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J Neurosurg / November 1, 2013

DOI: 10.3171/2013.9.JNS122056

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AANS, 2013

Arteriovenous malformations (AVMs) of the brain are a leading cause of nontraumatic intracranial hemorrhage among children and young adults. The prevalence of AVMs is 10500 AVMs per 100,000 persons (0.01%0.5% of the population),1,5,38 yet these le-sions are the most common cause of neurological impair-ment or death among patients younger than 20 years.42,53

The goal of AVM treatment is to obliterate flow through the abnormal vessels and, therefore, eliminate the threat of intracranial hemorrhage, while maintaining normal circulation and preserving neurological function.

Each of the current treatment modalities (microsurgical resection, endovascular therapy, and radiosurgery) has its own benefits and risks. Microsurgical resection is cura-tive for small, superficial lesions in noneloquent parts of the brain with low complication rates.32,34,39 However, af-ter decades of advances and refinement, the techniques of microsurgical resection will probably not improve sub-stantially, and resection will always be invasive. Although endovascular therapy can be considered minimally inva-sive and in theory can obliterate flow, it is technically possible in only a few cases; its main use is as an adjunct to resection or radiosurgery.16,41,59

Durable thrombosis in a rat model of arteriovenous malformation treated with radiosurgery and vascular targeting

Laboratory investigation*Rajesh Reddy, M.B.B.s., F.R.a.C.s.,1 T. T. hong duong, Ph.d.,3 jaCoB M. FaiRhall, M.B.B.s.,1 RoBeRT i. sMee, M.B.B.s.,2 and MaRCus a. sToodley, Ph.d., F.R.a.C.s.31Prince of Wales Medical Research Institute; 2Department of Radiation Oncology, Prince of Wales Hospital; and 3Australian School of Advanced Medicine, Macquarie University, Sydney, Australia

Object. Radiosurgical treatment of brain arteriovenous malformations (AVMs) has the significant shortcomings of being limited to lesions smaller than 3 cm in diameter and of a latency-to-cure time of up to 3 years. A possible method of overcoming these limitations is stimulation of thrombosis by using vascular targeting. Using an animal model of AVM, the authors examined the durability of the thrombosis induced by the vascular-targeting agents lipo-polysaccharide and soluble tissue factor conjugate (LPS/sTF).

Methods. Stereotactic radiosurgery or sham radiation was administered to 32 male Sprague-Dawley rats serving as an animal model of AVM; 24 hours after this intervention, the rats received an intravenous injection of LPS/sTF or normal saline. The animals were killed at 1, 7, 30, or 90 days after treatment. Immediately beforehand, angiography was performed, and model AVM tissue was harvested for histological analysis to assess rates of vessel thrombosis.

Results. Among rats that received radiosurgery and LPS/sTF, induced thrombosis occurred in 58% of small AVM vessels; among those that received radiosurgery and saline, thrombosis occurred in 12% of small AVM vessels (diameter < 200 m); and among those that received LPS/sTF but no radiosurgery, thrombosis occurred at an inter-mediate rate of 43%. No systemic toxicity or intravascular thrombosis remote from the target region was detected in any of the animals.

Conclusions. Vascular targeting can increase intravascular thrombosis after radiosurgery, and the vessel occlu-sion is durable. Further work is needed to refine this approach to AVM treatment, which shows promise as a way to overcome the limitations of radiosurgery.(http://thejns.org/doi/abs/10.3171/2013.9.JNS122056)

Key WoRds arteriovenousmalformations radiosurgery vasculartargeting vasculardisorders

1

Abbreviations used in this paper: AVM = arteriovenous malfor-mation; LPS = lipopolysaccharide; sTF = soluble tissue factor.

* Drs. Reddy and Duong contributed equally to this work.

See the corresponding editorial, DOI: 10.3171/2013.6.JNS131080.

This article contains some figures that are displayed in color on line but in black-and-white in the print edition.

R. Reddy et al.

2 J Neurosurg / November 1, 2013

Radiosurgery has the benefits of low initial risk and can be administered on an outpatient basis. For treatment of AVMs of 3 cm in diameter or smaller, obliteration rates of 70%80% have been obtained.35,40 The limitations of radiosurgery include a delay to obliteration and decreased effectiveness for larger AVMs.2,10,12,13,18,25,29,43,44,46,61 For AVMs larger than 3 cm, all current treatment modalities have drawbacks, and very large AVMs are often untreat-able. Potential advances in radiosurgery could offer a treatment option for these cases. Radiosurgical planning and delivery methods are improving along with advances in computing and technology.1922,50

The effectiveness of radiosurgery could also be im-proved with the addition of biological methods. One such approach is enhancement of thrombosis. In the field of cancer research, various techniques are used to stimulate thrombosis within tumor vessels, resulting in tumor ne-crosis. These vascular-targeting techniques rely on con-stitutive molecular or physical differences between tumor vessels and normal vessels. The concept of using similar techniques to induce thrombosis within AVM vessels is at-tractive, but this approach is limited by the fact that AVM vessels do not differ enough from normal vessels to enable selective thrombosis. This limitation could be overcome if the radiosurgery-induced molecular changes within the targeted vessels were sufficient to differentiate them from normal vessels. We have been investigating this approach in an animal AVM model and have recently demonstrated proof of principle of a radiosurgery and vascular-targeting agent strategy to enhance early thrombosis.47

The aim of this study was to further evaluate the durability and long-term effectiveness of thrombosis ob-tained by using radiosurgery and vascular targeting.

Methods

Approval for animal experimentation was obtained from the Animal Care and Ethics Committee of the University of New South Wales. Animal experimenta-tion was performed in accordance with Animal Care and Ethics Committee guidelines and the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.

Thirty-two male Sprague-Dawley rats were used as the animal model of AVM. This model has been shown to share hemodynamic, angiographic, morphological, and molecular characteristics with human AVMs.23,31,33,49,55,56 The AVM model creation has been described in de-tail.23,47,54,60 Briefly, rats were anesthetized, and an ante-rior cervical approach was used to expose the left com-mon carotid artery and external jugular vein. Blood flow through the common carotid artery was measured by us-ing a 1-mm Doppler ultrasonic probe (Transonic Systems Inc.). The caudal external jugular vein (at its junction with the subclavian vein) was ligated, and it was anastomosed (end-to-side) to the common carotid artery with continu-ous 10-0 nylon sutures. Blood flow was then measured through the proximal common carotid artery and the ar-terialized vein by using 1-mm and 2-mm Doppler ultra-sonic probes, respectively (Transonic Systems Inc.).

The 32 rats were divided into 3 treatment groups: vas-

cular-targeting agent plus radiotherapy (LPs/sTF+RTX), vascular-targeting agent plus sham radiotherapy (LPs/sTF-RTX), and saline plus radiotherapy (saline+RTX). For each of the 4 time points (1, 7, 30, and 90 days after treatment), the LPs/sTF+RTX group contained 3 rats (to-tal 12), the LPs/sTF-RTX group contained 3 rats (total 12), and the saline+RTX group contained 2 rats (total 8).

At 6 weeks after creation of the AVM model, ra-diosurgery was administered. A custom-made stage was attached to the X-Knife LINAC device (Radionics). Radiation (25 Gy) was delivered to the model nidus with the 90% isodose line encompassing the fistula. The con-tralateral carotid arteries and jugular vein received a ra-diation dose less than 4 Gy.

At 24 hours after administration of radiosurgery, ei-ther LPS/sTF or saline was injected into the femoral vein of each rat that had received radiosurgery. At 24 hours after sham radiosurgery, LPS/sTF was injected; LPS was given at 0.1 mg/kg body weight, and sTF was given at 0.4 mg/kg body weight. The LPS and sTF were suspended in 1 ml 0.9% sterile saline; injection of the suspension was followed by 1.5 ml 0.9% sterile saline to flush the injec-tion line. Rats in the saline group received only 1.5 ml of 0.9% sterile saline.

At 1, 7, 30, or 90 days after injection, angiograms (via a mini-laparotomy and iliac artery cut down) were obtained to assess the nidus and draining vein; imme-diately afterward, rats were killed by paraformaldehyde perfusion fixation. After perfusion, vascular tissue from the neck region bilaterally was harvested for analysis. Tissue samples were embedded in paraffin. Sections of 10-m thickness were taken from the proximal carotid artery, carotidjugular anastomosis, distal carotid artery, arterialized feeding vein, infracranial AVM nidus, con-tralateral infracranial tissue, contralateral carotid artery, and draining vein. For confirmation of the presence of antemortem thrombi in the vessel lumen, sections were stained with H & E or with Martius scarlet blue.

The end points examined were fistula blood flow (measured by Doppler flowmetry), draining vein diameter (measured angiographically), and intravascular thrombo-sis rates (assessed histologically). Thrombosed and pat-ent vessels were counted on 5 representative high-power fields of sections from each vascular region and from each of the harvested organs by 2 observers blinded to the animal treatment group.

Using H & Estained sections and Martius scar-let bluestained sections, the same 2 observers counted thrombosed and patent vessels on 5 representative high-power fields from the AVM nidus. The results of each ob-server were compared; if results differed, those sections were reexamined by both observers together, and the number of thrombi was decided. We performed ANOVA using SPSS version 13 software (SPSS Inc.). A value of p < 0.05 was considered statistically significant.

Results

Blood Flow and AngiographyThe Doppler measurements of flow through the fis-


Vascular targeting for brain AVM treatment

3

tula obtained before treatment and at 1, 7, 30, and 90 days after treatment are shown in Table 1. Doppler assessment of the blood flow was performed at 2 points during the ex-periment. Before fistula formation, blood was measured through the common carotid artery, and after treatment (before the animal was killed), blood flow was measured through the arterialized vein by using a 1- to 4-mm ultra-sonic probe (Transonic Systems). Results of Doppler flow measurement analyses were not statistically significant.

Thrombosis RatesIntravascular thrombosis was detected in rats in all

3 groups (Fig. 1). Average thrombosis rates were high-est (58%) among animals in the LPS/sTF+RTX group, second highest (43%) among those in the LPS/sTFRTX group, and lowest (13%) among those in the saline+RTX group. Among animals in the control saline+RTX group, an initial absence of thrombosis was followed by a steady increase in thrombosis over time.

Statistical analysis of pooled data from the experi-mental groups revealed an association between LPS/sTF (with or without radiation) and development of thrombi (p = 0.03). At 90 days, thrombosis rates among rats in the LPS/sTF+RTX group were significantly higher than rates among rats in the other 2 groups (p < 0.01).Histological Analyses

Histological examination demonstrated the presence of antemortem thrombi and postmortem clots within the vessel lumens. In addition to staining the sections with hemotoxylin and eosin (Fig. 2), to confirm the presence of fresh, mature, and old fibrin antemortem thrombi, we also used Martius scarlet blue stain (Fig. 3). Occlusion was oc-casionally seen in larger vessels; however, most thrombi were seen in small vessels (less than 200 mm). The an-temortem thrombi were a composition of fibrinplatelet bands and erythrocyte-rich accumulations. In addition, lines of Zahn were occasionally seen; the paucity of this finding could be explained by thrombi occurring in the smaller vessels, with less area for lamination to occur. Among rats in the saline+RTX control group, very few thrombosed vessels were found; no thrombosis was seen at day 7 (Fig. 2 A2), but thrombosis gradually and steadi-ly increased over time, as did evidence of radiation effect, such as intimal hypertrophy and subendothelial prolifera-

tion (Fig. 2 B2 and C2). In contrast, among rats in the LPS/sTFRTX sham radiation group, rates of thrombosis were higher (Fig. 2 A1, B1, and C1). Among rats in this group, rates of thrombosis varied according to time, al-though not with a progressive increase. Among rats in the LPS/sTF+RTX full-treatment group, rates of formation of fibrin and erythrocyte thrombi were the highest; some variation occurred over time (Fig. 2 A3, B3, C3; Fig. 3A and B). The highest rate of thrombosis was observed at the final time point (Day 90).

Discussion

As the leading cause of nontraumatic intracranial hemorrhage among children and young adults, intracra-nial AVMs are clinically significant.42,53 Hemorrhage accounts for a considerable short-term mortality rate of 10%29%26,52 and a risk for permanent disability of 14%85%.3,38 The classification of AVMs according to size, pattern of venous drainage, and neurological eloquence of adjacent brain enables stratification of treatment risk.4 Low-grade AVMs (Grade III) account for approximate-ly 40% of AVMs and can be treated with low risk.4,15 One-third of AVMs are high grade (Grade IVV), and over 90% of these are untreatable.9,45 Of the intermediate-grade AVMs (Grade III), many are not treatable without high risk. New treatments are needed for these patients.

A promising new therapeutic technique is the pro-TABLE 1: Doppler flow through arterialized jugular vein of rats, before and after treatment*

Blood Flow (ml/min)LPS/sTF+RTX LPS/sTF-RTX Saline+RTX

Time (days) Pre-Tx Post-Tx Change Pre-Tx Post-Tx Change Pre-Tx Post-Tx Change

1 43 6.56 99 25.53 56 35 5.51 128 37.9 93 100 19 72 21.21 -287 53 12.67 176 39.26 123 72 20.07 296 46 224 67 13 113 7.7 46

30 21 6.5 17 9 -4 NM NM 0 NM NM 090 29 4.36 249 25.4 220 105 9.71 291 41.63 186 44 12 16 11.31 -28

* NM=flownotmeasuredbecauseofequipmentfailure;Tx=treatment. Indicatesdaysposttreatment. ValuesarepresentedasthemeanSEM.

Fig. 1. Intravascular thrombosis in theAVMnidusmodel (percent-ageofvessels),by treatmentgroup, invesselssmaller than200mm at4 timepoints.At90days, thrombosis in rats in theLPS/sTF+RTXgroupdifferedsignificantly fromthat inrats in theLPS/sTF-RTX and saline+RTXgroups(p

R. Reddy et al.


motion of intravascular thrombosis by use of molecular therapies. Similar strategies have been used for cancer therapy, in which deliberate stimulation of intravascular thrombosis has been investigated as a way to achieve tu-mor necrosis.11,14,17,30,51 This vascular targeting can use a ligand-directed strategy, combining a targeting moiety and an effector moiety. The targeting moiety is an anti-body or peptide that binds to a marker that is selectively expressed on tumor vessel endothelium, and the effector moiety induces thrombosis.51 Vascular targeting can also use a nonligand strategy, which involves administration of agents that directly interact with the target cells be-cause of 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 cura-tive. For selective thrombosis of AVM vessels with either a ligand-directed or a nonligand strategy, endothelial sur-face molecules that highly discriminate between AVM

vessels and normal vessels are required. The differences in molecular characteristics and ultrastructure between AVM vessels and normal vessels have been investigated. Expression of interstitial cell adhesion molecule-1, vas-cular cell adhesion molecule-1, and E-selectin is higher in human AVM endothelium than in normal cerebral vessel endothelium.55 In addition, AVM endothelial cells demonstrate loss of tight junctions, abnormal cytoplas-mic processes, and production of Weibel-Palade bodies.57 However, unlike vascular targeting in cancer research, in which inherent differences between tumor and normal endothelial cells are sufficient to enable selective target-ing, no marker has been detected in AVMs that would be sufficiently discriminating to allow production of throm-bosis within AVM vessels only.28,48,49,5558

We have proposed that stereotactic radiosurgery could be used as a priming technique, selectively altering the sur-face molecule expression of the endothelial cells in AVMs without affecting normal brain vessels.27,48,55 In this para-digm, radiosurgery is not being used as the main treatment modality; it is simply being used to create discriminating endothelial changes within AVM endothelial cells.

In a previous study, we showed that radiosurgery and vascular targeting can induce selective intravascular thrombosis in an animal model of AVM.47 In that study, we used a nonligand approach and administered LPS/sTF systemically 1 day after radiosurgery. Among its many effects, LPS directly stimulates endothelial expression of tissue factor, interstitial and vascular cell adhesion mol-ecules, and E-selectin.6,7,24,37

Tissue factor expression induced by LPS is insufficient to initiate coagulation; however, the coadministration of sTF, which binds to the endothelial surface after association with coagulation factor VIIa, allows the surface density of

Fig. 3. Evidenceofsmallvesselocclusionfromantemortemthrom-bus in representative sections from irradiated rat tissue treated withLPSandsTFandstainedwithMartiusscarletblue.Stainingwasblindlyappliedto5representativehigh-powerfieldimageson3independentAVM nidus tissues. A: At 30 days after treatment.Originalmagni-fication40;Bar=200m. B:At90daysafter treatment.Originalmagnification20;Bar=500m.

Fig. 2. IntravascularthrombosisratesassessedhistologicallywithH&EstaininAVMnidustissue.Stainingwasblindlyap-pliedto5representativehigh-powerfieldimageson3independentAVMnidustissues.Thrombosedandpatentvesselswerecountedbyobserversblindedastotheanimaltreatmentgroup:LPS/sTF-RTX (A1, B1, and C1),saline+RTX(A2, B2, and B3),and LPS/sTF+RTX (A3, B3, and C3)at7days(A1A3),30days(B1B3),and90days(C1C3)aftertreatment.EvidenceofsmallvesselthrombosisisseenintheLPS/sTF-RTXandtheLPS/sTF+RTXtreatmentgroupsatDays7,30,and90butnotinthesaline+RTXgroup.Originalmagnification40;Bar=200m.



5

tissue factor to reach sufficient levels to initiate thrombus formation. This effect is also dependent on the endothe-lial cell membrane altering its structure so that phospha-tidylserine is on the external surface.8 Externalization of phosphatidylserine occurs in tumor vessels, which allows the LPS/sTF treatment to produce thrombosis selectively in those vessels. It has been shown that radiation causes phos-phatidylserine externalization in lung endothelium,36 and we have shown that radiosurgery causes externalization of phosphatidylserine in the endothelial cells in the AVM ves-sels (J. M. Fairhall et al., personal communication, 2010).

The previous study showed that radiosurgery with vascular-targeting therapy is feasible, but that study did not address the durability of the induced thrombosis. The results of the study reported here demonstrate that the in-duced thrombosis is durable, and even increases, up to at least 3 months after treatment. The highest thrombosis rates were observed for rats in the LPS/sTF+RTX group. Thrombosis was localized to vessels within the tissue vol-ume treated with radiosurgery.

Thrombosis did occur in rats in the LPS/sTFRTX group. It is possible that sufficient endothelial changes are caused by the shear stress from the high blood flow in these vessels23 to enable the LPS/sTF treatment to induce thrombosis. Among rats in this group, thrombosis dura-bility appeared less robust; rates of vessel occlusion at the later time points were lower.

As with the earlier study, the radiosurgery and vascu-lar-targeting treatment successfully created small vessel thrombosis but did not result in occlusion of larger vessels. This finding accounts for the lack of blood flow changes detected by using Doppler flow measurements and angi-ography. Possible explanations for the lack of large vessel thrombosis include a difference in the endothelial response to radiation and, more likely, sufficiently high flow through these vessels to prevent thrombus formation.

This work supports the concept of radiosurgery and vascular targeting as a possible new treatment method for AVMs. We are currently working to identify other discrim-inating endothelial surface molecular changes that could be targeted by using a ligand-based technique. Although the nonligand approach reported here produced significant thrombosis, using LPS in humans raises safety concerns. A ligand approach has many theoretical advantages, in-cluding the ability to use different effector moieties and the ability to attach multiple effector molecules to a single antibody. Using nanoparticles containing large concentra-tions of an effector moiety attached to a targeting antibody is an attractive approach that is worth exploring.

In this study, we did not achieve complete occlusion of the model AVM, and large vessels were not throm-bosed. In addition to using a ligand-based treatment, oth-er possible methods to overcome these limitations include increasing the dose of the targeting agent, using multiple treatments, increasing the effector concentration by us-ing nanoparticle technology, and combining the vascular-targeting treatment with endovascular techniques.

Conclusions

The results of this study support the concept of us-

ing radiosurgery as a primer to enable vascular-targeting treatment for AVMs. Durable microvessel thrombosis was achieved after radiosurgery and administration of LPS/sTF. The thrombosis was localized, and no systemic effects were seen. These findings confirm that the effect of radiosurgery used to induce thrombosis can be en-hanced and localized by using vascular-targeting agents. Although more work is needed before human clinical tri-als can be conducted, confirmation of this proof of prin-ciple warrants investigation of more vascular-targeting agents.

Disclosure

The authors report no conflict of interest concerning the mate-rials or methods used in this study or the findings specified in this paper.

This study was funded in part by grants from the Neurosurgical Society of Australasia and the Brain Foundation. Dr. Reddy was sup-ported by a grant from the Royal Australasian College of Surgeons.

Author contributions to the study and manuscript preparation include the following. Conception and design: Smee, Stoodley. Acquisition of data: Reddy. Analysis and interpretation of data: Reddy. Drafting the article: Duong, Reddy. Critically revising the article: all authors. Reviewed submitted version of manuscript: all authors. Approved the final version of the manuscript on behalf of all authors: Duong. Statistical analysis: Reddy, Fairhall. Administrative/technical/material support: Duong. Study supervision: Stoodley.

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Manuscript submitted November 19, 2012.Accepted September 10, 2013.Please include this information when citing this paper: published

online November 1, 2013; DOI: 10.3171/2013.9.JNS122056.Address correspondence to: T. T. Hong Duong, Ph.D., The

Australian School of Advanced Medicine, 2 Technology Place, Macquarie University, Sydney, NSW 2109, Australia. email: [email protected].

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