The purpose of this study was to analyze immunohistochemical characteristics of the cell population in a radiosurgically obliterated cerebral arteriovenous malformation (AVM) after recurrent hemorrhage. Immunohistochemical reactions were carried out on paraffin-embedded histological sections for von Willebrand factor (FVIII), CD34 endothelial antigen (CD34), vimentin and alpha-smooth-muscle actin (SMA) cytoskeletal proteins. Histopathological analysis revealed that the majority of AVM channels were replaced by hypocellular scar tissue. However some of them still disclosed thrombus organization by granulation tissue seven years after radiosurgery. FVIII and CD34 reactions revealed vessel neoformation in thrombuses. Proliferating spindle-shaped cells with SMA and vimentin expression were identified in the granulation tissue. These histopathological findings suggest the role of re-canalization in recurrence of hemorrhage following radiosurgical obliteration of the AVM. Contribution of myofibroblastic elements in the vessels' occlusion after radiosurgery is also demonstrated. Neurol Res 2003; 25: 718-721]
Keywords: AVM; radiosurgery; re-bleeding; re-canalization; immunohistochemistry; myofibroblast
INTRODUCTION
Arteriovenous malformations of the brain have been regarded as hamartomatous lesions originating from embryonic maldevelopment1,2. Histologically, they are characterized by clusters of abnormal arteries and arterialized veins with irregular vessel wall structure, and without intervening capillary bed. The first successful treatment of an AVM with radiosurgery was reported by Steiner et al. in 1972(3). Since then, it has become an effective and widespread primary or alternative method in the management of selected cerebral AVMs, especially for those with difficult access for surgery or embolisation4-6. Angiography has been regarded as the standard method to confirm AVM obliteration after radiosurgery7. Although we could find two reports on recurrent hemorrhage after angiographic confirmation of AVM occlusion8,9, immunohistochemical analysis of rebleeding after radiosurgically obliterated AVM has not yet been published. The purpose of this study was to analyze the phenotypic nature of cell population contributing to the obliterative process of the AVM, and in the recurrence of hemorrhage after radiosurgery.
PATIENT AND METHODS
A 25-year-old male patient was admitted with headaches, nausea, vomiting and confusion to another hospital in 1993. CT scan showed a left frontal intracerebral hemorrhage (ICH) bulging into the ventricular horn. Angiography revealed a tiny arteriovenous malformation in the region of the left caudate nucleus with a prominent draining vein towards the deep venous system. Secondary hydrocephalus developed, necessitating ventricular drainage. The AVM was treated with LINAC radiosurgery at an outside institution one month following the onset of the symptoms. The lesion was irradiated with 25 Gy at the 80% isodose line. Because of the persistence of raised intracranial pressure a left occipital ventriculo-peritoneal shunt was inserted. The patient recovered successfully. Occlusion of the AVM nidus was confirmed by control angiography two years later. Seven years after the initial stroke cognitive problems, headaches and memory disturbances occurred. A space occupying, hemorrhagic tumor-like lesion was discovered at the place of the original ICH. A growing mass with contrast enhancement and morphological heterogeneity was observed on serial control CT and MR images (Figure 1A). Repeated angiography did not reveal either a persistent nidus or an early draining vein, but a faint capillary 'blush' at the original locus of the AVM was noticed. Positron emission tomography (with C-11 labeled Methionine) demonstrated increased uptake of the tracer in the same area (Figure 1B). The diagnosis was not clear at that time. Signs of raised intracranial pressure developed due to the mass effect of the lesion. The patient's condition deteriorated, and the lesion was removed totally by emergency surgery craniotomy (Figure 1C). The macroscopic impression was of a hemorrhagic tumor-like tissue at the time of operation (Figure 1D).
The resected specimen was fixed in 10% neutral buffered formaldehyde, processed routinely, and embedded in paraffin. Besides the routine hematoxylin-eosin, van Gieson and Masson's trichrome staining, immunohistochemical reactions were carried out for FVIII, CD34, vimentin and SMA antigens. Biotin-streptavidin-peroxidase complex methods were performed according to standard protocols on 5 [mu]m thick sections. The following antibodies were used in this study: anti-FVIII (Rabbit Anti-Human poiyclonal, DAKO A/S, Denmark) and anti-CD34 (monoclonal Qbend/10, BioGenex, San Ramon, CA, USA) to highlight endothelial layer of the vessels, anti-vimentin (monoclonal V9, BioGenex), and anti-SMA (monoclonal asm-1, Novocastra, Newcastle-upon-Tyne, UK) to characterize cytoskeletal antigens in modified fibroblastic cells.
RESULTS
Histopathological examination of the surgical pathology material revealed a blend of obliterated AVM vessels, recent and elder secondary hemorrhages from recanalization of thrombuses. The majority of AVM vessels were completely replaced by hypocellular, hyalinized scar tissue, without identifiable aperture. At these channels only the contours were discernible. However, some of the AVM vessels showed irregularly stratified wall structure, and although organizing thrombuses obliterated the lumina, they were still recognizable. The thrombuses were in different stages of organization with neovascularization (Figure 2A). Around the newly formed vessels recent and older hemorrhages with hemosiderin production were demonstrated. Intense granulation tissue reaction surrounded and propagated into the thrombuses (Figure 2B). They were rich in thin walled capillaries, fibrocytes, fibroblasts, hemosiderinladen macrophages, and were infiltrated by lymphocytes.
Immunohistochemistry demonstrated prominent FVIII reactivity in the endothelial cells of newly formed rudimentary buds and channels in the obliterating thrombuses of the nonhyalinized AVM vessels (Figure 3A). These neoformations possessed thin, fragile wall structure, and were frequently surrounded by extensive hemorrhages. The rudimentary buds usually consisted of a single FVIII positive cell layer without any further recognizable wall elements. Endothelial cells showing remarkable CD34 positivity were observed in these regions as well, which was most striking in the vessel neoformations of thrombuses (Figure 3B). Areas of the AVM replaced by hyalinized scar tissue did not contain FVIII or CD34 positive cells. The proliferating granulation tissue around and within the thrombuses was abundant in spindle-shaped cells expressing marked vimentin positivity (Figure 3C). The shape of these spindle-shaped cells was identical to fibrocytes or activated fibroblasts. Cells with similar morphological characteristics demonstrated definite SMA immunreactivity as well (Figure 3D). In the hyalinized AVM regions scattered fibrocytes with faint vimentin positivity, but without SMA expression were detected.
DISCUSSION
Histological characteristics of pathological vessels in cerebral AVMs have been described extensively by previous authors. Following Virchow's fundamental work on pathological description of brain vascular lesions10, the current histopathological classification of cerebral vascular malformations into AVMs, cavernous angiomas, teleangiectasias and venous anomalies was elaborated by McCormick11. Since the role of radiosurgery has been increasing continuously in the neurosurgical realm, unknown complications like tumor induction by low radiation, or re-bleeding of an obliterated AVM might occur in the future.
The goal of radiosurgery is complete nidus occlusion in AVMs. This result is achieved pathologically through a thrombo-obliterative process in the vessels evoked by the ionizing energy of irradiation12,13. Angiographic and MRI examinations have documented that 65%-87% of AVMs are obliterated, and that 75% shrink in volume, 2-5 years after radiosurgery14. However it is not clear from the different series whether there is a significant change in the risk of hemorrhage during the latency period. The risk is higher for larger AVMs and for older patients, and it is lower when higher doses of radiation are used4?15. The totally obliterated nidus and the lack of an early draining vein verified on control angiogram had been previously considered as a cure after radiosurgical treatment of an AVM16. Recently a few cases have been reported with recurrent hemorrhage after angiographie confirmation of AVM occlusion8'9, but they did not supply immunohistological data about the background pathological changes.
Although the annual number of treatments has quadrupled in five years, the histopathological background to radiobiological and pathophysiological effects of radiosurgery has not been fully characterized. A few observations have been published on pathomorphological changes in partially obliterated AVMs after radiosurgery17-19. Blood vessels within the analyzed AVMs showed progressive changes leading to narrowing or occlusion of the lumen. These investigations demonstrate that the obi iterative process starts with endothelial destruction followed by subendothelial and perivascular spindle-shaped cell proliferation in subtotally obliterated AVM vessels after radiosurgery. This proliferation may be connected with thrombus formation. The immunohistochemical characteristics of the proliferating spindle-shaped cells in these studies were identical to myofibroblasts, originally described in granulation tissue during wound healing by Gabbiani et al20. Kondziolka et al.21 anticipated that a similar response would occur in AVMs that proceed to complete obliteration.
Results of the present study correlate with the previous observations that besides thrombus formation, spindle-shaped cells take part in the obliteration and shrinking of the AVM vessels after radiosurgery. The double vimentin and SMA expression in similar cells supports their myofibroblastic nature in the granulation tissue. The single vimentin demonstration in the hyalinized scar tissue areas suggests that resting fibrocytes replace the activated myofibroblasts at the end-stage of the obi iterative process. Scar tissue is a hypocellular, hypometabolic tissue undergoing hyalinization, and constitutes a rigid conglomerate, which might be responsible for stabilization of AVM occlusion following radiosurgery. However, until thrombus organization has not been completed in the vessels, neovascularization may supply a potential source for rebleeding. Yamamoto et al.19 has demonstrated contrast and gadolinium enhancement of the radiosurgically treated AVM nidus by CT and MRI as long as eight years after negative control angiography. This phenomenon might be attributed either to radiation related increased permeability of the AVM vessels' wall, or recanalization and granulation tissue formation, which is rich in capillaries and small arteries. Because of their small caliber relative toother vessels of the AVM, it is unlikely that the presence of recanalized channels could be detected by angiography17,19. The limit to visualize a blood vessel by angiography is 1 mm in diameter21.
The contrast enhancing mass seen seven years later at the site of the treatment might be radiologically so called 'radiation necrosis', however we would prefer the term 'radiation-induced changes'. In a pathological sense necrotic tissue means 'dead tissue' which results in resolution of the tissue at last, i.e. cyst formation. But at the presented case histopathology demonstrated vigorous, proliferating granulation tissue at the site of the 'capillary blush' or 'radiation necrosis' which contained large amount of capillaries, and it might have been responsible for the 'blush' and contrast enhancement.
Regarding the subtotal thrombus organization in spite of the negative control angiography after radiosurgery, the role of PET examination should be considered to assess metabolic activity of AVMs. This sophisticated method detects sensitively metabolic changes of proliferating tissues, like granulation tissue in the presented case. Therefore it might play an important role in the follow up management of radiosurgically treated AVMs, combined with anatomical data of angiography and MRI22.
ACKNOWLEDGEMENTS
The authors wish to thank Serge Goldman, MD, PhD, ULB, Hopital Erasme for the PET investigation. This study was supported by the Loterie Nationale, the Ministere de la Politique Scientifique, and th Fonds National de la Recherche Scientifique, Belgium, the Hungarian Ministry of Health & Welfare (ETT-2003).
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Gyorgy T. Szeifert*, Isabelle Salmon, Danielle Baleriaux, Jacques Brotchi and Marc Levivier
Centre Gamma Knife, Universite Libre de Bruxelles, Hopital Academique Erasme, Brussels, Belgium * National Institute of Neurosurgery, Budapest, Hungary
Correspondence and reprint requests to: Prof. Marc Levivier, MD, PhD, Centre Gamma Knife, Universite Libre de Bruxelles, Hopital Erasme, Route de Lennik 808, B-1070 Brussels, Belgium.
[Marc.Levivier@ulb.ac.be] Accepted for publication March 2003.
Copyright Forefront Publishing Group Oct 2003
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