECTIVE DOSE AFTER ICH Results Western Analysis of Hp levels Hp expre ssion was readily detectable in brain homogenates serum, and liver homogenates (Figure 3 1 ). However, amounts of Hp in serum and liver were as actin as a loading control revealed Hp le vels in the liver were on average higher at 24 hours post surgery and decreased at 48h at all doses expect for 30mg/kg. Levels of Hp at 24 hours post surgery were 2.15 0 .46, 3.83 1.79, 4.44 1.78, and 3.47 1.65 times higher actin for 0, 5, 15 or 30mg/kg epicatechin respectively (Figure 3 1). Statistical analysis revealed that 15mg /kg at 24h was significant ( P =0.023) as compared to vehicle, with both 5 and 30mg/kg failing to reach significance ( P =0.062 and 0.9 respectively). Levels of Hp in li ver homogenates at 48 hours were 0.75 0.39, 1.48 actin for 0, 5, 15, or 30mg/kg epicatechin respectively (Figure 3 1). Statistical analysis revealed neither 5 nor 15mg/kg reach significance ( P =0.10 and 0.12 respectively) as compared to vehicle at 48 hours post surgery. However 30mg/kg did reach significance ( P =0.037) at 48 hours. Comparison between similar treatment groups at 24 and 48 hours revealed that with the exception of 30mg/kg mice all mice demonstrated significantly elevated Hp at 24 hours post craniotomy compared to 48 hour animals. Brain levels of Hp were significantly lower as compared to serum and liver levels at all doses and time points. Once again using densitometric analysis actin as a
40 loading control revealed that Hp was detectable in brain homogenates, however at no dose or time point did the expression reach significance over vehicle treated animals. Serum levels of Hp were significant at all doses and time points with P <0.05. Comparison between similar doses and 24 and 48 hours revealed that levels were not significantly different between time points with the exception of 30mg/kg that demonstrated higher levels at 24 hours. It should be noted that no loading contro l was used for the analysis so the data is in raw densitometric units. However, bicinchoninic acid assay was run on all serum samples and 10g of total protein was loaded for all serum samples T his allowed for crude comparison between treatment groups. qRT PCR Analysis of endogenous Hp, CD163, and B iliverdin reductase B (Blvrb) expression Real time reverse transcription quantitative PCR ( qRT PCR ) results demonstrated that endogenous Hp expression within the brain is at relatively low levels at all doses and time points. Levels were normalized to TATA binding protein Expression of Hp within in the brain was significant at 5 mg/kg at 24 hours po s t surgery ( P =0.0076) however 15 and 30mg/kg did not reach significance at 24 hours as compared to vehicle. Brain Hp expression levels at 24h were 0.71 0.0 3 0 .78 0.03, 0.79 0.06, and 0.74 0.22 relative to TBP for 0, 5, 15, or 30mg/kg epicatechin respectively Expression between epicatechin doses at the same time point did not reveal any significant increase correlated with an increase in dose. Expression of Hp failed to reach statistical significance at 48 hours for all doses compared to vehicle. Additionally comparison of expression of Hp with dose across time points revealed that at 48 hours, all doses incl uding vehicle were significant compared to 24 hours expression levels. All expression levels wer e normalized to TATA binding protein (Fig ure 3 2).
41 Expression levels of CD163 within the brain were not significant at all doses and time points as compared to vehicle with the exception of 30m/kg at 24 hours (p=0.018). Additionally comparison of expression of CD163 with dose across time points demonstrated that at all doses with the exception of 30mg/kg 48 hours had higher expression levels as compared to 24 ho urs (Fig ure 3 2). Endogenous expression levels of Blvrb within the brain were significantly elevated at 24 hours for all doses excluding 15mg/kg. However all expression levels at 48 hours were not significant as compared to vehicle. Comparison of express ion of Blvrb with dose across time points revealed that all doses at 48 hours had significantly higher levels of Blvrb. Additionally comparison across doses in similar time points revealed no significant increase in Blvrb with dose (Fig ure 3 2). Pretreatm ent with E picatechin Low est E ffective D ose to I nduce Hp P rior to ICH Wildtype and Nrf2 / C57BL/6 mice were orally administered 0.2mL 0 or 15mg/kg of epicatechin dissolved in 1% methylcellulose 24 hours prior to experimental induction of autologous ICH. T his dose and timing were chosen as it represented the lowest effective dose to upregulate Hp in the brain and periphery at 24 hours ( F igure 3 1). Dosing at 24 hours was chosen to mitigate the direct antioxidant effects of epicatechin and investigate the pr otective effects through Hp and Nrf2. Mice were survived for 72 hours and then sacrificed. Behavioral testing was performed every 24 hours post ICH. Mice were transcardially perfused with PBS and liver collected prior to perfusion with PFA for western anal ysis of Hp levels. Brains were harvested after perfusion with PFA, post fixed and cryosectioned for histological and immunohistochemical staining.
42 Epicatechin E ffects on Histological O utcomes Epicatechin administered 24 hours prior to ICH significantly re duced lesion volume at 72 hours, in WT mice, 15.27 7.02 and 7.04 2.73 mm 3 for vehicle and epicatechin treated mice respectively (Figure 3 3). The protective effect of epicatechin was ablated in Nrf2 / mice, lesion volumes were similar regardless of tre atmen t, 10.01 4.97 and 9.65 5.84 mm 3 for vehicle and epicatechin treated mice Nrf2 / respectively. Vehicle treated WT and all Nrf2 / mice had similar lesion volumes. Additionally, animals receiving autologous ICH sham demonstrated significantly smalle r lesion s as compared to all animal groups receiving ICH. Mortality during surgery was 2 1 %. Western Analysis of Hp Post ICH Hp was readily detectable in all liver homogenates post ICH. Administration of epicatechin 24 hours prior to ICH significantly incre ased Hp levels in the liver in WT animals (0.31 0.58 and 0.47 0 actin for vehicle and epicatechin treated WT mice respectively). The effect was ablated in Nrf2 / mice with no significant difference in Hp levels in treated versus vehicle. Vehicle treated WT and all Nrf2 / mice demonstrated similar le vels of Hp expression within the liver (Fig ure 3 7). Functional Outcomes Hp was inducible in a time and dose dependent manner and WT mice treated with 15mg/kg demonstrated significantly reduced lesion volumes as compared to vehicle treated WT mice and all Nrf2 / mice. However at no time point did NDS become significant between any group receiving ICH (Fig ure 3 4). Wildtype animals receiving needle insertion had significantly reduced NDS compared to all animals receiving ICH. The same trend was demonstrate d with grip strength and adhesive removal (Fig ure 3 5 and Figure 3 6).
43 Discussion Intracerebral hemorrhage is a devastating neurological event that is associated with high early mortality and disability as compared to other forms of stroke. The release o f blood and free Hb into the brain parenchyma generates a cascade of oxidative events that culminate in neuronal and glial cell death. Free Hb is thought to be the primary in itiator of the oxidative cascade. O ne important mechanism to mitigate this toxicit y is through binding to Hp and subsequent metabolism of the complex via CD163. quickly overwhelmed with even minor hemoly tic events. 6 Current treatments for ICH focus on palliative care, mechanical evacuation of the hematoma and/or physical rehabilitation; there is a serious lack of treatments that focus on the underlying molecular cause of pathology. 3 The purpose of this study was to determine the minimum effective dose of the natural compound epicatechin to induce Hp expression and elucidate the mechanism behind the upregulation of Hp, and test whether the minimum effective dose could provide neuroprotection through upregulation of Hp. To establish a minimum effective dose we administered three different doses of epicatechin in wildtype animals receiving sham operation and compared Hp expression in brains, liver, and the serum to wildtype vehicle treated animals receiving sham. Previous studies have shown that Hp is inducible with the natural compound sulforaphane when administered after ICH indu ction 7 Deletion of transcriptional factor Nrf2 ablates the protective action of epicatechin and leads to exacerbated injury in both ischemic and hemorrhagic stroke m odels 56 57 However, there have been no studies investigating the role of Hp in epicatechin induced protection in I CH.
44 In this study, we obtained results that were consistent with those of previous studies. Levels of Hp in the brains of vehicle treated mice were found to be significantly lower compared to both serum and liver levels (Fig ure 3 1). In addition, the lev el of Hp RNA expression within the brain of vehicle treated mice was found to be low and detectable but significantly below that of Blvrb (Fig ure 3 2). This supports the findings that Hp synthesis within the brain is very limited and not high enough to com pensate for severe hemoly tic events. Both the qRT PCR and western analysis of brain levels of Hp corroborate each other. However, we found that brain levels of Hp protein and RNA showed a trend toward toxicity at 30mg/kg with decreased levels at both 24 an d 48 hours post surgery. This trend toward brain toxicity highlights the importance of determining the lowest effective dose to mitigate toxic effects of epicatechin. In addition, as opposed to the clear dose dependent induction of Hp in the liver and seru m, the levels of Hp did not show a clear dose dependent increase in RNA levels within the brain. This suggests that the low level of Hp expression is regulated more through the inflammatory signals rather than under the control of epicatechin within the br ain, however this does not appear to be the case in the periphery (Fig ure 3 1). While the qRT PCR data showed a lack of clear dose dependent increases in Hp, the western blot analysis of brain Hp levels showed a trend towards a dose response, but at no do se or time point was the level significant compared to vehicle (Fig ure 3 2). One explanation for this trend is from peripheral Hp leaking across the blood brain barrier. The size of the Hp protein and previous studies suggest that Hp is able to cross the i ntact blood brain barrier 6 This aspect makes any compound that increases Hp in the periphery a potential therapeutic in events of hemolysis like ICH, because of the increased Hp in the brain prior to hemorrhage as well as flowing in with
45 extravasated blood serving to increase the brains Hb binding capacity. Another possibility for the trend towards a dose response is the contamination of the homogenate from Hp that fa iled to be washed out of vessels. While this is a possibility due to the large concentration of Hp within circulation, it is not likely the case here as the time and volume of PBS the mice were perfused with would have diluted or washed out the remaining H p in the vessels. No studies have been conducted indicating the mechanism of epicatechin induced upregulation of Hp, we tested the hypothesis that epicatechin is able to upregulate Hp through transcriptional factor Nrf2. The administration of epicatechin in sham operated animals, was able to induce upregulation of Hp in a time and dose dependent manner, in both the liver and serum samples. Overall, we observed a dose response at all doses in serum and liver samples, however the increase in Hp expression only became significant at 15mg/kg 24 hours post surgery. The large increase in expression of Hp compared to vehicle treated animals at 24 hours suggests that epicatechin is a potent activator of Hp expression in the periphery and that the mechanism of act ion is not likely direct action, as the stoichiometry is far too low to explain the dramatic increase. No study has investigated the effect of epicatechin pretreatment to induce Hp on the outcome post ICH. We tested the hypothesis that epicatechin mediat ed neuroprotection is mediated through upregulation of Hp via transcriptional factor Nrf2. Pretreatment of WT mice 24 hours prior to ICH with 15mg/kg significantly reduced lesion volumes as compared to vehicle treated wildtype and Nrf2 / mice. Additionall y, the protective effect was ablated in Nrf2 / treated with epicatechin, both Nrf2 / treatment groups had similar lesion volumes (Fig ure 3 3). This data is supportive of previous
46 studies that demonstrated Nrf2 is essential for the neuroprotection in ICH. Most importantly, this data supports previous findings that the mechanism of epicatechin neuroprotection is mediated through transcriptional factor Nrf2. The choice to administer epicatechin 24 hours prior to ICH demonstrated reduced lesion volumes in WT mice but not Nrf2 / mice. Our data provides further evidence that epicatechin neuroprotection is mediated through Nrf2 instead of direct antioxidant actions because 24 hours after administration the majority of the epicatechin would have been metabolized. However, this does not rule out the possibility of long lived active metabolites that could confer protection through direct antioxidant pathways Although this is unlikely, as demonstrated by the lesion volumes in Nrf2 / mice treated with epicatechin. A potential future direction for this study would be the investigation of epicatechin and its metabolite levels in the brain and the periphery using various analytical techniques that would allow for identification of metabolites and concentration within br ain tissue Pretreatment with epicatechin 24 hours prior to ICH was able to induce Hp expression and elevate Hp levels in WT mice up to 72 hours post surgery or 96 hours post epicatechin administration. Haptoglobin levels in epicatechin treated WT mice w ere significantly elevated as compared to vehicle treated WT and all Nrf2 / mice receiving ICH and vehicle treated WT shams. This suggests that a single acute dose is able to increase the reservoir of Hp to the point that it remains elevated up to 72 hour s after ICH, this is important because it indicates that a single acute oral dose is able to significantly increase the amount of Hp and therefore the Hb binding capacity. The reduced lesion volume in WT epicatechin treated mice in combination increased Hp up to 72 hours suggests that the mechanism of neuroprotection is through the upregulation of Hp via Nrf2. However, one limitation of this study is the fact that WT vehicle and all
47 Nr2 / mice displayed similar levels of Hp expression. This suggests that H p expression can be regulated by mechanisms other than Nrf2. This is supported by previous studies that demonstrate that Hp can also be regulated through interleukin 6. A potential future direction would be to investigate the role of epicatechin in inducin g Hp through other transcriptional factors. In addition, all Nrf2 / mice demonstrated a nonsignificantly smaller lesion volume. This data is contrary to other studies conducted in Nrf2 / mice, which demonstrated that Nrf2 is necessary for protection and knockouts display enlarged lesion volumes in both ischemic and hemorrhagic models. A likely source for this slight variation is in surgical technique between days. Another potential reason may involve some form of toxicity of the vehicle, which is mediated through Nrf2, however, this is unlikely as Nrf2 is responsible for cytoprotective pathways. The fact that none of the functional outcomes reached significance at any time point or dose highlights one of the limitations of conducting preclinical research in rodent models. One cause of this is the fact that while the epicatechin dosing regimen can significantly reduce histological outcomes it cannot confer enough protection to increase functional outcomes. Previous studies in ischemic stroke with epicatech in suggest that 15mg/kg is able to provide neuroprotection and improve functional outcomes. Another limitation that this data highlights is the potential for spontaneous recovery. Spontaneous recovery could be that rodents, in particular mice, have vastly different brain compositions regarding the white matter of humans and small animals and also the faster resolution of edema and inflammation in rodents. However, we only investigated functional outcomes up to 72 hours post surgery, while hematoma resolutio n has begun by 72 hours, it is possible that the increased Hp as a result of
48 epicatechin has its effects at farther time points after surgery. A potential future direction would be to inve stigate functional outcomes after 7 days or after 28 days. In this study, we used needle insertion without any infusions of inert fluids, while this controls for the deficits induced by the needle and is an accepted method of controlling for ICH. Simply inserting a needle is not the best method for controlling for the ad ditional effects of the blood that is injected. However, extensive studies as to the best solution to inject so as to minimize destruction of tissue or increased inflammation have not been performed. A future direction for this study would be the determina tion of a composition of fluid that most closely matches brain ionic concentration and minimizes inflammation.
49 Figure 3 1 Epicatechin upregulates Hp in a dose dependent manner. A ) Effect of epicatechin on haptoglobin level s in the brains of sham operated WT mice with respective B actin controls. B ) The effect of epicatechin on the haptoglobin protein levels in the livers of sham mice with respective B actin. Epicatechin was given orally at 0, 5, 15, and 30 mg/kg (n=4 per do se). Mice were sacrificed at 24 hours (n=8) and 48 hours. (n=8). C ) the effect of epicatechin on Hp levels in serum D ) Relative densitometric analysis of Hp ex actin. MeanSEM. B. A. C. D.
50 Figure 3 2. Real time reverse transcription quantitative PCR analysis of brain mRNA expressio n levels of Hp, CD163, and Biliv erdin reductase B (Blvrb) relative to TATA binding protein mRNA expression level.
51 F igure 3 3. Epicatechin treatment reduces lesion volume. A ) Cresyl violet (CV) stained brain sections obtained after autologous blood induced ICH in vehicle or 15mg/kg epicatechin in WT mice (n=4 for both groups). CV stained brain section obtained after autologous blood induced ICH in 15mg/kg epicatechin dosed Nrf2 / mice (n=5). CV stained brain section obtained after autologous blood induced ICH in vehicle dosed Nrf2 / mice CV s tained needle insertion sham controls (n=3). B ) Bargraph of lesion volume. Infusion of 30L of blood produces a large hematoma of similar size in vehicle WT and Nrf2 / mice, and epicatechin treated Nrf2 / mice. MeanSEM. B.
52 F igure 3 4. Neurological deficit score after ICH. An investigator blinded to dosing assessed the neurologic deficits of mice with a 24 point neurological deficit scoring system at 24, 48, and 72 hours No significant differences in NDS were observed betwee n vehicle and epicatechin treated mice. Mean SEM.
53 Figure 3 5. Adhesive removal time after ICH in treated animals. A ) An investigator blinded to dosing assessed the adhesive removal time of mice at 24, 48, and 72 ho urs No significant differences in grip strength were observed between Hp expressing mice and control mice. B ) Bargraph of adhesive removal time for right or left forepaw assessed at 24, 48 and 72 hours. No significant difference in removal time was observ ed cross group same limb. A significant difference was observed in all groups receiving ICH between left and right forelimb removal time. MeanSEM. A. B.
54 Figure 3 6. Grip strength after ICH in epicatechin treated anim als. An investigator blinded to dosing assessed the grip strengths of mice with a forelimb force gauge system at 24, 48, and 72 hours. No significant differences in grip strength were observed between vehicle and epicatechin treated mice
55 A. Hp chain actin B. Figure 3 7. Epicatechin significantly upregulates Hp post ICH in WT animals but not Nrf2 / animals. A) Representative blot of Hp in livers of epicatechin or Vehicle treated WT and Nrf2 / actin cont rols. B) Histogram of relative densitometric analysis of Hp levels in livers of treated mice and shams. Liver Hp Relative Density
56 CHAPTER 4 AAV INDUCED HP OVEREXPRESSION IN WILDTYPE MOUSE BRAINS Results Hp V ector P lasmids T rans fect HEK 293 C ells in vitro and I nduce Hp E xpression Human embryonic kidney cell cultures were trans fected with plasmids expressing Hp, Hp eGFP or Hp V5. Cultures were allowed to grow to confluence to insure efficient production of the transgene Hp was readily detectable in all samples of HEK cells transf ected with Hp expressing plasmids while eGFP trans fected cells did not display detectable levels of Hp in homogenate or media (Fig ure 4 1) AAV I nduced O verexpression of Hp in Animals S ubjected to ICH Neonatal WT C57BL/6 mice were injected intracerebrov entricularly with AAV1 expressing Hp, Hp V5, Hp eGFP or eGFP A dditional control mice received 1x PBS or no injection but were cryoanesthetized. Mice injected with AAV eGFP that were sacrificed at 3 months without receiving ICH displayed efficient and stab le expression of the transgene (Fig ure 4 2). The mean lesion volumes wer e as follows 2.03 0.65, 4.32 1.39, 4.47 3.18, 13.28 10.98, 3.33 1.06, and 8.71 5.42mm 3 for AAV Hp, AAV HP V5, AAA Hp eGFP, eGFP, 1x PBS, and nav e animals respectively (Fig ure 4 3 ). All mice treated with Hp expressing AAVs showed reduced lesion volumes compared to eGFP and nave animals H owever, animals receiving 1x PBS also demonstrated re duced lesion volumes (Figure 4 3 ). Among Hp constructs, AAV Hp mice displayed the mos t reduced lesion volume compared to Hp eGFP or Hp V5. Mice who received AAV Hp had significantly reduced lesion volume compared to non injected mice.
57 F unctional Outcomes AAV1 expressing Hp was able to significantly reduce ICH lesion volume 3 months after ICV injection in neonatal C57BL/6 mice. All Hp expressing constructs reduced lesion volume compared to non injected or nave animals receiving ICH. All Hp expressing constructs also demonstrated reduced neurological deficits compared to controls H owever only AAV Hp injected mice at 48 hours reached statistical signifi cance compared to nave animals. The mean scores for neurological deficits in the AAV Hp injected mice averaged between the two observers were 7.0 1.0 0 and 9.75 2.21. This significance in NDS between AAV Hp injected mice and nave mice was ablated at 72 hours post surgery (Fig ure 4 4 ). Forelimb grip strength of all mice receiving Hp expressing constructs failed to reach significance at any time point post compared to eGFP, nave or PBS a nimals receiving ICH. Average grip strength for all groups with the exception of AAV Hp receiving mice returned to baseline values within 24 hours after surgery (Fig ure 4 5 ). The sensorimotor adhesive removal also failed to produce significant results foll owing ICH among all groups and time points. However, a significant difference in removal time between right and left limbs was observed in all groups and at all time points post ICH ( F ig ure 4 6) Discussion The binding of free hemoglobin following a hemo lytic event in the brain is of paramount importance in order to prevent devastating oxidative stress and neuronal loss 57 bind free hemoglobin is several thousand fold lower than the binding capacity of the periphery 6 While natural compounds have been shown to upregulate Hp the event is only transient lasting as long as the compound or its active metabolites persist s 7 The ability of natural
58 compounds, such as epicatechin, to upregulate Hp is medi ated through complex signaling cascades such as transcriptional factor Nrf2. H owever, the upregulation of this cascade and the subsequent upregulation of Hp can be delayed by several hours before any effects are seen 57 84 Therefore, a mechanism that can produce large amounts of Hp within the brain relatively rapidly and for a prolonged period of time is key to develop ing a potential therapeutic for ICH and other intracranial hemolysis events. Gene therapy with AAV represents one possible solution to the problem that pharmacologic approaches often have in regards to the delayed and transient effects of upregulating Hp a nd other cytoprotective molecules within the CNS. The purpose of this study was to determine if induction of widespread Hp overexpression within the brain via neonatal ICV injection could provide upregula t ed Hp expression for several months and confer im proved histological and functional outcomes P revious studies have suggested that hypohaptoglobinemia exacerbates injury in ICH possibly because of the buildup of free Hb and subsequent oxidative stress 7 The irreversible binding of Hp to Hb and subsequent catabolism leads to a sharp decline of Hp upon major hemo lytic events, with levels of Hp not returning to normal for 3 7 days 19 However, few studies have investigated the role of Hp overexpression in the brain as a way to increase the Hp reservoir to combat hypohaptoglobinemia and the supraphysological levels of free hemoglobin following ICH. Adeno associated virus serotype 1 was chosen because when injected ICV in post natal day 0 pups, it transduced to a large portion of neurons and maintained stable expression for an extended period.
59 In this study, our results were consisten t with previous studies that have suggested that Hp plays a pivotal role in mitigating neurotoxicity following ICH. We found that all constructs expressing Hp were able to reduce lesion following ICH. This finding supports the hypothesis that the binding o f free Hb by Hp is key to preventing excessive secondary damage following ICH. The lesion volumes in all Hp overexpressing mice showed smaller lesion volumes than those mice from the previous study were treated with epicatechin to induce upregulation. This evidence suggests that while having an increased reservoir of Hp is beneficial in both cases, having that reservoir already within the brain prior to the hemoly tic event confers additional protection. Additionally, the protection conferred in Hp overexpre ssing mice was most likely not due to some form of preconditioning by the virus or cryoanesthetization because the eGFP expressing mice and the mice receiving no post natal day 0 injections displayed large hematomas with volumes on par with those observed Nrf2 / and vehicle treated mice from the previous study. However, it should be noted that mice receiving PBS post natal day 0 injections displayed reduced lesion volumes; this could be because of the PBS preconditions the mice for the insult or that PBS p ost natal day 0 is neuroprotective. This finding is most likely due to experimenter error because the mice received ICH 3 months after ICV injection This experimenter error most likely occurred while performing ICH surgeries. Compared to epicatechin tre ated WT animals, all Hp overexpressing animals displayed reduced lesion volumes. Importantly, the mice expressing AAV Hp showed the most dramatic reduction in lesion volume while in the mice expressing the tagged Hp the reduction was not as dramatic. This highlights a potential limitation of using
60 tagged proteins, as the tag may interfere with the binding or activity of Hp. Previous studies have demonstrated that subtle changes in Hp tertiary and secondary structure produce a molecule that has very differen t characteristics. One such study found that modified Hp had lost almost all of its Hb binding capacity, but was essential for B cell maturation 85 The potential for the labeled Hp to have altered function is a serious issue that sh ould be investigated further. The failure of almost all of the groups and time points to show significant improvement in functional outcomes even in Hp overexpressing mice highlights the pitfalls of utilizing rodent model for ICH. As mentioned above, thi s could be due to spontaneous recovery due to differences in brain anatomy of rodents and their ability to heal at an expedited rate or to the fact that the overexpression of Hp has its primary effects on functional outcomes at later time points. A potenti al future study would be to survive the animals for an extended period following ICH and monitor functional outcomes as well as Hp levels from both endogenous synthesis and AAV induced expression. Several important future studies that arise from these f indings are to further confirm the role of Hp in neuroprotection foll owing ICH. One study to confirm this would be to conduct the study in Hp / animals via post natal day 0 ICV injection and induction of ICH in order to determine if the level of AAV Hp ex pression is enough to rescue the knockouts and to confirm that the mechanism of protection is in fact mediated through Hp. Additionally, the injection and efficient overexpression of Hp within the brain of adult mice would serve as an intriguing prospectiv e study, as ICV in neonatal humans is not feasible and neonates are not the at risk population. If feasible injection in adult brains
61 would better serve the goal of preventative medicine by supplying those individuals at risk for ICH with a large reservoir of Hp able to bind free Hb and mitigate toxicity. This could have profound effects on rates of mortality and morbidity following ICH. Another potential study would be to investigate the effect of Hp genotype on outcomes following ICH and whether the use o f Hp1 1 in Hp2 2 transgenic mice to see if both groups could in fact be rescued from the harmful effects following hemolytic events within the brain.
62 30 kDa 40 kDa 50 kDa 60 kDa Figure 4 1. Hp plasmid constructs are able to transfect HEK cells and in duce expression of transgene. Blot probed for Hp protein in media.
63 Figure 4 2. AAV1 is able to transduce neurons after neonatal injection and induce expression of transgene out to 3 months. Mice were intracerebro ventricularly injected with AAV1 eGFP on P0 and allowed to survive 3 months before sacrifice and brain collection. Green is eGFP and blue is DAPI. 20x magnification.
64 A Hp eGFP Hp Hp V5 PBS Nave B AAV eGFP Figure 4 3. Overexpression of Hp is able to reduce lesion volume. A ) Cresyl violet (CV) stained brain sections obtained after autologous blood induced ICH in AAV Hp eGFP, AAV Hp, AAV Hp V5, AAV eGFP, nave, and PBS animals respectively. B) Bar graph of lesion volume. Infusion of 30L of blood produces a large hematoma of similar size in control animals. All Hp expressing animals display reduced lesion volumes. MeanSEM.
65 Figure 4 4. Neurological deficit score after ICH in Hp overexpressing mice. An investigator blinded to dosing assessed the neurologic deficits of mice with a 24 point neurological deficit scoring system at 24, 48, and 72 hours No significant differences in NDS were observed between groups with the exception of AAV Hp and nave animals at 48 hours post surgery. Mean SEM.
66 Figure 4 5. Grip strength after ICH in Hp overexpressing mice. An investigator blinded to dosing assessed the grip strengths of mice with a forelimb force gauge system at 24, 48, and 72 hours. No significant diff erences in grip strength were observed between Hp expressing animals and controls. MeanSEM
67 Figure 4 6. Adhesive removal times after ICH in Hp overexpressing mice. A) An investigator blinded to genotype assesse d the adhesive removal time of mice at 24, 48, and 72 hours post ICH. No significant differences in removal times were observed between Hp expressing mice and control mice. B) Histogram of adhesive removal time for right or left forepaw assessed at 24, 48 and 72 hours. No significant difference in removal time was observed cross group same limb. A significant difference was observed in all groups receiving ICH between left and right forelimb removal time. MeanSEM. A. B.
68 CHAPTER 5 CONCLUSIONS In conclusion, e picatechin is able to induce Hp expression in a time and dose dependent manner and the increased expression is mediated through the Nrf2 makes it a potential therap eutic to help combat the complex mechanisms behind the pathology of ICH and therefore improve functional outcomes. The administration of a single acute dose of epicatechin is able to significantly reduce lesion volume in WT animals, but this protection is ablated in Nrf2 / mice. However, going forward to a dose that confers improved functional and histological outcomes at 72 hours or even later time points post surgery will need to be determined to increase the therapeutic potential of epicatechin. The abi lity of epicatechin to reduce lesion volumes and upregulate Hp by activating Nrf2, makes it a powerful tool that could be added to the current preventative regimen of individuals at risk. Overexpression of Hp via AAV1 was able to significantly reduce les ion volume in WT mice following ICH. AAV1 was able to effectively transduce neuronal cultures to overexpress Hp. The fact that AAV overexpression of Hp was able to significantly reduce lesion volumes suggests that gene therapy targeting expression of Hp wi thin the brain has extremely high potential to serve as a promising ther apeutic for intracranial hemolytic events, an arena that currently is limited to mostly palliative care. In the future, studies will need to be conducted to determine a time point at w hich overexpression of Hp within the brain confers improved functional outcomes following ICH and if that time point represents promise for reducing morbidity. The ability to
69 overexpress Hp within the brain holds great therapeutic promise in the tr eatment of ICH and other hemolytic events within the brain. Compared to epicatechin treated WT animals, all Hp overexpressing animals displayed reduced lesion volumes. Importantly, the mice expressing AAV Hp showed the most dramatic reduction in lesion volume whi le in the mice expressing the tagged Hp this reduction was not as dramatic. This finding highlights a potential limitation of using tagged proteins, as the tag may interfere with the binding or activity of Hp. Previous studies have demonstrated that subtle changes in Hp tertiary and secondary structure produce a molecule that has very different characteristics. One such study found that modified Hp had lost almost all of its Hb binding capacity, but was still essential for B cell maturation. 85 The potential for the labeled Hp to have altered function is a serious issue that should be investigated further. Overall, the data from both the epicatechin and AAV induced expression of Hp suggest that haptoglobin plays a pivotal role in the mitigation of hemoglobin derived neurotoxic events following ICH. The dramatic reduction in lesion volume seen in all AAV Hp expressing mice compared to epicatechin treated mice suggest s that having a reservoir of Hp present within the CNS prior to insult provides improved outcomes and disparity in lesion volumes between the epicatechin treated mice and AAV Hp expressing mice suggests that a large reservoir of Hp in the CNS serves as powerful agent against the deleterious effects of hemoglobin and ICH. Thus preloading the CNS with large amounts of Hp synthesized within the CNS is a more powerful therapeu tic than increasing peripheral Hp levels with only a slight increase in CNS Hp levels.
70 However, both epicatechin and AAV Hp were able to reduce lesion volume compared to control animals suggesting that any agent or therapy that is able to upregulate Hp per ipherally or centrally has the potential to mitigate the neurotoxicity of hemolysis event within the CNS and improve outcomes. All together, the data suggests haptoglobin plays a very important role in the mitigation of secondary injury following ICH, by the removal of free Hb. Increased Hp levels resulted in reduced lesion volumes post ICH. This suggests that the increased levels of Hp within the CNS are able to increase the Hb clearance capacity of the CNS post ICH. Additionally both the pharmacologic approach and the gene therapy approach for Hp upregulation within the CNS demonstrated a marked ability to reduce lesion volume. Therefore, this suggests that Hp itself is an important mediator of secondary injury following ICH and that it is necessary to mitigate the toxicity of free Hb. This suggests that Hp is a potential therapeutic for the treatment of hemolytic events within the CNS, and that both pharmacological and gene therapy approaches could be used to increase Hp levels within the CNS and have similar beneficial results on the outcomes following hemolytic events within the CNS.
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79 BIOGRAPHICAL SKETCH Sean Robbins was born in Jacksonville, Florida 1988 to Jon and Karen Robbins. His family moved to Deland, Florida when he was three. He grew up in Deland and educated through the Volusia county school system before attending the University of Florida. While at the UF, he pursued b 2007 to August 2013.