I. Erol Sandalcioglu

Differential angiogenesis function of CCM2 and CCM3 in cerebral cavernous malformations

OBJECT Loss-of-function mutations in CCM genes are frequently detected in familial cerebral cavernous malformations (CCMs). However, the current functional studies of the CCM genes in vitro have been performed mostly in commercially purchased normal cell lines and the results appeared discrepant. The fact that the cerebral vascular defects are rarely observed in CCM gene-deficient animals suggests the requirement of additional pathological background for the formation of vascular lesions. Consistent with these data, the authors assumed that silencing CCM genes in the endothelium derived from CCMs (CCM-ECs) serves as a unique and valuable model for investigating the function of the CCM genes in the pathogenesis of CCMs. To this end, the authors investigated the role and signaling of CCM2 and CCM3 in the key steps of angiogenesis using CCM-ECs. METHODS Endothelial cells (ECs) derived from CCMs were isolated, purified, and cultured from the fresh operative specimens of sporadic CCMs (31 cases). The CCM2 and CCM3 genes were silenced by the specific short interfering RNAs in CCM-ECs and in control cultures (human brain microvascular ECs and human umbilical vein ECs). The efficiency of gene silencing was proven by real-time reverse transcriptase polymerase chain reaction. Cell proliferation and apoptosis, migration, tube formation, and the expression of phosphor-p38, phosphor-Akt, and phosphor-extracellular signal-regulated kinase-1 and 2 (ERK1/2) were analyzed under CCM2 and CCM3 silenced conditions in CCM-ECs. RESULTS The CCM3 silencing significantly promoted proliferation and reduced apoptosis in all 3 types of endothelium, but accelerated cell migration exclusively in CCM-ECs. Interestingly, CCM2 siRNA influenced neither cell proliferation nor migration. Silencing of CCM3, and to a lesser extent CCM2, stimulated the growth and extension of sprouts selectively in CCM-ECs. Loss of CCM2 or CCM3 did not significantly influence the formation of the tubelike structure. However, the maintenance of tube stability was largely impaired by CCM2, but not CCM3, silencing. Western blot analysis revealed that CCM2 and CCM3 silencing commonly activated p38, Akt, and ERK1/2 in CCM-ECs. CONCLUSIONS The unique response of CCM-ECs to CCM2 or CCM3 siRNA indicates that silencing CCM genes in CCM-ECs is valuable for further studies on the pathogenesis of CCMs. Using this model system, the authors demonstrate a distinct role of CCM2 and CCM3 in modulating the different processes of angiogenesis. The stimulation of endothelial proliferation, migration, and massively growing and branching angiogenic sprouts after CCM3 silencing may potentially contribute to the formation of enriched capillary-like immature vessels in CCM lesions. The severe impairment of the tube integrity by CCM2, but not CCM3, silencing is associated with the different intracranial hemorrhage rate observed from CCM2 and CCM3 mutation carriers. The activation of p38, ERK1/2, and Akt signal proteins in CCM2- or CCM3-silenced CCM-ECs suggests a possible involvement of these common pathways in the pathogenesis of CCMs. However, the specific signaling mediating the distinct function of CCM genes in the pathogenesis of CCMs needs to be further elucidated.

Implications of Dll4-Notch signaling activation in primary glioblastoma multiforme

BACKGROUND Glioblastoma multiforme (GBM) is a highly aggressive brain tumor characterized by massive neovascularization, necrosis, and intense resistance to therapy. Deregulated Notch signaling has been implicated in the formation and progression of different malignancies. The present study attempted to investigate the activation status of Dll4-Notch signaling in primary human GBM and its association with vascular and clinical parameters in patients. METHODS Major components of Dll4-Notch signaling were examined by real-time reverse-transcription polymerase chain reaction (PCR), Western blotting, and immunohistochemistry in GBM (n = 26) and control (n = 11) brain tissue. The vascular pattern (VP) and microvascular density (MVD) were analyzed after laminin immunostaining. O6-Methylguanine-methyltransferase (MGMT) promoter methylation in GBM samples was detected by methylation-specific PCR. RESULTS The mRNA levels of Dll4, Jagged1, Notch1, Notch4, Hey1, Hey2, Hes1, and VEGF were 3.12-, 3.58-, 3.37-, 5.77-, 4.89-, 3.13-, 6.62-, and 32.57-fold elevated, respectively, in GBM samples, compared with the controls. Western blotting revealed a 4-, 3.7-, and 45.6-fold upregulation of Dll4, Notch1, and Hey1, respectively, accompanied by a downregulation of PTEN expression and an increase in the expression of p-Akt and VEGF. Immunostaining located the immunoreactivity of Dll4 and Notch1 in endothelial cells, microglia/macrophages, tumor cells, and astrocytes. Furthermore, the upregulation of Dll4-Notch signaling components was correlated to a low MVD and was potentially related to a classic VP, tumor edema, and MGMT promoter methylation. CONCLUSIONS The upregulation of Dll4-Notch signaling components was found in a subset of GBM samples and was associated with some angiogenic and clinical parameters. These findings highlight this signaling pathway as a potential therapeutic target for patients with GBM who show an activation of Dll4-Notch signaling.

The venous angioarchitecture of sporadic cerebral cavernous malformations: a susceptibility weighted imaging study at 7 T MRI

Background and purpose To test the hypothesis that sporadic cerebral cavernous malformations (CCMs) are systematically associated with venous malformations (VMs) using susceptibility weighted imaging (SWI) at 7 Tesla (T) field MRI. Methods A prospective unselected series of 20 patients with symptomatic or asymptomatic sporadic CCM diagnosed using 1.5 T MRI was additionally scanned using high resolution (250 µm2 in-plane) SWI at 7 T MRI. Imaging data were analysed to examine the presence and formation of CCM associated venous vessel structures. Interobserver agreement was assessed using kappa statistics. Results In the 20 patients harbouring 23 CCMs, a solitary or multiple venous drainage was found in all lesions. A ‘typical’ VM was found in seven lesions. In the other cases, associated abnormal venous structures were also depicted although they appeared structurally different. Excellent interobserver agreement was achieved (95% confidence interval 0.92 to 0.99). Conclusions Our data support previous assumptions that sporadic CCMs are systematically associated with local venous abnormalities involving larger outflow vessels. However, the typical appearance of a VM was not confirmed in all cases. The role of the venous environment in the pathomechanism of CCMs remains unclear.

Spontaneous cerebellar hemorrhage—experience with 57 surgically treated patients and review of the literature

The treatment of spontaneous cerebellar hemorrhage is still discussed controversially. We analyzed a series of 57 patients who underwent surgical evacuation of a cerebellar hematoma at our department. Preoperative clinical and radiological parameters were assessed and correlated with the clinical outcome in order to identify factors with impact on outcome. The overall clinical outcome according to the Glasgow Outcome Scale at the last follow-up was good (GOS 4–5) in 27 patients (47%) and poor (GOS 2–3) in 16 patients (28%). Fourteen patients (25%) died. The initial neurological condition and the level of consciousness proved to be significant factors determining clinical outcome (p = 0.0032 and p = 0.0001, respectively). Among radiological parameters, brain stem compression and a tight posterior fossa solely showed to be predictive for clinical outcome (p = 0.0113 and p = 0.0167, respectively). Overall, our results emphasize the predictive impact of the initial neurological condition on clinical outcome confirming the grave outcome of patients in initially poor state as reported in previous studies. The hematoma size solely, in contrast to previous observations, showed not to be predictive for clinical outcome. Especially for the still disputed treatment of patients in good initial neurological condition, a suggestion can be derived from the present study. Based on the excellent outcome of patients with good initial clinical condition undergoing surgery due to secondary deterioration, we do not recommend preventive evacuation of a cerebellar hematoma in these patients.

Evaluation of Hardware-related Geometrical Distortion in Structural MRI at 7 Tesla for Image-guided Applications in Neurosurgery

RATIONALE AND OBJECTIVES Geometrical distortion is a well-known problem in structural magnetic resonance imaging (MRI), leading to pixel shifts with variations up to several millimeters. Because the main factors of geometrical distortion are proportional to B(0), MRI spatial encoding distortions tend to increase with higher magnetic field strength. With the increasing prospects of utilizing ultra-high-field MRI (B(0) ≥ 7 Tesla) for neuroimaging and subsequently for image-guided neurosurgical therapy, the evaluation and correction of geometrical distortions occurring in ultra-high-field MRI are essential preconditions for the integration of these data. Hence, we conducted a phantom study to determine hardware-related geometrical distortion in clinically relevant sequences for structural imaging at 7 T MRI and compared the findings to 1.5 T MRI. MATERIAL AND METHODS Hardware-related geometrical distortion was evaluated using a MRI phantom (Elekta, Sweden). Both applied scanner systems (Magnetom Avanto 1.5 T and Magnetom 7 T, Siemens Healthcare, Erlangen, Germany) were equipped with similar gradient coils capable of delivering 45 mT/m of maximum amplitude and a slew rate of 220 mT/m/ms. Distortion analysis was performed for various clinically relevant gradient echo and spin echo sequences. RESULTS Overall, we found very low mean geometrical distortions at both 7 T and 1.5 T, although single values of up to 1.6 mm were detected. No major differences in mean distortion between the sequences could be found, except significantly higher distortions in turbo spin-echo sequences at 7 T, mainly caused by B(1) inhomogeneities. CONCLUSION Hardware-related geometrical distortions at 7 T MRI are relatively small, which may be acceptable for image coregistration or for direct tissue-targeting procedures. Using a subject-specific correction of object-related distortions, an integration of 7 T MRI data into image-guided applications may be feasible.

Intraoperative MRI and Functional Mapping

The integration of functional and anatomical data into neuronavigation is an established standard of care in many neurosurgical departments. Yet, this method has limitations as in most cases the data are acquired prior to surgery. Due to brain-shift the accurate presentation of functional as well as anatomical structures declines in the course of surgery. In consequence, the acquisition of information during surgery about the brains current functional state is of specific interest. The advancement of imaging technologies (e.g. fMRI, MEG, Intraoperative Optical Intrinsic Signal Imaging--IOIS) and neurophysiological techniques and the advent of intraoperative MRI all had a major impact on neurosurgery. The combination of modalities such as neurophysiology and intraoperative MRI (ioMRI), as well as the acquisition of functional MRI during surgery (ifMRI) are in the focus of this work. Especially the technical aspects and safety issues are elucidated.

Non-enhanced MR imaging of cerebral aneurysms: 7 Tesla versus 1.5 Tesla.

Purpose To prospectively evaluate 7 Tesla time-of-flight (TOF) magnetic resonance angiography (MRA) in comparison to 1.5 Tesla TOF MRA and 7 Tesla non-contrast enhanced magnetization-prepared rapid acquisition gradient-echo (MPRAGE) for delineation of unruptured intracranial aneurysms (UIA). Material and Methods Sixteen neurosurgical patients (male n = 5, female n = 11) with single or multiple UIA were enrolled in this trial. All patients were accordingly examined at 7 Tesla and 1.5 Tesla MRI utilizing dedicated head coils. The following sequences were obtained: 7 Tesla TOF MRA, 1.5 Tesla TOF MRA and 7 Tesla non-contrast enhanced MPRAGE. Image analysis was performed by two radiologists with regard to delineation of aneurysm features (dome, neck, parent vessel), presence of artifacts, vessel-tissue-contrast and overall image quality. Interobserver accordance and intermethod comparisons were calculated by kappa coefficient and Lins concordance correlation coefficient. Results A total of 20 intracranial aneurysms were detected in 16 patients, with two patients showing multiple aneurysms (n = 2, n = 4). Out of 20 intracranial aneurysms, 14 aneurysms were located in the anterior circulation and 6 aneurysms in the posterior circulation. 7 Tesla MPRAGE imaging was superior over 1.5 and 7 Tesla TOF MRA in the assessment of all considered aneurysm and image quality features (e.g. image quality: mean MPRAGE7T: 5.0; mean TOF7T: 4.3; mean TOF1.5T: 4.3). Ratings for 7 Tesla TOF MRA were equal or higher over 1.5 Tesla TOF MRA for all assessed features except for artifact delineation (mean TOF7T: 4.3; mean TOF1.5T 4.4). Interobserver accordance was good to excellent for most ratings. Conclusion 7 Tesla MPRAGE imaging demonstrated its superiority in the detection and assessment of UIA as well as overall imaging features, offering excellent interobserver accordance and highest scores for all ratings. Hence, it may bear the potential to serve as a high-quality diagnostic tool for pretherapeutic assessment and follow-up of untreated UIA.

Proliferation Activity Is Significantly Elevated in Partially Embolized Cerebral Arteriovenous Malformations

Background: The natural history of cerebral arteriovenous malformations (AVMs) is yet to be determined. It has been shown that angiogenic factors are involved in the pathogenesis of AVMs, in particular in partially embolized lesions. This study was conducted to investigate the expression of angiogenic and proliferative factors in relation to different clinical conditions and treatment modalities. Methods: Immunohistochemistry was performed for 145 consecutive cases of cerebral AVMs. The specimens were stained with antibodies against VEGF, bFGF, Ki 67, CD 34 and CD 31. Expression was correlated with clinical presentation (haemorrhage, seizures or other symptoms), AVM localization, size, eloquence and venous drainage, as well as with preoperative AVM embolization. Results: Whereas no correlation was found between the expression of angiogenic factors and different clinical conditions, we observed a significantly increased proliferation activity as shown by Ki 67 expression in patients with intracerebral haemorrhage (p = 0.02) and in patients with preoperative embolization (p = 0.02). Conclusions: Increased proliferation activity in partially embolized AVMs supports a ‘no-touch’ strategy and clinical observation in high-risk AVMs and demands complete AVM elimination in treatable lesions.

Associated Aneurysms in Supratentorial Arteriovenous Malformations: Impact of Aneurysm Size on Haemorrhage

Background: Associated aneurysms (AAs) are presumed to represent an additional risk factor for intracranial haemorrhage from cerebral arterio-venous malformations (AVMs). To date, efforts to capture their natural history, as well as to identify aneurysms with the potential capability of regression after AVM treatment remain incomprehensive. As the aneurysm size represents an important aspect for the treatment indication of incidental saccular aneurysms, this factor has rarely been encountered for the treatment of AAs so far. The present study aims to determine the angiographic and clinical characteristics of AAs with special focus on aneurysm size and their consequences for treatment. Methods: Patients with cerebral AVMs, treated in our department between 1990 and 2013, were analyzed retrospectively. Only patients with supratentorial AVMs and flow-related AAs of the feeding arteries were evaluated. Thus, patients harboring AVMs of the cerebellum and the brain stem and patients with intranidal, venous or remote aneurysms were excluded. Treatment strategies were assessed with special attention on bleeding source and on AA size. Results: In 59 of 409 patients (14%) with supratentorial AVMs, a total of 85 AAs of the feeding arteries were identified. 14 of 59 individuals (24%) presented with multiple AAs. Of 85 AA, 58 aneurysms (68%) were classified as proximal and 27 aneurysms (32%) as distal. The most common location of AAs was the middle cerebral artery (MCA, 39%), followed by the internal carotid artery (ICA, 27%) and the anterior cerebral artery (ACA, 21%). The mean AA size was 4.4 mm ± 3.4 mm. Intracranial haemorrhage was found in 21 of 59 patients (36%) with coexisting AAs. Among these, 10 individuals (17%) suffered from rupture of an AA, accounting for nearly half of all bleedings in this subgroup. Among those patients bearing a single AA, the size of ruptured aneurysms differed significantly from those unruptured (6.6 mm vs. 4.4 mm, p = 0.0046). Nineteen patients (32%) received treatment of 22 AAs, whereas sole AVM treatment was adopted in 26 patients (44%) and conservative management in 14 patients (24%). The main reasons to leave AAs untreated were the small AA size (<5 mm), poor clinical state or treatment denial by the patients. Conclusions: The aneurysm size of AAs in AVM influences the risk of haemorrhage. Therefore, the treatment of larger (diameter ≥5 mm) AAs should be considered, even if a treatment indication of the associated AVM is not given.

In vitro characterization of the angiogenic phenotype and genotype of the endothelia derived from sporadic cerebral cavernous malformations.

BACKGROUND:Cerebral cavernous malformation (CCM) is mainly a disorder of endothelial cells. Although the endothelial function of CCM genes has been characterized in familial CCMs, little attention has been paid to the pathological alterations of the endothelium in sporadic CCMs. OBJECTIVE:We assumed that the endothelia derived from sporadic CCMs present genotypic and/or phenotypic alterations and exhibit unique responses to the pathogenic stimuli. METHODS:Endothelial cells were prepared from fresh operative specimens of sporadic CCMs with a single lesion (CCM-ECs, n = 20). The expression of VEGF and its receptors and CCM1-3 genes were detected by real-time reverse transcriptase polymerase chain reaction (RT2-PCR). Endothelial cell proliferation, migration, sprouting, and tube formation were compared between CCM-ECs and control endothelial cells after different angiogenic stimuli and after silencing CCM1. RESULTS:RT2-PCR revealed a highly activated VEGF system in CCM-ECs without significant alteration in CCM1-3 gene expression. Accordingly, CCM-ECs exhibited great growth potential under normal culture conditions and a significantly high proliferation activity in response to various angiogenic stimuli including hypoxia, fetal calf serum, and vascular endothelial growth factor treatment. A considerably higher mobility, spontaneous sprouting and extensive tube-branching were exclusively detected in CCM-ECs. In comparison with control endothelia, CCM-EC resisted apoptotic stimuli and showed distinct responses to activating angiogenesis after silencing CCM1. CONCLUSION:Distinct genotypic and phenotypic features occur in CCM-EC independently from the deficiency in CCM1-3 gene expression. The distinct responses of CCM-EC to different pathogenic stimuli suggest that CCM-EC is a valuable in vitro model for further study of CCMs.