Vascular Targeting Enhancement of Radiosurgery for

Cerebral Arteriovenous Malformations

Rajesh Reddy M.B. B.S. F.R.A.C.S.

Prince of Wales Medical Research Institute

University of New South Wales

Submitted as requirement for the degree of Master of Surgery

University of New South Wales

July 2010

2 Abstract

Objective: Radiosurgical treatment of brain arteriovenous malformations (AVMs) has the significant shortcomings of being limited to lesions smaller than 3 cm diameter and of a latency to cure of up to 3 years. Stimulation of thrombosis using vascular targeting is attractive as a possible means of overcoming these limitations. This study examined the effect of the vascular targeting strategy lipopolysaccharide (LPS) and soluble tissue factor (sTF) in an animal model of AVM treated with radiosurgery.

Methods: Stereotactic radiosurgery (SRS, 20 animals) or sham radiation (12 animals) was administered to an animal model of AVM in 32 male Sprague-Dawley rats. Twenty- four hours after intervention, animals received an intravenous injection of LPS/sTF conjugate or normal saline. Animals were sacrificed at 1, 7, 30, or 90 days after treatment. Angiography was performed immediately prior to sacrifice and model AVM tissue was harvested for histological analysis to assess vessel thrombosis rates.

Results: There was no systemic toxicity or intravascular thrombosis remote from the target region in any of the animals. SRS combined with saline resulted in thrombosis of 12% of small AVM vessels (diameter < 200µm). Thrombosis occurred in 58% of small vessels in animals that received SRS and LPS/sTF. An intermediate thrombosis rate of

43% was observed in animals given the LPS/sTF agent but no radiation.

Conclusion: This study demonstrated that vascular targeting can increase intravascular thrombosis after radiosurgery and that the vessel occlusion is selective and durable.

i Declaration

Originality Statement ‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgment is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project’s design and conception or in style, presentation and linguistic expression is acknowledged.’

Rajesh Reddy

July 2010

ii

Acknowledgments

It is hard to write an acknowledgement without sounding cliched or resorting to well worn aphorisms. Be that as it may, the graditude and thanks I express are sincere.

The obstacles and trials I faced whilst undertaking this Masters project during my neurosurgical training were challenging.

It would not have been possible without the guidance, tutelage, and support of my supervisor - Marcus Stoodley. His constant presence, accessibility, and friendship, helped me perserve through times and thoughts that I might not be able to accomplish what I sought to achieve. He is a source of respect and admiration for his ability to excel as an academic and a surgeon.

I am also indebted to the key members of his lab. Jacob Fairhall, for his friendship, humour, and generally sharing the pain in the duality of research and surgical training.

James Tu and Sarah Hemley, also for their friendship as well as always being around to answer my many questions about lab work.

My partner, has also contributed to this work in many ways - the value of her relentless reminders as to complete the various stages of my project and thesis is unquestionable.

More so she has been at my side to encourage me, accept my mistakes, and make me feel proud whenever I have tried my best.

And lastly, my parents. They gave me my name, they gave me my education, and above all else they gave me my life. Words cannot describe my feelings to them and my appreciation of the efforts they have put into giving me the life I have now.

iii This study was funded in part by grants from the Neurosurgical Society of Australasia and the Brain Foundation.

Dr Reddy was supported by a grant from the Royal Australasian College of Surgeons

iv

Table of Contents

Vascular Targeting Enhancement of Radiosurgery for Cerebral Arteriovenous Malformations Abstract ...... i Objective: ...... i Methods:...... i Results:...... i Conclusion:...... i Declaration ...... ii Originality Statement ...... ii Acknowledgments...... iii Figures...... vii Tables ...... ix Introduction ...... 1 Clinical Overview ...... 1 The History of AVMs ...... 2 The Pathology of AVMs ...... 3 AVM Classification...... 10 AVM Treatment ...... 11 Radiosurgery ...... 12 History of Radiosurgery...... 12 Physics of Radiosurgery...... 13 Biology of Radiosurgery ...... 15 Radiosurgery Techniques and Current Devices...... 17 Role of Stereotaxy...... 21 The Effects of Radiosurgery on AVMs...... 23 Potential for Improvement of Radiosurgery...... 24 Vascular Targeting ...... 25 Potential for Vascular Targeting Strategies in AVMs ...... 27 Hypotheses ...... 30 Aims ...... 31 Materials and Methods...... 32 Overall experimental plan ...... 32

v Ethics and Animal care ...... 33 Morbidity and Mortality...... 34 AVM Model Creation ...... 34 Radiosurgery Dosimetry ...... 37 Radiosurgery Delivery Protocol...... 39 Administering TF and LPS ...... 40 Angiography...... 41 Perfusion Fixation ...... 43 Tissue Processing ...... 44 Staining - Haematoxylin and Eosin...... 45 Staining - Martius/scarlet/blue (MSB) ...... 45 Blood flow measurement ...... 46 Light Microscopy and Quantification of thrombosis ...... 46 Data and statistical analysis...... 47 Results ...... 48 Blood flow...... 48 Angiography findings...... 48 Histology ...... 49 Thrombosis rates ...... 51

Discussion ...... 65 R Conclusions ...... 74 References ...... 75 R

vi

Figures

Figure 1: A: Schematic diagram of the vasculature of the animal model, demonstrating proximal and distal carotid arteries (red outlined vessels) and jugular veins (blue vessels). B: Schematic diagram of the animal model of AVM, site of carotid-jugular anastomosis (inset) and AVM formation (purple vessels) 6 weeks after carotid-jugular fistula creation...... 32

Figure 2: Intraoperative image of the animal model of AVM, demonstrating proximal and distal carotid artery, jugular vein, and site of carotid-jugular anastomosis...... 37

Figure 3: Anaesthetised rodent in custom made stage attached to the head of the linear accelerator...... 38

Figure 4: Anaesthetised rodent in custom made stage. Insets demonstrate images from planning phase to localize the AVM nidus and plan treatment...... 39

Figure 5: Animal model of AVM, demonstrating carotid-jugular anastomosis (1) which creates an arterialised feeding vein (2), a nidus(3), and a draining vein (4). A: schematic diagram of model. B: xray image from carotid angiogram demonstrating the model AVM...... 43

Figure 6: Representative section from irradiated tissue treated with targeting agent from 90 day time point. Evidence of large vessel antemortem thrombus (H&E; original magnification, × 10)...... 50

Figure 7: Intravascular thrombosis in the model AVM nidus (percentage of vessels), by treatment group in vessels smaller than 200 µm (LPS, lipopolysaccharide; sTF, soluble tissue factor; RTX, radiosurgery)...... 51

Figure 8: Representative section from irradiated tissue treated with saline from 7 day time point. No evidence of thrombus formation, nor evidence of radiation effect (H&E; original magnification, × 40)...... 52

Figure 9: Representative section from irradiated tissue treated with saline from 30 day time point. No evidence of thrombus formation. Evidence of radiation effect, with proliferation of subendothelial tissue and intimal hypertrophy (H&E; original magnification, × 40)...... 53

Figure 10: Representative section from irradiated tissue treated with saline from 90 day time point. Evidence of thrombus formation and evidence of radiation effect (H&E; original magnification, × 40)...... 54

vii Figure 11: Representative sections from non-irradiated tissue treated with LPS and sTF from 7 day time point. Evidence of thrombus formation. No evidence of radiation effect (H&E; original magnification, × 40) ...... 55

Figure 12: Representative section from non-irradiated tissue treated with LPS and sTF from 30 day time point. Evidence of thrombus formation. No evidence of radiation effect (H&E; original magnification, × 40) ...... 55

Figure 13: Representative section from non-irradiated tissue treated with LPS and sTF from 90 day time point. Evidence of partial vessel occlusion from antemortem thrombus – potential evidence of recanalisation. No evidence of radiation effect (H&E; original magnification, × 40) ...... 56

Figure 14: Representative section from irradiated tissue treated with LPS and sTF from 7 day time point. Evidence of small vessel occlusion from antemortem thrombus, and from post- mortem clot. No evidence of radiation effect (H&E; original magnification, × 40)...... 57

Figure 15: Representative section from irradiated tissue treated with LPS and sTF from 30 day time point. Evidence of small vessel thrombosis. (H&E; original magnification, × 40)...... 58

Figure 16: Representative section from irradiated tissue treated with LPS and sTF from 30 day time point. Evidence of small vessel occlusion and radiation effect (H&E; original magnification, × 20) ...... 59

Figure 17: Representative section from irradiated tissue treated with LPS and sTF from 90 day time point. Evidence vessel thrombosis (H&E; original magnification, × 20)...... 60

Figure 18: Representative sections from irradiated tissue treated with LPS and sTF from 30 day time point. Evidence of small vessel occlusion from antemortem thrombus (MSB; original magnification, × 40) ...... 61

Figure 19: Representative sections from irradiated tissue treated with LPS and sTF from 90 day time point. Evidence of small vessel occlusion from antemortem thrombus (MSB; original magnification, × 20) ...... 62

viii

Tables

Table 1: Doppler flow (mL/min) through the arterialised jugular vein before and after treatment...... 48

Table 2: Intravascular thrombosis in the model AVM nidus (percentage of vessels), by treatment group in vessels smaller than 200 μm...... 51

ix

x Introduction A new method for treating brain arteriovenous malformations (AVMs) is needed. The research reported here is aimed at contributing towards developing such a treatment. An overview of AVMs is provided, followed by discussions of radiosurgery and vascular targeting, which are the subject of the experiments reported in this thesis.

Clinical Overview AVMs of the brain are a leading cause of non-traumatic intracranial haemorrhage in children and young adults. AVMs are abnormalities of intracranial vessels, consisting of fistulous connections between the arterial and venous vasculature without an intervening capillary bed. A tangled collection of vessels is formed within the brain parenchyma and is referred to as the ‘nidus’.

Whilst reports of AVM prevalence range from only 10 – 500 per 100,000, (0.01% to

0.5% of the population),[1-3] AVMs are the most common cause of neurological impairment or death in patients younger than 20 years.[4-6] The gender distribution of patients is equal. Lesions are usually solitary but rarely may be multiple, and also may be associated with other types of vascular malformation, such as venous and arterial aneurysms.[7]

AVMs may present with seizures, headaches or focal neurological deficits. However, the clinically most important and most common presentation is due to haemorrhage, with an incidence of 2-4% per year.[2, 8]

Haemorrhage may be intraparenchymal, intraventricular or subarachnoid,[9, 10] and carries a considerable short term mortality rate of 10-29%,[8, 11] and a risk of permanent morbidity ranging from 14-85%.[2, 12] Ondra et al did not did not find that AVM haemorrhage was more common in patients who had suffered a prior haemorrhage,[2] 1 however more recent studies have found the risk of haemorrhage is higher when a patient has had a previous bleed.[13-15]

The clinical problem of AVM, in addition to individual suffering, is also a significant impact on the healthcare system. It is a condition that has potential long term risks of recurrent intracranial haemorrhage, permanent neurological deficits, epilepsy, and attracts expensive interventional treatments and need for rehabilitation services.

The History of AVMs Acute neurological events such as “apoplexy,” some of which would have been attributable to AVMs, have been described since antiquity. The documentation of such events has been seen in the writings of Hippocrates and Galen,[16] and there is even evidence of such events in the Edwin Smith Papyrus.[17] Whilst subsequent anatomists and physicians were aware of the gravity of apoplexy as described by Galenic doctrine, it was not until significantly later that the underlying pathology would be identified.

William Hunter has been credited[18] for providing the early concepts of AVM in his

1762 monograph documenting extracranial AVMs. A century later Rokitansky first comprehensively described angiomas of the intracranial cavity, though he considered these as highly vascular tumours. Virchow, based on his extensive pathological studies, in his 1862 publication ‘Die Kranhaften Geschwulste’[19] detailed an attempt at classifying AVMs. Virchow also speculated that whilst a small number of angiomas were neoplastic, the majority represented a vascular anomaly.

In the 1920s Cushing[20], [21], Bailey[20], [22] and Dandy[23] further refined the understanding of CNS vascular anomalies, and set the scene for our current understanding of AVMs.

2 Another landmark event in the management of AVM also occurred in the 1920’s with advent of cerebral angiography.[24], [25] Benefiting from the advances in angiography,

Olivecrona was one of the first neurosurgeons to methodically excise AVMs. More importantly, Olivecrona described the use of cerebral angiograms to systematically assess and characterise the angioarchitecture of AVMs.[26], [27]

Progress continued with developments in neuroradiology, such as the computed tomography (CT) scanner, magnetic resonance imaging (MRI) and functional mapping.

Additional advances in surgical tools such as the operating microscope, image guided stereotaxy, microvascular techniques, and endovascular adjuncts have improved the outcomes in treatment of AVMs. Despite these advances, a population of patients remains for whom no effective treatment can be undertaken safely. AVMs that cannot be effectively treated will be described below in the section on AVM classification.

The Pathology of AVMs Thus far, therapeutic decision-making has been based on an understanding of the natural history, clinical setting, and the gross anatomical pathology of AVMs. Fundamentally, however, treatment strategies are aimed at either removing or occluding the abnormal vessels.

Whilst the macroscopic anatomy, histopathology, and angioarchitecture of AVMs are now well known, it is only recently that attention has been paid to study the ultrastructural, molecular, and genetic basis of AVMs.

Normal Vascular Morphology The vascular system, despite some regional and organ differences, has a common histological organization. Vessels consist of three histologically distinct layers, the tunica intima, the tunica media, and the tunica adventitia. Each layer contains variable

3 amounts of smooth muscle cells and elastin. The variability in cellular constituents is determined early in development and is based on the physiological function that the vessel serves to an organ or tissue.[28]

The tunica intima, the thinnest constituent layer, consists of a single layer of endothelial cells mounted on a basement membrane. The basement membrane contains the extracellular matrix proteins fibronectin, laminin, and Type IV collagen, which provide structural support. Below this exists a sub-endothelial fibro-elastic connective tissue layer and an organized layer of internal elastic lamina that provides flexibility and stability for endothelial cells. Also in close apposition to the endothelial cells are perivascular cells or pericytes.

The tunica media contains predominantly smooth muscle cells and elastin fibers. The media is rich in extracellular matrix protein, Type III collagen, and various vascular smooth muscle cell proteins. These cell layers tend to be more highly organized in larger arteries. In most organs the media also contains an external elastic lamina that provides structural support. The external elastic lamina is not present in cerebral vessels.

The last and outermost layer of the blood vessel is the tunica adventitia that is almost entirely composed of fibro-elastic connective tissue, principally of longitudinally oriented Type III collagen fibres. Both lymphatic and nerve plexi are present in the adventitia along with the vasa vasorum in larger vessels.

Elastic arteries are present near the heart and other organs and are associated with the movement of large volumes of blood. These types of arteries, such as the aorta, are composed of many layers of perforated elastic membrane and are therefore particularly adapted to accommodate large changes in blood volume. The highly elastic nature of the

4 walls of these blood vessels functions to effectively dampen the large oscillations in blood flow and provide for a more homogeneous movement of blood away from the heart.

Most arteries in the body are classified as muscular. The function of muscular arteries is to ensure the rapid and complete distribution of blood to all organs and tissues. While the walls of these arteries are predominantly muscular in nature, histological studies show that they contain discontinuous elastic fibres within the large layer of smooth muscle cells.

The continued bifurcation of arteries leads to the smallest vessels of the arterial tree: arterioles, whose function is to reduce flow from the larger vessels. This reduction in flow is necessary to prevent damage to the capillaries.

The arterial vascular system transitions to the venous system through the capillary network. The capillary is marked histologically by a lack of smooth muscle; consisting of a single layer of endothelial cells, a basement membrane, and enveloped by pericytes.

The majority of nutrients, solutes, and water are exchanged between blood and the surrounding tissues across the surface area of the capillary bed.

The return of blood to the heart via the venous system begins with its movement into the postcapillary venules, which coalesce to form larger veins. The transition from capillary to vein is marked by the gradual re-appearance of smooth muscle cells in the tunica media of the vessel wall and a layer of collagen and elastic fibres in the adventitia.

The size of the veins increases as the vessels approach the heart, and some veins such as those located in the lower limbs of the body contain uni-directional semi-lunar valves

5 that prevent blood from pooling in the extremities. Both small- and medium-sized veins acquire a greater level of smooth muscle cells in their tunica media while large veins have an increased amount of connective tissue in this layer.

Cerebral Vascular Morphology Cerebral blood vessels (like peripheral blood vessels) are composed of a tunica intima, media, and adventitia. However, there are a number of properties by which the cerebral vasculature differs from the systemic vasculature. These include the existence of the blood-brain barrier, blood-cerebrospinal fluid barrier, and a valveless venous system.[29]

Cerebral endothelial cells (and their interconnections of tight junctions) are the main anatomic site of the blood brain barrier, assisted by the surrounding pericytes and ensheathing astroglial processes.[29] Cerebral endothelial cells also show differences in their morphology and growth characteristics, ET-1 release, and responsiveness to a variety of mediators.[30]

Cerebral arteries have a well-developed internal elastic lamina, a paucity of elastic fibers in the media, little adventitial tissue, and lack an external elastic lamina.[31] It has also been shown that intracranial vessels lack vasa vasorum, which due to the vessel wall thickness, indirectly suggests that these vessels gain their metabolic requirements from sources other than the main vascular lumen.[31]

AVM Morphology AVMs are considered congenital abnormalities consisting of a vascular ‘shunt’ directly from the arterial to draining venous circulation without an intervening capillary bed.[32]

Although macroscopically AVMs may vary considerably in size and appear to be a disorganized, abnormal tangle of vessels, all AVMs have three primary components: feeding arteries, a nidus, and draining veins.[27, 33, 34]

6 Arterial feeders vary in number and may arise from superficial supply alone, deep supply alone, or combined deep and superficial vessels.[35] The superficial supply vessels include cortical branches of the anterior, middle, and posterior cerebral arteries, and/or arteries arising from the vertebrobasilar system. The deep supply vessels arise from choroidal or perforating arteries.[34, 35]

The nidus is the central portion of the AVM in which the arteriovenous shunting occurs as the multiple feeding arteries converge, and from which dilated veins emerge.[27]

The venous drainage incorporates one or more enlarged veins, which may drain into superficial dural venous sinuses, into the deep venous structures, or into both systems.[27]

Typically, no functionally important brain tissue is found within the AVM nidus,[36, 37] and it is thought that there is functional displacement to the margin of the AVM, as distinct from cortical reorganization that occurs secondary to acute cerebral lesions.[38]

The surrounding parenchyma may appear normal or demonstrate gliosis, as a result of the AVM shunt "stealing" blood away from this tissue.[39] The adjacent tissue may also contain haemosiderin as a result of prior haemorrhage.[27]

Histology and Ultrastructure of AVMs AVM vessels are abnormal, with abrupt transitions between vessels that may range from relatively well differentiated arteries and veins to malformed vessels apparently neither artery nor vein.[39] The size of the vessel lumen varies, and ectactic segments and aneurysm formation are also seen.[27, 34, 39]

AVM arteries have variable amounts of smooth muscle and fibroblast proliferation which contribute to the irregular thickening of the walls.[27, 39] The elastic lamina shows

7 irregularities, and may be duplicated or absent. [27, 39, 40] Veins have an arterialised appearance due to thickened walls as result of fibroblast proliferation and increased cellularity.[27]

Irregular nodules of hyalinized intima and smooth muscle project into the vessel lumen, islands of sclerotic tissue, endothelial thickening, hypertrophy of the media, or even focal disappearance of the media altogether make distinction between arteries and veins difficult.[33, 39, 40]

Within the arteries and veins, evidence of prior thrombosis and recanalization may be evident.[27] Of uncertain significance, beta-amyloid protein deposition has also been reported in AVMs.[27, 41]

Just as the macroscopic and microscopic angioarchitecture of AVMs shows differences from normal vasculature, so too does the ultrastructure. Electron microscopic analysis of vessels from the AVM nidus demonstrates an underlying matrix of disorganized collagen bundles and smooth muscle cells.[42] Intact pericytes are present in AVM vessels, and the feeding arteries of the AVM showed that the tunica intima, media, and adventitia have normal ultrastructure.[42]

AVM endothelial cells show resemblance with normal vasculature, though in areas of

AVMs that had been embolised, the cells are disrupted. Interendothelial cell junctions show similar tight junctions when AVM vessels are compared to normal vessels.[42]

Tu et al.[37] identified a number of differences between AVM perinidal vessels compared to normal cerebral vessels. Abnormalities were demonstrated in the AVM perinidal blood brain barrier, with no evidence of basement membranes nor astrocytic foot processes. Endothelial cells were seen to have areas of separatation, fenestrated

8 processes, cytoplasmic vesicles and vacuoles, and lysosomes. Inflammatory cells and reactive tissue were found surrounding the perindal capillaries. Weibel-Palade bodies

(membrane-bound cytoplasmic organelles that contain selectins and von Willebrand factor)[43] were also detected in these vessels.[37]

Molecular Characteristics of AVMs Although it is generally believed that the majority of cerebral AVMs are congenital abnormalities, the clinical findings and behaviours of some lesion suggest an acquired component exists.[40] The detail of AVM pathogenesis is currently unknown, however identification of abnormal patterns of angiogenic and growth factors, as well as vessel structural and adhesion molecules suggest that AVMs might arise result of a disturbance of angiogenesis and vasculogenesis.[29, 40]

Vascular endothelial growth factor (VEGF) acts directly on endothelial cells to promote a range of important angiogenic processes, such as endothelial cell proliferation, migration, adhesion, and tube formation. A number of groups have shown strong VEGF expression in AVM endothelium and subendothelium with little or no expression in normal cerebral vessels.[44-49]

Angiopoetins, and their receptors (Tie-1 and Tie-2), have been shown to play a role in the later stages of vascular development.[50, 51] Sato et al. demonstrated that experimental embyros deficient in Tie 2 produce the formation of abnormal enlarged vessels without intervening normal capillaries, and that these abnormal vessels resemble human AVMs.[50]

Hashimoto et al. found that endothelial cell expression of VEGF-R and angiopoetin receptors in endothelial cells is significantly higher in surgically resected brain AVMs

9 than in normal vessels, suggesting that upregulatation of VEGF and Tie in AVMs indicates some ongoing angiogenesis.[48, 52-54]

Investigation of the expression of luminal surface and adhesion molecules, has shown mixed results. Storer et al. did not find any differences of expression of thrombotic molecules; thrombomodulin, tissue factor, and von Willebrand Factor in AVMs compared to normal cerebral vessels.[55]Cell adhesion molecules (CAMs), such as platelet endothelial cell adhesion molecule- 1 (PECAM-1), are important for leukocyte adhesion and transendothelial migration, as well as vasculogenesis.[56-58] Studies of

PECAM-1 expression have shown equivocal findings.[44, 59-61]

Hashimoto et al. demonstrated that intercellular CAM-1was upregulated in AVMs.[49]

Whilst Storer et al also demonstrated increased expression of intercellular CAM-1, as well as vascular CAM-1, this difference was not found to be statistically significant.[59]

Selectins are another group of important inflammatory mediators whose expression has been studied. E-selectin has been found to be significantly upregulated on the endothelium of AVMs.[59]

Identification of molecular characteristics will help to address a number of issues. It would be useful to determine the pathogenesis of AVMs, if these changes are as result of the abnormalities of the lesion, or arise as result of the haemodynamic forces of an abnormal circulation. The discovery of unique molecular characteristics would also allow new interventions and pharmacological methods to be developed to treat AVMs.

AVM Classification The classification of AVMs according to size, pattern of venous drainage, and neurological eloquence of adjacent brain allows stratification of treatment risk.[62] A

10 number of grading systems to classify AVMs have been described. The current most widely accepted system used is the Spetzler-Martin Grading Score.[61] They described a five point grading system, summing points assigned to three variables: size, location in eloquent brain, and the presence of deep venous drainage. Points are assigned for the

AVM size; 1 point if the nidus is less than 3 cm, 2 points if the nidus is 3-6 cm, and 3 points if the nidus is greater than 6 cm. If the location is eloquent, 1 point is assigned. If there is deep venous drainage, then 1 point is assigned.

The aim of the Spetzler-Martin grading system is to estimate the risk of surgery and determine operability. Low grade AVMs (I-II) account for approximately 40% of

AVMs and can be treated with low risk.[62, 63] One third of AVMs are high grade (IV-

V), and over 90% of these are untreatable without unacceptably high risk.[64, 65] Of the intermediate grade AVMs (III), many of these are also not treatable without high risk.

New treatments are needed for these patients.

AVM Treatment The goal of AVM treatment is to obliterate flow through the abnormal vessels and therefore eliminate the threat of intracranial haemorrhage, whilst maintaining normal circulation and preserving neurological function. Each of the current treatment modalities (microsurgical resection, endovascular therapy, and radiosurgery) has their own benefits and risks. Surgical resection is curative for small, superficial lesions in non-eloquent brain with low morbidity.[66-68] However, following decades of advances and refinement, the techniques of microsurgical resection are unlikely to improve substantially and surgical resection will always be an invasive treatment option. Whilst endovascular therapy can be considered minimally invasive, and in theory can achieve

AVM obliteration, this is technically possible in only in small number of cases and its main use is as an adjunct to surgical resection or radiosurgery.[69-71] 11 Radiosurgery has the benefits of low initial risk and can be administered on an outpatient basis. Obliteration rates of 70-80% have been obtained in treating AVMs of 3 cm diameter or smaller.[72, 73] The limitations of radiosurgery include latency to obliteration and decreased efficacy with larger AVMs.[74-84]

For AVMs larger than 3 cm, all current treatment modalities have drawbacks and very large AVMs are often untreatable. Potential advances in radiosurgery could offer a treatment option in these cases.

Radiosurgery Radiosurgery is a treatment modality that refers to a single-session, precise delivery of a therapeutically effective high dose of ionizing radiation to an imaging-defined target volume of tissue.[85] Presently, in the treatment of certain diseases the results are comparable if not better than surgical intervention. Additionally, whilst in some respects surgical approaches and techniques are reaching their limits, radiosurgery is in its relative infancy. As research expands the knowledge of radiobiology and the effects on a molecular level, improvements may be developed to enhance the efficacy of radiosurgery.

History of Radiosurgery All living organisms are exposed to ionizing radiation on a continuous and daily basis.

Although humans have evolved in an environment of ionizing radiation contributed to by cosmic rays, radons, and other terrestrial radionuclides, the effects of ionizing radiation were not known until human-made sources were developed.[86],[87],[88]

The first harmful effect of radiation was reported in 1901.[89] Antoine Henri Becquerel, who discovered the natural radioactivity of uranium, and shared the 1903 Nobel Prize for physics with Marie and Pierre Curie;[90] sustained a skin burn, attributed to the vial

12 of radium he carried in his vest pocket. The first radiation induced leukaemia was described in 1911, and patterns of other radiation induced injuries were noted.

The observation that radiation caused injury to healthy tissue, soon led to the hypothesis that it could be used to destroy diseased tissue. Before the physics and biology of radiation were clearly understood, physicians began to use radiation to treat various forms of cancer, leading to the arrival of radiotherapy as a treatment modality.[91, 92],[93]

The founding father of Radiosurgery was Swedish neurosurgeon Lars Leksell. Leksell envisaged a minimally invasive treatment approach to ameliorate the difficulties in surgical technique, blood loss, anaesthesia related complications, and associated poor outcomes of conventional neurosurgical operations. In 1949 he first described a stereotactic guiding device that, in 1951, was coupled to a dental x-ray machine. X-ray beams were directed to the target point by rotating the x-ray tube along the arc of the stereotactic frame.[94],[95] Using this technology he was able to irradiate the Gasserian ganglion for the treatment of trigeminal neuralgia in a series of patients.[95],[96],[97]

The initial use of radiosurgery was seen as a means to deliver small precise lesions deep in the brain for the treatment of functional disorders.[98] Whilst, Leksell later examined the use of cross fired protons, as well as x-rays from early linear accelerators

(LINACs),[97],[99] further refinement led Leksell and Larsson to use Cobalt-60 as a photon radiation source.[100],[101] In 1967, the first gamma knife (using 60Co) was used to treat a young boy with a craniopharyngioma.[102],[103]

Physics of Radiosurgery Ionization is the basic initiator of the events that cause radiation damage to living tissues. The influence of ionizing radiation on biological systems results in the

13 transference of energy into the system according to fundamental physical principles.

The energy deposited results in ionization or excitation.

Excitation occurs if alpha or beta particles or gamma photons transfer sufficient energy to remove one of the electrons from an inner to an outer orbital shell. Ionization occurs if alpha or beta particles or gamma photons transfer sufficient energy to remove one of the electrons from the target atom.

The absorption of energy from ionizing radiation produces damage to molecules by either direct or indirect actions. For direct action, the damage occurs as a result of ionization of atoms on key molecules in the biological system. This causes inactivation or functional alteration of the molecule. Indirect action involves the production of reactive free radicals whose toxic damage to the key molecule has a biological effect.

Ionizing radiation arises from either of electromagnetic or particulate origins. Examples of electromagnetic radiation are X-rays and gamma rays, which differ in their source.

X-rays are produced when electrons change orbits within an atom, or electrons from an external source are deflected around the nucleus of an atom. They can be produced artificially, by mechanically making electrons strike a target, which causes the electrons to give up their kinetic energy as x-rays.

Gamma ray emission is associated with alpha, beta, and positron decay, produced by nuclear disintegration of radioactive isotopes.

Types of particulate radiation include electrons, protons, alpha particles, neutrons, and heavy charged ions. The clinical relevance of particulate sourced ionizing radiation is in protons. Protons have a large mass, and when they stop abruptly, depending on their energy; in the process of sudden deceleration, most of their energy is given up. This 14 tends to cause ionization just before the proton stops. This region of enhanced ionization, termed the Bragg peak, means that proton beams exert their effects in a relatively compact region. Hence, protons have advantages over other types of particulate radiation enabling the dose to be conformed to the treatment volume.

Biology of Radiosurgery As eloquently put by Schwartz, a photon is a photon is a photon.[104] Hence, the radiobiological response of tissue to radiation is the same regardless of whether the source is a gamma knife, linear accelerator, or cyclotron.

The main biological effects of radiation take place at the level of DNA. Irradiation of tissue results in the transfer of energy which results in a variety of lesions in DNA, including DNA-protein cross-links, cross-linking of DNA strands, oxidation and degradation of bases, and cleavage of sugar-phosphate bonds.[105] Radiation may also cause breaks of the DNA which may be either single- or double-strand breaks.

However, in order to achieve cell death, radiation must produce double-strand breaks in the DNA, as human cells have a high capacity to repair single-strand damage.[106],[107],[108],[109],[110]

In addition to the DNA damage, breaks may also occur to chromosomes when cells are irradiated. Lethal aberrations arise as broken ends of chromosomes combine with broken ends of different chromosomes resulting in grossly distorted and nonviable formations.[106] These abnormal combinations are most readily seen during mitosis.

Another mechanism of radiation-induced lethality occurs with the interaction of radiation with water in tissues to generate free radicals. Free radicals are highly reactive chemical entities that lack a stable number of outer shell electrons, and are therefore

15 unstable and damage cells. The generation of free radicals depends upon the degree of oxygenation/hypoxia in the target tissues.[107]

Accordingly, the sensitivity to radiation damage is greatest during the G2-M phase of the cell cycle, and as a rule, rapidly dividing cells are more sensitive to radiation damage.

Conventional radiotherapy exploits differences between abnormal (e.g. tumour) cells and normal cells to achieve efficacy. There are processes that occur after radiation that can alter the biological effect of radiation.[111] Most important, is the cell’s ability, the enzymatic mechanisms for healing intracellular injury, to repair sub-lethal damage.

Second, is reoxygenation, whereby oxygen and other nutrients are redistributed to viable cells following radiation injury. Also of significance is the variability of a cell’s radiosensitivity over the cell cycle.

Conventional radiotherapy usually entails treating large fields, and is fractionated.

Fractionation is the delivery of the radiation dose in increments separated temporally.

This allows the differences discussed above, to be used to limit the risk of injury to normal tissue whilst maximizing the damage done to abnormal tissue, thereby increasing the therapeutic ratio.[112]

Radiosurgery is a different approach from that of conventional fractionated radiotherapy. By definition, it is the use of a high dose of precisely focussed radiation delivered in a single session.[94],[100],[113] Unlike fractionated radiotherapy, it does not aim to exploit differences between normal and abnormal tissue, but limits its effect on surrounding normal tissue by the highly focussed nature of radiosurgical ionizing beams.[112]

16 Whilst fractionated radiotherapy is most effective in killing rapidly dividing cells, radiosurgery can affect the target cells irrespective of their mitotic activity.[112]

Radiosensitivity is a limitation of conventional radiotherapy, as in utilizing a therapeutic ratio to damage abnormal cells more than surrounding tissue, the daily delivered dose must be low.[114],[115] As result, some tumours are regarded as radioresistant to fractionated radiotherapy doses. However, radiosurgery overcomes radiation resistance with its use of high dose ionizing radiation.[113]

Radiosurgery Techniques and Current Devices The aim of radiosurgery is to deliver a high dose of radiation focussed on a small target, and have a steep fall-off to minimize radiation exposure to the surrounding tissue.[100],[116]

As mentioned previously, a number of sources of ionizing radiation can be utilized to perform radiosurgery, giving rise to different devices.

Gamma Knife Modern gamma units consist of a central hemispherical steel shell containing a core of

192 cylindrical 60Co sources. The 60Co gamma ray sources are 1mm in diameter and

20mm in length and align radially to converge on a common focal point, the isocenter.

60Co is a man-made radioisotope, produced by neutron activation of the naturally occurring stable 59Co. Submitted to a very high neutron flux in a nuclear reactor, 59Co atom nuclei progressively capture a neutron and transform themselves into 60Co atom, whose nucleus is still in an excited state.[99] During the return to a stable state, each

60Co source emits photon energy by undergoing radioactive decay to 60Ni. In the process of decay, 60Co emits one electron and two gamma rays.[117]

17 The basic principle of the gamma knife is that the radiation dose along any single beam is very low, but the dose at the intersection point of all of the beams is very high. Each

60Co radiation beam undergoes primary collimation within the casting through a two- stage tungsten collimator.[117],[118] Final collimation is achieved through one of several helmets. Each helmet contains 201 precisely positioned collimators that align with the primary collimators. Varying the collimator apertures of the helmets can define an approximately spherical dose distribution at an isocenter of 4, 8, 14, or 18 mm.[117]

Each source in the helmet can be plugged with tungsten blocks to turn off certain beams, thereby altering the shape of the dose distribution. This produces dose plans that conform to the irregular three-dimensional volumes of lesions.

Further conformality of the dose distribution is achieved by the construction of multiple isocenters to create a planned treatment volume that appropriately matches the irregularly shaped target volume. A number of isocenters of varying beam weighting, collimator sizes, and stereotactic coordinates can be used such that the final dose distribution conforms to the size, shape, and number of target lesions while minimizing radiation to surrounding tissue.

Linac LINAC has become the preferred device for conventional radiotherapy, and is commonplace with units available at most tertiary referral centres. Investigation into the application of LINAC for radiosurgery was prompted by the high cost of the gamma knife and shielding required for a unit, and the ubiquity of LINACs.[119] In 1982,

Barcia-Salorio used LINAC for radiosurgical treatment of a carotid-cavernous fistula.[120] Following this, Colombo,[121],[122] Betti and Derechinsky,[123] and others have described and further developed LINAC based radiosurgical systems.

18 Like gamma knife radiosurgery, LINAC-based radiosurgery consists of a collimated radiation beam focused on a stereotactically identified target. The linear accelerator functions to accelerate electrons using high-frequency electromagnetic waves that travel and combine themselves in a copper wave guide to create a travelling electric field. An electron gun, connected to the wave guide, injects an electron bunch into it, and these electrons are accelerated by the travelling electric field.

Accelerated electrons are directed toward a heavy metal alloy target (usually in tungsten) on a small spot, to convert the electron kinetic energy into x-ray braking radiation. The heavy metal alloy spot is the source from which x-rays are emitted in every space direction. The x-ray beams are then shaped according to clinical needs with different kinds of collimators.[97],[117],[118]

Unlike the gamma knife unit, a LINAC has only one source of radiation. Consequently, by using a gantry that rotates over the patient thereby producing an arc of radiation with the isocentric target at its focus, multiple static beams are used to converge on the target volume. With improvements in LINAC units, simultaneous gantry and couch movement can create multiple noncoplanar intersecting arcs that maximize the dose at isocenter whilst maintaining a sharp dose falloff to the adjacent tissue.[117]

Further advances to enhance radiosurgery include the application of intensity modulated radiotherapy (IMRT) and robotic radiosurgery.

The radiation beams produced by standard LINAC machines have a uniform intensity or fluence across the field. In order to conform the radiation beams to the shape of the lesion to be treated, simple measures have been used in the past, such as using wedges or compensating filters.[124] IMRT entails the creation of beams that are of non-uniform

19 or varying intensity across the field. IMRT can produce more conformal dose distributions than before for irregular shaped targets. IMRT requires advanced planning, verification, and delivery techniques, all of which require the computing power that recent advances have allowed.[125]

Robotic radiosurgery has similarly become feasible as result of the improvements in technology and computing. A lightweight LINAC can be mounted on a robotic arm, derived from industrial robotics, can be targeted along six different axes.[117] An example of this is the CyberKnife system, which with real time imaging, allows any patient movement to be translated into a change in target position that the robotic arm can adjust to re-aim the beam toward the new target position. With a mobile LINACs capability of positioning along multiple points in space, each with multiple approach angles, robotic radiosurgery is not restricted to isocentric delivery and allows multicentric beams of high conformity.[117],[126] Another advantage of the CyberKnife is that it allows radiosurgical treatment of the whole body rather than just the head and neck.[126]

Heavy Particle The principle of charged particle radiation is that a particle radiation beam will deposit much of its energy at a depth determined by the charge and kinetic energy of the beam and by the physical characteristics of the absorbing tissue.[127] The absorbed radiation dose deposited by the charged particles increases significantly at the end of the particle range, giving rising to a maximum dose at depth which is greater than the entrance dose, which is termed the Bragg-peak. Beyond the Bragg-peak the dose falls to nearly zero, thereby sparing normal tissue.[127],[128]

20 The Bragg-peak is the major advantage of particulate irradiation over photon irradiation.

With photon beams the absorbed dose decreases with the thickness of tissue traversed.

In comparison, charged particle beams have an initial region of low dose as the beam penetrates through normal tissue, followed by the Bragg-peak at the target region. To increase the photon radiation dose to the target involves increasing the radiation exposure to the normal tissue along the beam path. However, modulating a charged particle beam to produce a similar increased target dose will not increase the radiation exposure of normal tissue to the same extent.

Charged particle beams can be modulated in a number of ways. The use of differing charged particle beam energies will vary the depth and extent of the Bragg-peak effect on the target tissue. Charged particle beams may also be collimated by varying apertures, much like IMRT for LINAC radiosurgery.[127],[129]

The disadvantage of using charged particles for radiosurgery is the requisite infrastructure and cost. An accelerator is required to produce the charged particles, either a large linear accelerator or a cyclotron, both of which require a large area and significant expense.[97]

Role of Stereotaxy Regardless of the device used, the most important facet of radiosurgery is the precise localization of the target in three-dimensional space.

Whilst reports of image guidance in surgery are documented less than a year after

Roentgen’s discovery of x-rays, with its use in removing penetrating foreign objects,[130] the principles underlying stereotaxy preceded this innovation.

21 The origins of Stereotaxy can be traced to the 17th century teachings of Descartes,[131] and his concept of defining the location of any point in space in reference to its relation to three perpendicular intersecting planes. The most definitive description of a stereotactic method and device is attributed to Horsley and Clarke.[132],[119],[117] From

1906 they used stereotactic apparatus to study the deep cerebellar nuclei and cerebellar function in the monkey.[119],[133],[134]

Leksell introduced a new concept: the arc-quadrant.[119] His instrument consisted of a fixation device fastened to the patient’s skull and a moveable arc-quadrant that attached to the fixation device. The arc-quadrant (which defines a sphere) moved so that a probe inserted perpendicular to the sphere’s tangent, at a distance equal to the radius, was always at the desired intracranial target point.[94],[119]

With advances in design over the intervening years, use of stereotaxis has become common place in cranial and spinal neurosurgical procedures. The basic principle of nearly all current stereotactic equipment is firm fixation of the stereotactic reference apparatus to the patient’s body. In standard operative cases, the reference apparatus is attached directly via skull pins or to the spine, or indirectly to the Mayfield 3 point fixation headrest which in turn attaches to the head via skull pins. Registration is performed, which is the identification of homologous structures on the patient and their corresponding imaging.[130] Mathematical processes transform the coordinate system from the imaging study (CT or MRI) to the coordinate system of the stereotactic device.[119] Once registered, instruments introduced into the surgical field can be visualized, on computer screen, in three dimensions. This allows the surgeon to choose the least damaging route to the desired operative site target, as well as allowing the identification of intracranial structures.

22 Stereotaxy has been further adapted to the fundamental non-invasive nature of radiosurgery. Rather than have apparatus attached to the skull, masks that are specifically moulded to the external contour of the patients face can be used. These masks allow a high degree of accuracy and positioning reproducibility, and are used by the stereotactic device as the reference point.[117]

The Effects of Radiosurgery on AVMs Kondziolka et al.[113] summarized the effects of radiosurgery on AVMs as causing a proliferative vasculopathy. Beginning with injury to endothelial cells, the subendothelium and perivascular spindle cells then proliferate. Collagen fibres undergo hyaline degeneration causing the vessels walls to become thickened,[135] which eventually results in luminal narrowing and closure. Ultimately, the mass of the AVM is replaced by scar tissue.[113]

Important ultrastructural changes have been described. Tu et al.[136, 137] examined surgically resected human AVM tissue which had previously been treated with radiosurgery. Their findings included damage to the endothelial cells and subendothelial fibroblasts. Neoproliferation of smooth muscle cells was seen in the tunica media.[136]

A mechanism contributing to wall contraction and subsequent obliteration could be due to myofibroblasts shown to exist within AVM vessels following radiosurgery.[135, 138, 139]

Weibel-Palade bodies have also been found in the smooth muscle cells of the tunica media, reinforcing the role of vascular smooth muscles cells in response to radiation injury.[136]

Though not specifically investigating the effect of radiosurgery on AVMs, a number of groups have investigated molecular changes on endothelium following radiosurgery.

23 Induction of inflammatory reaction involving the migration of polymorphonuclear leukocytes and other blood formed elements into the irradiated area was demonstrated to be the cause of endothelium disruption and separation from the vessel wall.[140] As leukocyte adhesion and recruitment is mediated by the increased expression and presentation of endothelial cell adhesion molecules (CAM), it is of note that

Prabhakarpandian et al.[140] and Sharp et al.[141] also demonstrated that intercellular adhesion molecule (ICAM)-1 expression is increased in irradiated human endothelial.

Increased expression of ICAM-1 has also been demonstrated in the brains of mice that were irradiated.[142]

Identifying constitutive molecular or physical differences between AVM vasculature and normal vessels, could hold the key to developing new treatment paradigms.

Potential for Improvement of Radiosurgery Radiosurgical planning and delivery methods are improving alongside the advances in neuroimaging, computing, and technology.[117, 126, 143-145]

Consideration has also been given to develop strategies for radioprotection and of healthy brain, which could allow for more aggressive radiosurgical treatment and thus increasing the therapeutic ratio. Options that have been investigated include the use of chemical radioprotective agents, neural stem cell implants, or the shielding of critical structures.[112, 113]

There are also biological methods that could potentiate the efficacy of radiosurgery.

Early work in this area was in the field of oncology, with the development of radiosensitizers and radioenhancers with the aim of increasing the toxicity of radiation in cancer cells whilst limiting the injury to adjacent healthy tissue.[146]

24 Other techniques investigated in the radiosurgical treatment of cancers are the use of viral vector-based multigene therapy and the use of nanotechnology and nanoparticles to further enhance tumour dose during radiosurgery.[146]

The innovations demonstrated in the use of radiosurgery for the treatment of cancer, is mirrored by similar innovations in the development of novel pharmacological approaches.

Vascular Targeting Pharmacological treatments are now the cornerstone of cancer treatment. Though

Ehrlich first coined the term "chemotherapy" in the early 1900s, his definition of using chemicals to treat disease was largely in the treatment of infectious disease.[147] The era of cancer chemotherapy began in the 1940s with the first use of nitrogen mustards and folic acid antagonist drugs.[147, 148]

Cancer chemotherapy has benefited from genome sequencing, which suggest that many of the signalling networks that regulate cellular activities such as proliferation and survival are radically altered in cancer cells.[147] This has led to the development of

"targeted therapy", where molecular and genetic characteristics of tumours are utilized to damage cancer cells whilst causing less harm to normal cells.

Molecular targeted therapies in cancer chemotherapy can be divided into non-vascular and vascular categories. Non-vascular targets include oncogenes, cell surface receptors, and second messenger systems.[149]

Denekamp described in the early 1980s, that comprised tumour blood flow led to tumour regression.[150-152] Targeting tumour vasculature is appealing, as the vessels provide oxygen and nutrients requisite for tumour survival and growth.[149-155] Tumour

25 vascular targeting approaches can be considered as anti-angiogenic or anti-vascular.

Anti-angiogenic approaches aim to prevent neovascularization, whereas anti-vascular approaches use vascular disrupting agents to cause the selective shutdown of established tumour vasculature.[149, 153]

These vascular targeting techniques rely on constitutive molecular or physical differences between tumour vasculature and normal vessels. Tumour vasculature demonstrates increased tortuosity and variable vessel diameter, and often an immature phenotype.[149, 153] Vessel walls may be poorly developed with a discontinuous endothelial cell lining, and there may be poor connections between pericytes and endothelial cells.[153, 156-158] Vessel wall irregularities also include abnormal basement membrane, irregularly shaped endothelial cells and abnormal vascular smooth muscle cells.[149, 153]

Various techniques are used to stimulate thrombosis within tumour vessels. The major classes of vascular targeting agents (VTAs) that are being developed are ligand-directed

VTAs and non-ligand-directed (or small molecule) VTAs.[149, 159]

Ligand-directed approaches use ligands that bind selectively to components of tumour blood vessels to target agents that then occlude those vessels.[159] Endothelial cell damage and/or thrombosis is then achieved by coupling the vascular targeting moiety with a toxin or pro-coagulant.[149] Hence, ligand directed VTAs have two main components; a targeting, and an effector moiety that are linked. The targeting moiety may be an antibody, peptide, or growth factor that is directed against a marker or receptor that is selectively up-regulated on tumour cells and not on normal endothelial cells.[149, 159]

26 In contrast, small molecule VTAs do not localize selectively to tumour vessels. This targeting strategy exploits the pathophysiological differences between tumour and normal tissue endothelium to achieve selective occlusion of tumour vessels. Some of these pathophysiological differences include increased proliferation and increased permeability of tumour vessels compared with normal tissue endothelial cells.[159]

Potential for Vascular Targeting Strategies in AVMs Applying similar principles as cancer treatment vascular targeting to induce thrombosis within cerebral AVM vessels is a conceptually attractive approach. Unlike the treatment of tumours, success of this strategy would not depend on death of every cell in the lesion: intravascular occlusion alone is sufficient for AVM cure.

Intimate contact with blood makes AVM endothelium an accessible target for a pharmacological agent. In addition, as described earlier, the endothelium of AVM has different molecular properties than the endothelium of normal cerebral vasculature, which should enable it to be targeted.

However, there are limitations of this approach at present. A targeting moiety that is sufficiently discriminatory to permit a ligand-directed approach against a marker or receptor that is selectively expressed on AVM endothelium has not been identified.

Similarly, use of a small molecule VTA is limited by the fact that AVM vessels are not sufficiently different from normal vessels to allow selective thrombosis.

The successful application of vascular targeting would require a "priming‟ technique that selectively alters the surface molecule expression of the endothelial cells in AVMs without affecting normal brain vessels. This technique would also need to be precise, as inadvertent occlusion of vessels supplying normal brain would be potentially catastrophic. 27 It has already been demonstrated that irradiation causes phenotypic changes on endothelium cells.[55, 136, 137, 139-141] Hence, the stand-out choice for priming would be stereotactic radiosurgery. Stereotactic radiosurgery would also allow for precise spatial localisation, such that endothelial changes are restricted to vessels within the radiosurgery target volume.

Storer et al. have confirmed proof of principle that radiosurgery induces molecular changes that are sufficient to use a vascular targeting to discriminate targeted vessels from normal vessels.[160] Using an animal model of AVM, twenty four hours after stereotactic radiosurgery or sham radiosurgery, trial VTAs were administered intravenously. The trial VTAs used were lipopolysaccharide (LPS), soluble tissue factor

(sTF), a combination of both, or sterile saline. Tissue obtained four days after VTA administration demonstrated enhanced early thrombosis in the model AVM tissue in the treatment group that received radiosurgery and LPS + sTF.

The basis of this small molecule VTA strategy is based on the tumour targeting research of Thorpe and Ran.[161, 162] This group demonstrated in tumour vasculature that there is translocation of phosphatidylserine from the internal to the external endothelial membrane leaflet.[162] He et al. demonstrated that a similar translocation of phosphatidylserine to the external leaflet of the membrane occurs in endothelial cells after radiation.[163] LPS and sTF interact with externalised phosphatidylserine to produce stimulation of thrombosis.[160, 164]

Although the vascular targeting approach of Storer et al. yielded promising results,[160] further investigation is warranted. Their study showed that it was possible to produce enhanced early thrombosis, however, consideration needs to be given to the durability of the thrombus formed.

28 Questions that need to be addressed include: is this thrombus stable, does it lead to vascular occlusion in the long term, is the occlusion occurring in small and large vessels, and is it susceptible to recanalisation?

29

Hypotheses

1. Radiosurgery induces changes in endothelial characteristics in AVM vessels;

2. A vascular targeting agent can be used to exploit these changes to induce

localized thrombosis following radiosurgery in an animal AVM model;

3. The thrombosis achieved is durable and long lasting.

30

Aims

1. To utilize a vascular targeting agent to induce localized thrombosis in an animal

model of AVM treated with radiosurgery;

2. To assess the long term durability of thrombosis achieved.

31

Materials and Methods

Overall experimental plan An animal model of AVM was used in 32 male Sprague-Dawley rats, seen in Figure 1.

Figure 1: A: Schematic diagram of the vasculature of the animal model, demonstrating proximal and distal carotid arteries (red outlined vessels) and jugular veins (blue vessels). B: Schematic diagram of the animal model of AVM, site of carotid-jugular anastomosis (inset) and AVM formation (purple vessels) 6 weeks after carotid-jugular fistula creation.

Six weeks after creation of the carotid-jugular fistula, radiosurgery was administered using a specially-developed stereotactic frame. The region of the anastomosis and nidus was irradiated with a dose of 25Gy, whilst contralateral carotid arteries and jugular veins received a dose of around 2 to 4 Gy. Twenty four hours after irradiation, rats received either: LPS and sTF (treatment group), normal saline (saline control), or LPS and sTF but no radiation (sham radiation control). In the treatment group and the sham radiation control groups, there were three animals for each time point. In the saline control group, there were two animals for each time point. The animals were then sacrificed at 1, 7, 30, or 90 days after treatment. At the chosen time points, the animals 32 were sacrificed by perfusion fixation. Immediately prior to sacrifice an angiogram was obtained to assess the nidus and draining vein. Following perfusion, vascular tissue from the neck region was harvested for analysis.

The endpoints examined included fistula blood flow measured using a Doppler flow meter, the draining vein diameter measured angiographically, and intravascular thrombosis rates assessed histologically.

Ethics and Animal care Approval for animal experimentation was obtained from the Animal Care and Ethics

Committee of the University of New South Wales (ACEC reference number 04/77).

Animal experimentation was performed in accordance with ACEC guidelines and the

Australian Code of Practice for the Care and Use of Animals for Scientific Purposes

(7th Edition, 2004).

Male Sprague-Dawley rats were obtained at approximately 6 weeks of age, with weights ranging from 170 to 300g. Standard rat cages were used to house no more than three animals per cage and bedding was changed once to twice weekly. Food and water were allowed ad libitum.

Animal procedures were not performed in the presence of other animals and attempts were made to allow acclimatization to new surroundings prior to any procedures.

Animals were housed singly for one week post-operatively. After a procedure, observations were carried out daily for the first week and then weekly thereafter.

Observations included weight, assessment of motor function, behaviour and wound health. It was planned to euthanize the animal in the case of significant morbidity or

33 weight loss greater than 20% of pre-procedure body weight. However, no animal met these criteria.

Morbidity and Mortality The procedures were generally well tolerated. There was no evidence of significant lasting neurological morbidity in any of the animals. No animals had to be euthanized prematurely in the post-procedure period.

One animal death occurred at the end of the fistula creation procedure due to local anaesthetic cardiotoxicity. Three deaths occurred initially during the radiosurgery, most likely as result of over anaesthesia. Following this, the anaesthetic protocol was revised, resulting in no further morbidity. There was one death at 48hrs post radiosurgery, which was most likely due to radiation induced tracheitis.

All animals that died, were autopsied with examination of the brain and major organ systems.

AVM Model Creation Male Sprague-Dawley rats from 6-12 weeks old were used in this experiment.

Anaesthesia was induced using a Perspex induction chamber (20 cm x 25 cm x 30 cm), with a mixture of isoflurane 4% and oxygen (2 L/min). The animal was then transferred to the operating rig, upon which general anaesthesia was maintained with the animal self-ventilating isoflurane via a nose cone.

The depth of anaesthesia was assessed using the respiratory rate and by checking the hind limb withdrawal to pain (with the use of blunt forceps). No procedure was commenced until there was a consistent absence of response to pain. Observations of respiratory rate, core temperature and pain response were recorded every 5 minutes for

34 the first 20 minutes and fifteen minutely thereafter. For the duration of the procedure, there was continuous monitoring of rectal core temperature and of oxygen saturation via trancutaneous pulse oximetry on a hind limb. A heating blanket was used for the duration of the procedure.

The animal was positioned supine and the forelimbs taped in an abducted position. The operation site (anterior cervical region) was shaved and prepared with povidone-iodine.

The procedure was performed in a sterile field using aseptic technique.

An anterior cervical midline incision was made from the interramal vibrissae to the manubrium. A combination of sharp and blunt dissection was used to identify the sternohyoid and left sternocleidomastiod muscles. Dissection was continued into the groove formed by these two muscles to the left omohyoid muscle. This was then divided to provide exposure of the left common carotid artery (CCA).

The left CCA was dissected circumferentially of all connective and adipose tissue, from the entry into the neck to the bifurcation into internal and external carotid artery branches. Blood flow was measured through the CCA using a 1 mm Doppler ultrasonic probe (Transonic Systems Inc., Ithaca, NY).

The left external jugular vein (EJV) was then identified superficial and lateral to the sternocleidomastoid muscle, and also dissected circumferentially of all connective and adipose tissue, from its junction with the axillary/subclavian vein rostrally for a distance of approximately 2 cm. Care was taken to preserve tributaries to the EJV, though usually one or two near the junction with the subclavian vein were divided to enable adequate mobilization of the EJV.

35 The EJV was then ligated with 10/0 nylon suture at its junction with the subclavian vein. An aneurysm clip was then placed across the rostral EJV. The EJV was transected at a slight angle close to the proximal ligature and then further mobilized to allow approximation to the lateral side of the CCA.

Microclips were applied proximally and distally on the CCA, and a small arteriotomy made on the lateral aspect. An end-to-side anastomosis of the EJV to the CCA was performed using a continuous 10/0 nylon suture. The clips were sequentially removed from the EJV, distal CCA, and proximal CCA. Blood flow was measured through the proximal CCA and the vein using 1 and 2 mm Doppler ultrasonic probes (Transonic

Systems Inc., Ithaca, NY). Haemostasis was achieved with pressure, and the use of bilpolar electrocautery, where necessary.

Following the creation of the fistula, a sterile implantable microchip was inserted subcutaneously to allow subsequent identification of the animal. The wound was washed out with dilute povidone-iodine and saline, then closed in one layer with 3/0 silk suture to the panniculus carnosus and skin. Figures 1 and 2, illustrate the formation of the fistula and model AVM nidus.

36

Figure 2: Intraoperative image of the animal model of AVM, demonstrating proximal and distal carotid artery, jugular vein, and site of carotid-jugular anastomosis.

At the end of the procedure, the isoflurane was turned off, allowing the animal to breathe oxygen until the time of awakening. Once awake, the animal was placed in an individual cage, and returned to the holding facility in the Prince of Wales Medical

Research Institute.

Radiosurgery Dosimetry Prior work at the POWMRI Neurosurgery laboratory had been done to allow planning of the radiosurgery.[160]A single animal, six weeks following surgical creation of an arteriovenous fistula, was euthanized. The surgical wound was reopened to expose the arteriovenous anastomosis and simulated nidus. The vasculature was injected with a

37 contrast agent (OmnipaqueTM). The animal was then placed on a custom made stage, which was attached to the head of the X-KnifeTM linear accelerator device (Radionics,

Burlington, MA) and a CT was performed. The contrast filled anastomosis and nidus were visualized on the CT angiogram, allowing their three-dimensional locations in a coordinate system defined by the head ring to be determined. This information allowed planning of radiation arcs that deliver a dose of 25 Gy of gamma irradiation to the site of the nidus with the 90% isodose line encompassing the anastomosis. Figures 3 and 4 illustrate the planning and administration of radiosurgery to the rodent AVM model.

Figure 3: Anaesthetised rodent in custom made stage attached to the head of the linear accelerator.

38

Figure 4: Anaesthetised rodent in custom made stage. Insets demonstrate images from planning phase to localize the animal model AVM nidus and plan treatment.

Radiosurgery Delivery Protocol In the experimental animals, anaesthesia for radiosurgery was initially induced with a mixture of isoflurane 4% and oxygen (2 L/min), and maintained with an intramuscular injection of ketamine and midazolam. Twenty minutes prior to the procedure, ketamine

(40 mg/kg) and midazolam (1.5 mg/kg) were given intramuscularly. Once the righting response was lost the animals were given a further booster dose of ketamine (20 mg/kg) and midazolam (0.75 mg/kg) to ensure an adequate duration of anaesthesia. Whilst sedated, animals breathed oxygen (2 L/min) via a nose cone.

The depth of anaesthesia was assessed using the respiratory rate and by checking the hind limb withdrawal to pain. Routine observations were made of respiratory and heart

39 rate. Animals remained sedated for around 30 to 40 minutes. Following the procedure, the animal was kept in a quiet area until recovery. The animal was returned to the holding room, once it was seen that there was no evidence of significant morbidity.

Once sedated, the animal was placed on the stage attached to the head ring of the X-

KnifeTM linear accelerator (Radionics, Burlington, MA). Correct positioning of the animal relative to the planned treatment location was confirmed by ensuring that the nidus was placed at the intersection of the three targeting laser beams that designate the centre of the radiation delivery arcs. The same treatment plan was used for all animals, giving a maximal dose of 25 Gy to the nidus in the subcranial region.

The sham radiation control group was treat identically, however were not irradiated.

Administering TF and LPS Twenty four hours following radiosurgery, animals received intravenous administration of either sterile saline, or LPS and sTF; or twenty four hours following sham radiosurgery received LPS and sTF.

The LPS was given as a dose of 0.1 mg/kg body weight. The dosage of sTF was 0.4 mg/kg body weight. The LPS and sTF were suspended in 1 mL 0.9% sterile saline, which was followed by a ‘push’ of 1.5 ml 0.9% sterile saline. The sterile saline control group received 1.5 ml of 0.9% sterile saline.

Following the general anaesthetic protocol (with isoflurane) and monitoring as described above, the animal was position supine and the hind limbs taped in an abducted position. The operation site (right inguinal region) was shaved and prepared with povidone-iodine. The procedure was performed in a sterile field using aseptic technique.

40 The femoral arterial pulse was palpated and a 1 cm longitudinal incision was made over this point. Sharp and blunt dissection was used to identify the femoral neurovascular bundle.

The femoral vein was dissected circumferentially and separated from the artery and nerve. The femoral vein was exposed from the origin of a profunda branch proximally to the origin of the superficial epigastric vein distally.

The distal femoral vein was ligated with 10/0 nylon suture at its junction to the superficial epigastric vein. Suture (10/0 nylon) was passed under the proximal femoral vein; tension on this was used to occlude the vessel. A small incision was made in the femoral vein and a 24 gauge cannula was passed into the lumen, directed proximally.

Intravenous administration of targeting agent was performed as a slow push over 5 minutes via the cannula. The cannula was removed, and the femoral vein distal to the origin of the profunda branch was ligated with 10/0 nylon.

Haemostasis was achieved with pressure and bipolar electrocautery. The wound was washed out with dilute povidone-iodine and saline, then closed in one layer with 3/0 silk suture to the panniculus carnosus and skin.

At the end of the procedure, the isoflurane was turned off, and 100% oxygen administered until the animal was awake. Once awake the animal was placed in an individual cage, and returned to the animal holding facility.

Angiography Immediately prior to sacrifice an angiogram was obtained to assess the nidus and draining vein.

41 Following the general anaesthetic protocol (with isoflurane) and monitoring as described above, the animal was positioned supine and the hind limbs taped in an abducted position. The operation site (left iliac region) was shaved and prepared with povidone-iodine. The procedure was performed in a sterile field using aseptic technique.

The iliac arterial pulse was palpated and a 2 cm longitudinal incision was made over this point. Sharp and blunt dissection was used to identify the common iliac artery.

Sutures (10/0 nylon) were passed under the proximal and distal common iliac artery; tension on these were used to occlude the vessel. A small incision was made in the artery, and a fine gauge angiography catheter passed into the lumen, directed towards the aortic arch.

Intra-arterial contrast (OmnipaqueTM) was administered, and an angiogram performed using image intensifier fluoroscopy (Apelem, Model APX-HF, Nîmes, France). A 5 cent piece was included in the x-ray field for scale purposes. The fluoroscopy screening was recorded on video camera for analysis later. Figure 5 illustrates a single x-ray image from a typical angiogram obtained.

42

Figure 5: Animal model of AVM, demonstrating carotid-jugular anastomosis (1) which creates an arterialised feeding vein (2), a nidus(3), and a draining vein (4). A: schematic diagram of model. B: x-ray image from carotid angiogram demonstrating the model AVM.

Once the angiogram was obtained the catheter was withdrawn, and the sutures used to ligate the common iliac artery. The wound was then packed, and the animal was perfuse-fixed.

Perfusion Fixation The animals were then sacrificed at 1, 7, 30, or 90 days by paraformaldehyde perfusion fixation.

Each animal was anaesthetised following the angiogram using the routine general anaesthetic protocol (with isoflurane). The animal was positioned supine and the hind limbs taped in an abducted position. The operation sites (cervical, thoracic, and upper abdominal regions) were shaved and prepared with povidone-iodine.

43 An anterior cervical midline incision was made from the interramal vibrissae to the manubrium. A combination of sharp and blunt dissection was used to provide exposure of the feeding left CCA and arterialized vein. Blood flow was measured through the arterialised vein using a 2 or 4 mm Doppler ultrasonic probe (Transonic Systems Inc.,

Ithaca, NY).

A bilateral thoracotomy was performed to expose the heart. A left ventricular puncture was used to cannulate the ascending aorta. Calcium heparin (5000 i.u. in 5 ml normal saline) was infused over 30 seconds. The right atrium was incised to allow outflow. The animals were then infused with at least 500 ml of perfusate into the aorta at a pressure of 120mm Hg over 20 minutes. The perfusate solution was 4% paraformaldehyde in

0.1M phosphate buffer.

Following perfusion, vascular tissue from the neck region bilaterally was harvested for analysis. The specimens were; proximal carotid artery, carotid-jugular anastomosis, distal carotid artery, arterialised feeding vein, infracranial AVM nidus, contralateral infracranial tissue, contralateral carotid artery, and draining vein. The head and brain of each animal was preserved in paraformaldehyde. The heart, lungs, and abdominal viscera were removed and grossly inspected for evidence of infarction or haemorrhage.

Tissue Processing Tissue specimens were post-fixed in paraformaldehyde for 48 hours. Specimens were then processed in a Tissue Tek VIP processing centre (Sakura Finetek, Tokyo, Japan).

Graded ethanol baths were used sequentially for 20 minutes each (50%, 70%, 80%,

95%, and two × 100%), followed by two xylene baths of 90 minutes each and then four paraffin baths of increasing time (30, 60, 60, and finally 90 minutes).

44 Tissue samples were embedded in paraffin. From specimens of the carotid-jugular anastomosis, arterialised feeding vein, and infracranial AVM nidus; sections of 10 μm thickness were cut using a microtome (Microm, Walldorf, Germany), placed on a water bath, floated onto slides and then dried in an oven at 37ºC for at least 3 days.

Staining - Haematoxylin and Eosin Sections were deparaffinized with xylene (2 baths of 2 minutes each) and rehydrated in alcohol (100% and 90% for 1 minute each). The sections were then rinsed in distilled water and placed in haematoxylin for 5 minutes. Slides were then rinsed in tap water, differentiated by two quick dips in 2% hydrochloric acid alcohol and then washed again in tap water. The slides were bathed in lithium carbonate for 15 seconds to perform bluing. Following a tap water wash for 5 minutes, sections were counterstained with eosin (alcoholic) for 2 minutes. Slides were dehydrated with ethanol (2 × 90% for 15 seconds followed by 2 × 100% for 15 seconds each) and then washed in xylene (2 baths of 2 minutes each). Gurr’s Depex mountant was used to mount coverslips, and slides were left to dry at 37ºC for at least 3 days.

Staining - Martius/scarlet/blue (MSB) Sections were deparaffinized with xylene (2 baths of 2 minutes each) and rehydrated in alcohol (100% and 90% for 1 minute each). After rinsing in distilled water, sections were immersed in Celestine blue for 5 minutes followed by a 1 minute tap water wash.

The slides were then placed in haematoxylin for 5 minutes. Slides were then rinsed in tap water, differentiated by two quick dips in 2% hydrochloric acid alcohol and then washed again in tap water. The slides were bathed in lithium carbonate for 15 seconds to perform bluing. Slides were then rinsed in 95% alcohol, stained with martius yellow solution for 2 minutes and then rinsed briefly with distilled water. After a 10 minute

45 immersion in crystal ponceau solution, slides were rinsed in distilled water and treated with 1% phosphotungstic acid for 5 minutes.

Another wash with distilled water for 30 seconds preceded the final staining with methyl blue solution for 2 minutes. Slides were then washed in distilled water for 1 minute, dehydrated with graded ethanol (2 × 90% for 15 seconds followed by 2 × 100% for 15 seconds each) and then washed in xylene (2 baths of 2 minutes each). Gurr’s

Depex mountant was used to mount coverslips, and slides were left to dry at 37ºC for at least 3 days.

Blood flow measurement Doppler flow assessment of the blood flow was performed at two points during the experiment. Prior to fistula formation, blood flow was measured through the CCA.

Following fistula formation, flow was measured through the pre-treatment arterialized vein. Following treatment, prior to sacrifice, blood flow was measured through the arterialized vein. Doppler ultrasonic probes (1, 2, and 4mm, Transonic Systems Inc.,

Ithaca, NY) were used for flow assessment.

Light Microscopy and Quantification of thrombosis Light microscopy for H&E and MSB stained sections was performed on a Leica DMR microscope (Leica Microsystems, Wetzlar, Germany). Digital images were captured with an Axiocam HRC digital camera (Zeiss, Oberkochen, Germany).

From the model AVM nidus, for each animal, five sections at varying depths of the specimen were taken and processed as above. Using H&E sections, thrombosed and patent vessels were counted on representative high-power fields from the five sections of the AVM nidus by two observers separately, who were blinded to the treatment

46 group. The results of each observer were compared, and where differences were found; the slides were re-examined together, and the number of thromboses seen was decided.

Data and statistical analysis Logistic regression analysis was performed with SPSS version 13 software (SPSS Inc.,

Chicago, IL).

47

Results

Blood flow The mean Doppler measurements of flow through the fistula obtained before treatment and after treatment are presented in Table 1. In the group of animals that received LPS and sTF without radiation, and the group of animals that received saline with radiation, at the 30 day time point no Doppler flow measurements could be taken due to malfunction of the Doppler probes. Analysis of the Doppler flow measurements did not show any statistically significant findings.

Table 1: Mean Doppler flow (mL/min) through the arterialised jugular vein before and after treatment.*

Timepoint (LPS/sTF) + RTX (LPS/sTF) - RTX Saline + RTX

Pre Rx Post Change Pre Rx Post Change Pre Rx Post Change Rx Rx Rx

1 Day 43 99 56 35 128 93 100 72 -28

7 Days 53 176 123 72 296 224 67 113 46

30 Days 21 17 -4 0 0

90 Days 29 249 220 105 291 186 44 16 -28

*LPS, lipopolysaccharide; sTF, soluble tissue factor; RTX, radiosurgery; Rx, treatment

Angiography findings As result of equipment malfunction and technical difficulties it was not possible to obtain angiograms, nor to obtain measurements from the angiograms in all of the study animals. Analysis of the results that were obtained did not show any significant changes in the fistula, or draining vein diameters in the different treatment groups.

48 Histology Five high powered fields were examined from H&E sections of each animal model simulated AVM nidus to count patent and thrombosed vessels. Histologic examination demonstrated the presence of antemortem thrombi and post-mortem clot within the vessel lumen. For the purpose of thrombi count, only the antemortem thrombi were included. In addition to H&E staining, Martius Scarlet Blue was used to stain the sections. As MSB stains selectively for fresh, mature, and old fibrin[165] it was used to confirm the presence of antemortem thrombi.

Although occasional occlusion was seen in larger vessels (see figure 6), the majority of thrombi seen were in small vessels (less than 200 μm in size). The features of antemortem thrombi seen were a composition of fibrin:platelet bands and erythrocyte- rich accumulations. In addition, occasional lines of Zahn were also seen – the paucity of this finding would be explained due to the thrombi occurring in the smaller vessels, with less area for lamination to occur. Calcium and cholesterol deposition was not seen in any of the animal model AVM tissue examined.

49

Figure 6: Representative section from irradiated tissue treated with targeting agent from 90 day time point. Evidence of large vessel antemortem thrombus (H&E; original magnification, × 10). Scale bar = 100µm.

50

Thrombosis rates Thrombosis rates by treatment group and time points are presented in Table 2, and graphically in Figure 7.

Table 2: Intravascular thrombosis in the model AVM nidus (percentage of vessels), by treatment group in vessels smaller than 200 μm.*

Vessel Thrombosis (LPS, sTF) + (LPS, sTF) - Saline + RTX

RTX RTX

1 day % thrombosed 56% 46% 0%

7 days % thrombosed 54% 40% 10%

30 days % thrombosed 53% 55% 17%

90 days % thrombosed 69% 30% 23%

*LPS, lipopolysaccharide; sTF, soluble tissue factor; RTX, radiosurgery

51 80

70

60

50 Saline + RTX 40 LPS, sTF - RTX 30 LPS,sTF + RTX 20

10

0 1 day 7 days 30 days 90 days

Figure 7: Intravascular thrombosis rates in the model AVM nidus (by percentage of vessels thrombosed), by treatment group in vessels smaller than 200 µm (LPS, lipopolysaccharide; sTF, soluble tissue factor; RTX, radiosurgery).

In the control group that received saline and radiosurgery, there were very few irradiated vessels. Initially there was no thrombosis (Figure 8).

52

Figure 8: Representative section from irradiated tissue treated with saline from 7 day time point. No evidence of thrombus formation, nor evidence of radiation effect (H&E; original magnification, × 40). Scale bar = 200µm.

At later time points, a gradual steady increase in thrombosis over time was seen, as well as evidence of radiation effect, such as intimal hypertrophy and subendothelial proliferation (Figure 9 and 10).

53

Figure 9: Representative section from irradiated tissue treated with saline from 30 day time point. No evidence of thrombus formation. Evidence of radiation effect, with proliferation of subendothelial tissue and intimal hypertrophy (H&E; original magnification, × 40). Scale bar = 200µm.

54

Figure 10: Representative section from irradiated tissue treated with saline from 90 day time point. Evidence of thrombus formation and evidence of radiation effect (H&E; original magnification, × 40). Scale bar = 200µm.

Administration of the LPS and sTF with sham radiation resulted in higher rates of thrombosis (Figures 11, 12, 13). The rates of thrombosis in this treatment group varied according to time points, though not with a progressive increase.

55

Figure 11: Representative sections from non-irradiated tissue treated with LPS and sTF from 7 day time point. Evidence of thrombus formation. No evidence of radiation effect (H&E; original magnification, × 40). Scale bar = 200µm.

Figure 12: Representative section from non-irradiated tissue treated with LPS and sTF from 30 day time point. Evidence of thrombus formation. No evidence of radiation effect (H&E; original magnification, × 40). Scale bar = 200µm.

56

Figure 13: Representative section from non-irradiated tissue treated with LPS and sTF from 90 day time point. Evidence of partial vessel occlusion from antemortem thrombus – potential evidence of recanalisation. No evidence of radiation effect (H&E; original magnification, × 40). Scale bar = 100µm.

Animals in the full treatment group that received LPS and sTF after radiation showed the highest rates of formation of fibrin and erythrocyte thrombi, with some variation over time (Figures 14-19). The highest rate of thrombosis was observed at the final time point.

57

Figure 14: Representative section from irradiated tissue treated with LPS and sTF from 7 day time point. Evidence of small vessel occlusion from antemortem thrombus, and from post- mortem clot. No evidence of radiation effect (H&E; original magnification, × 40). Scale bar = 200µm.

58

Figure 15: Representative section from irradiated tissue treated with LPS and sTF from 30 day time point. Evidence of small vessel thrombosis. (H&E; original magnification, × 40). Scale bar = 200µm.

59

Figure 16: Representative section from irradiated tissue treated with LPS and sTF from 30 day time point. Evidence of small vessel occlusion and radiation effect (H&E; original magnification, × 20). Scale bar = 500µm.

60

Figure 17: Representative section from irradiated tissue treated with LPS and sTF from 90 day time point. Evidence vessel thrombosis (H&E; original magnification, × 20). Scale bar = 100µm.

61

Figure 18: Representative sections from irradiated tissue treated with LPS and sTF from 30 day time point. Evidence of small vessel occlusion from antemortem thrombus (MSB; original magnification, × 40). Scale bar = 200µm.

62

Figure 19: Representative sections from irradiated tissue treated with LPS and sTF from 90 day time point. Evidence of small vessel occlusion from antemortem thrombus (MSB; original magnification, × 20). Scale bar = 500µm.

The average rates of thrombosis in each group were 13% in the saline and radiosurgery group, 43% in the LPS/sTF and sham radiosurgery group, and 58% in the LPS/sTF and radiosurgery group. Logistic regression analysis of pooled experimental groups revealed an association between LPS/sTF (with or without radiation) and development of thrombi (p = 0.03). Thrombosis in the LPS/sTF plus RTX group was significantly higher than thrombosis in the other two groups at 90 days (p <0.01).

63 Macroscopic examination revealed no evidence of infarction, or of thrombi in the vasculature of the brain, lung, heart, liver, and other abdominal viscera.

64

Discussion

Intracranial AVMs are clinically significant as the leading cause of non-traumatic intracranial haemorrhage in children and young adults.[4, 5] Haemorrhage may be intraparenchymal, intraventricular or subarachnoid,[9, 10] and carries a considerable short term mortality rate of 10-29%,[8, 11] and a risk of permanent morbidity ranging from 14-85%.[2, 12]

The classification of AVMs according to size, pattern of venous drainage, and neurological eloquence of adjacent brain allows stratification of treatment risk.[62] Low grade AVMs (I-II) account for approximately 40% of AVMs and can be treated with low risk.[62, 63] One third of AVMs are high grade (IV-V), and over 90% of these are untreatable.[64, 65] Of the intermediate grade AVMs (III), many of these are not treatable without high risk. New treatments are needed for these patients.

The goal of AVM treatment is to eliminate the risk of haemorrhage by complete resection or obliteration of the AVM vessels. Partial removal or obliteration can increase the risk of haemorrhage.[64] The current treatment modalities are surgical resection, endovascular occlusion, or stereotactic radiosurgery.

Surgical resection of the AVM allows immediate protection from haemorrhage, however carries the risk of perioperative death or disability.[62, 63] Surgery is ideal for small, superficial AVMs in non-eloquent brain.[67, 68, 166] The risks of surgery are generally prohibitive for large AVMs, or AVMs located in eloquent regions of the brain.[64, 167]

65 Endovascular treatment of AVMs involves intra-arterial injection of occluding substances. Although endovascular treatment can achieve obliteration of small AVMs, it is used most commonly as an adjunct to surgery.[70, 71, 168]

Radiosurgery is a relatively non-invasive approach that avoids the immediate procedural risks of surgical resection or endovascular occlusion. Unlike radiosurgery for tumours, which aims to kill cells, the goal is vascular occlusion. The processes of AVM vessel occlusion after radiosurgery are being investigated, and appear to involve a combination of cellular proliferation and intravascular thrombosis.[55, 59, 136, 137] The major limitations of radiosurgery are that lesions greater than 3 cm respond poorly and the rates of complications increase proportionally to the diameter of the target, and that there is a latent period from treatment to obliteration of 2–3 years.[76, 77]

Despite technical advancements in these therapies over the last 3 decades, there remain significant limitations to each. There is a group of patients with large or critically located AVMs, who cannot be safely or effectively treated. It is unlikely that advances in surgical technique or endovascular therapy will be sufficient to reduce the risk associated with treatment of these AVMs.[64, 167]

In contrast, radiosurgery still has distinct potential for improvement. [95],[96],[97] Advances in computing and imaging technology have allowed improved lesion identification and localization.[119] Improvement has also occurred as result of the modulation of radiation effect using conformal beams, dose fractionation, and exploration of new energy modalities .[117] Understanding of radiobiology has led to the development of chemotherapeutic agents such as radiosensitizers, photosensitizers and vascular targeting agents as an addition means of modulating radiation effect.[145]

66 A broad hypothesis has been proposed: that the disadvantages of radiosurgery can be overcome with cellular and molecular strategies directed at improving the response to radiation. The differences in molecular characteristics and ultrastructure between AVM vessels and normal vessels have been investigated. Compared to normal cerebral vessel endothelium, human AVMs have increased expression of ICAM-1, VCAM-1, and E- selectin.[59] In addition, there is loss of tight junctions, abnormal cytoplasmic processes, and production of Weibel-Palade bodies.[37]

A potential strategy to increase AVM thrombosis after radiosurgery is to use vascular targeting to utilize these differences between normal vessels and AVM vessels to produce selective thrombosis. Vascular targeting has emerged in the field of cancer therapy, where deliberate stimulation of intravascular thrombosis has been investigated as a means to achieve tumour necrosis.[159, 169-172] Vascular targeting may use a ligand- directed strategy, combining a targeting moiety and an effector moiety. The targeting moiety is an antibody or peptide that binds to a marker that is selectively expressed on tumour vessel endothelium, and the effector moiety induces thrombosis.[159] Vascular targeting may also use a non-ligand strategy, with the administration of agents that directly interact with the target cells due to their molecular and ultrastructural differences from normal cells. Vascular targeting is conceptually attractive for the treatment of AVMs, because vascular thrombosis is the primary aim and is potentially curative. To achieve selective thrombosis of AVM vessels using either a ligand-directed or a non-ligand strategy, endothelial surface molecules that are highly discriminating between AVM vessels and normal vessels are required. Unlike, vascular targeting in cancer research where inherent differences between tumour and normal endothelial cells are sufficient to enable selective targeting, though it has been demonstrated that the

67 AVM endothelial phenotype differs from normal vasculature, no marker has been detected that would be sufficiently discriminating.[37, 47, 55, 59, 173-175]

However, the application of stereotactic radiosurgery as a priming technique, with its effects on endothelial cells and precise spatial localization, achieves selective alteration of the surface molecule expression of the endothelial cells in AVMs without affecting normal brain vessels.[37, 55, 136]

Storer et al demonstrated proof of principle that a vascular targeting approach can achieve selective thrombosis in an animal model of AVMs,[160] involving a non-ligand strategy using lipopolysaccharide (LPS) and soluble tissue factor (sTF).

LPS, a major component of the outer membrane of Gram-negative bacteria, has prothrombotic and proinflammatory effects. The endotoxin effects of LPS occur as result of binding to CD14 and TLR4 receptor complexes,[176] amongst others, which lead to the induction of cytokines, such as tumour necrosis factor-α from inflammatory cells.[177-180] LPS also directly stimulates endothelial processes, including the expression of TF, interstitial and vascular cell adhesion molecules, and E-selectin.[179-182]

TF expression induced by LPS is insufficient to initiate coagulation, however the coadministration of soluble TF which binds to the endothelial surface after association with coagulation factor VIIa, allows the surface density of TF sufficient to initiate significant thrombus formation.[183]

Nonirradiated fistula vessels over-express VCAM and may externalize phosphatidylserine.[184] Following radiosurgery, there is further translocation of phosphatidylserine from the internal to the external endothelial membrane leaflet.[163]

68 Critically, the interaction of LPS and TF with externalised phosphatidylserine is a powerful stimulator of thrombosis.[160]

The results of this study reiterated prior investigation into the use of a vascular targeting strategy to achieve selective thrombosis.[160] However, studying the effects of the treatment over longer time points has allowed some interesting observations to be made.

Analysis of Doppler flow measurements did not show any statistically significant changes in the AVM after treatment. The results are variable, suggesting that flow through the large vessels was irregularly affected. One explanation for this finding could be that there are processes occurring that affect flow in opposing manners. The initial Doppler flow reading was taken at the time of fistula creation, and as the AVM matures, the flow would increase such that the later reading should show higher flow.

However, if significant thrombosis is occurring (even only in small vessels), then the flow through the AVM would be decreased. This incongruity could explain the variation seen in flow through the large vessels.

Similarly, whilst limited by technical issues, the angiograms did not show any significant changes in the fistula, or draining vein diameters. This finding would also support the inconsistency of the Doppler flow measurements, that no consistent thrombosis was found macroscopically or radiologically in the feeding artery or draining vein.

The critical finding of this study was the histological assessment of thrombosis.

In the AVM model nidus, thrombosis rates are increased by radiation. As seen in Table

2 and Figure 7, the increase in thrombosis occurs in a gradual consistent fashion increasing over time. Thrombosis is also seen, following administration of the targeting

69 agent. In this treatment group, though the thrombosis rates are higher, there is less consistency, especially with a decreased rate observed in the 90 day time point.

An explanation for this observation is that as demonstrated previously,[184] nonirradiated fistula vessels over express VCAM and may externalize phosphatidylserine. This allows the LPS and sTF to cause thrombosis, however without radiosurgery to prime the endothelium the effect is not potentiated.

An alternative explanation could be recanalization, a phenomenon seen following vessel occlusion occurring as result of disease or iatrogenic means.[185, 186] Recanalization may occur following radiosurgery for treatment of human AVMs. This may also serve to explain the inconsistency and decrease in thrombosis rates seen the treatment group that received targeting agent but no radiation, see Figure 13.

The most significant and consistent increase in thrombosis rates was observed in the treatment group that received radiosurgery followed by administration of targeting agent. This confirms the durability of the thrombosis formed, especially in comparison with the group that received targeting agent without radiation. Given the only difference in these two treatment groups was radiosurgery, this further confirms its importance in achieving durable small vessel thrombosis.

Despite the promising results of this study, it is important to acknowledge potential limitations that are present. The first relates to the use of an animal model, and the validity of its comparison to human AVMs. The rat model of AVM used in this study is an acquired lesion with histological changes that stabilise following fistula creation, in comparison to the congenital lesions that are human AVMs. The basis of this animal

70 model for AVM is that some morphological, histological, and antigenic similarites have been demonstrated when tissue is compared to human AVM vessels.[184],[187]

As recently published by Tu et al.,[187] the model AVM vessels undergo dilatation and demonstrate heterogenous wall thickening with proliferation of smooth muscle cells, splitting of the elastic lamina, thickened endothelial layers, endothelial cushions, lack of tight junctions, and loss of endothelial continuity. The model AVM endothelium expresses P-selectin while VEGF and vWF are strongly expressed in the endothelial and subendothelial layers similar to human AVMs.[55, 59, 184]

The significant difference between the model AVM and human intracranial AVM are that the animal model AVM vessels are not cerebral vessels, which have previously been discussed to have a number of unique characteristics. There may also be interspecies differences in tissue response to radiation and targeting agent which would not allow direct extrapolation of the results to the treatment of human AVMs.

Other animal models of AVM have been described in the literature. De Salles et al.,[188] amongst other authors,[189] have studied the use of a porcine model. The swine’s rete mirabile, possesses several attributes of AVMs. It is a tangle of microarteries and arterioles fed and drained by large blood vessels. The rete is in close relationship to vital structures of the animal’s brain, which would be in important consideration in the administration of radiosurgery. However, the rete mirabile is a normal structure, hence lacks the pathological characteristics and molecular changes seen in AVMs.

An AVM model in sheep was described by Qian et al.[190] In their model, the circulatory flow is diverted from the carotid artery through both carotid retia to the contralateral carotid artery and jugular vein with retrograde flow following surgical creation of an

71 carotid-jugular anastomosis and ligation. Whilst, angiographic appearances of this model resembled those seen in human AVMs there is no information available on histological changes occurring in this model.

A more promising model has been described by Pietilä et al. in canines.[191] Canine cerebrovascular anatomy is similar humans and in this model, a fistula is created between the middle cerebral artery and the dorsal sagittal sinus using a segment of the superficial temporal artery. A vascularised muscle flap supplied by the superficial temporal artery was implanted into the ischaemic territory supplied by the middle cerebral artery. A number of histological changes were seen, such as; venous thickening and fibrosis, formation of a network of new vessels with a structure resembling a nidus, occasional thrombus formation and evidence of ischaemia and haemorrhage. This shares similarities to changes found in human AVMs.

The ideal model would be a large animal model that incorporates the anatomic arrangement of a human AVM (feeding artery, nidus, draining vein) with a high flow shunt, and is composed of cerebral vessels. However, lack of such a model will be a limitation for the foreseeable future.

Another limitation of the present study is the assessment of toxicity. Utilizing an animal model will always have limitations as detailed neurological assessments cannot be obtained. In the rodents used in this experiment, careful note was made of animal weight, assessment of motor function, and behaviour. The lack of negative behaviours

(such as self mutiliation or excessive grooming), and the maintenance of animal weight allowed the inference that no significant morbidity was associated with the procedures.

No gross neurological deficits were observed.

72 Prior to developing this treatment strategy in humans, more investigation would be needed to assess for thrombosis in other regions of the body. Whilst the thoracic and abdominal viscera were examined for macroscopic evidence of thrombosis or ischaemia, detailed microscopic assessment of these organs would be essential.

Similarly, it would also be useful to perform haematological assessment of coagulation profile before and after administration of the targeting agent to confirm that systemic effects are not present.

The confirmation of proof of principle as seen in this study is encouraging for future research in this area, in addition to addressing and improving upon the limitations discussed,

The use of AVM endothelial cell cultures can be used to investigate the cellular responses, without the need for an animal model.

Further investigations into the mechanisms of radiation and targeting agent induced changes in endothelial cell surface protein expression and the timing of these changes could identify factors that might be utilized to increase thrombosis.

Another approach directly related to this study would be to further investigate with increased numbers, later time points, and to analyse the effects of varying doses, or varied timing of administration of radiation and targeting agent.

73

Conclusions

The results of this study are promising. Durable micro vessel thrombosis was achieved following radiosurgery and coadministration of LPS/sTF. The thrombosis was localized, and no systemic effects were seen. This confirms that the effect of radiosurgery to induce thrombosis can be enhanced and localized using vascular targeting agents.

Whilst further work is needed before human clinical trials, confirmation of this proof of principle warrants investigation of more vascular targeting agents.

74

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