Hussein A. Zeineddine

Endothelial TLR4 and the microbiome drive cerebral cavernous malformations

Cerebral cavernous malformations (CCMs) are a cause of stroke and seizure for which no effective medical therapies yet exist. CCMs arise from the loss of an adaptor complex that negatively regulates MEKK3–KLF2/4 signalling in brain endothelial cells, but upstream activators of this disease pathway have yet to be identified. Here we identify endothelial Toll-like receptor 4 (TLR4) and the gut microbiome as critical stimulants of CCM formation. Activation of TLR4 by Gram-negative bacteria or lipopolysaccharide accelerates CCM formation, and genetic or pharmacologic blockade of TLR4 signalling prevents CCM formation in mice. Polymorphisms that increase expression of the TLR4 gene or the gene encoding its co-receptor CD14 are associated with higher CCM lesion burden in humans. Germ-free mice are protected from CCM formation, and a single course of antibiotics permanently alters CCM susceptibility in mice. These studies identify unexpected roles for the microbiome and innate immune signalling in the pathogenesis of a cerebrovascular disease, as well as strategies for its treatment.

RhoA Kinase Inhibition With Fasudil Versus Simvastatin in Murine Models of Cerebral Cavernous Malformations.

Background and Purpose— We sought to compare the effect of chronic treatment with commonly tolerated doses of Fasudil, a specific RhoA kinase (ROCK) inhibitor, and simvastatin (with pleiotropic effects including ROCK inhibition) on cerebral cavernous malformation (CCM) genesis and maturation in 2 models that recapitulate the human disease. Methods— Two heterozygous murine models, Ccm1+/−Msh2−/− and Ccm2+/−Trp53−/−, were treated from weaning to 4 to 5 months of age with Fasudil (100 mg/kg per day), simvastatin (40 mg/kg per day) or with placebo. Mouse brains were blindly assessed for CCM lesion burden, nonheme iron deposition (as a quantitative measure of chronic lesional hemorrhage), and ROCK activity. Results— Fasudil, but not simvastatin, significantly decreased mature CCM lesion burden in Ccm1+/−Msh2−/− mice, and in meta-analysis of both models combined, when compared with mice receiving placebo. Fasudil and simvastatin both significantly decreased the integrated iron density per mature lesion area in Ccm1+/−Msh2−/− mice, and in both models combined, compared with mice given placebo. ROCK activity in mature lesions of Ccm1+/−Msh2−/− mice was similar with both treatments. Fasudil, but not simvastatin, improved survival in Ccm1+/−Msh2−/− mice. Fasudil and simvastatin treatment did not affect survival or lesion development significantly in Ccm2+/−Trp53−/− mice alone, and Fasudil benefit seemed limited to males. Conclusions— ROCK inhibitor Fasudil was more efficacious than simvastatin in improving survival and blunting the development of mature CCM lesions. Both drugs significantly decreased chronic hemorrhage in CCM lesions. These findings justify the development of ROCK inhibitors and the clinical testing of commonly used statin agents in CCM.

Peripheral plasma vitamin D and non-HDL cholesterol reflect the severity of cerebral cavernous malformation disease

AIM To correlate cerebral cavernous malformations (CCMs) disease aggressiveness with peripheral blood biomarkers hypothesized mechanistically. PATIENTS & METHODS A prospective case-control study enrolled 43 CCM patients, where 25-(OH) vitamin D, HDL and non-HDL cholesterol, CRP plasma levels and leukocyte ROCK activity were correlated with parameters of disease aggressiveness reflecting chronic and acute domains. RESULTS Patients with one or more features of chronically aggressive disease (early age at symptom onset, two or more symptomatic bleeds, high lesion burden) had significantly lower 25-(OH) vitamin D and non-HDL cholesterol levels in comparison to patients without these features. CONCLUSION Validation of these biomarkers and their potential treatment modulation may influence the clinical care of patients with CCM disease.

Vascular permeability and iron deposition biomarkers in longitudinal follow-up of cerebral cavernous malformations

OBJECTIVE Vascular permeability and iron leakage are central features of cerebral cavernous malformation (CCM) pathogenesis. The authors aimed to correlate prospective clinical behavior of CCM lesions with longitudinal changes in biomarkers of dynamic contrast-enhanced quantitative permeability (DCEQP) and quantitative susceptibility mapping (QSM) assessed by MRI. METHODS Forty-six patients with CCMs underwent 2 or more permeability and/or susceptibility studies in conjunction with baseline and follow-up imaging and clinical surveillance during a mean 12.05 months of follow-up (range 2.4-31.27 months). Based on clinical and imaging features, cases/lesions were classified as stable, unstable, or recovering. Associated and predictive changes in quantitative permeability and susceptibility were investigated. RESULTS Lesional mean permeability and QSM values were not significantly different in stable versus unstable lesions at baseline. Mean lesional permeability in unstable CCMs with lesional bleeding or growth increased significantly (+85.9% change; p = 0.005), while mean permeability in stable and recovering lesions did not significantly change. Mean lesional QSM values significantly increased in unstable lesions (+44.1% change; p = 0.01), decreased slightly with statistical significance in stable lesions (-3.2% change; p = 0.003), and did not significantly change in recovering lesions. Familial cases developing new lesions during the follow-up period showed a higher background brain permeability at baseline (p = 0.001), as well as higher regional permeability (p = 0.003) in the area that would later develop a new lesion as compared with the homologous contralateral brain region. CONCLUSIONS In vivo assessment of vascular permeability and iron deposition on MRI can serve as objective and quantifiable biomarkers of disease activity in CCMs. This may be applied in natural history studies and may help calibrate clinical trials. The 2 techniques are likely applicable in other disorders of vascular integrity and iron leakage such as aging, hemorrhagic microangiopathy, and traumatic brain injury.

Micro-computed tomography in murine models of cerebral cavernous malformations as a paradigm for brain disease.

BACKGROUND Cerebral cavernous malformations (CCMs) are hemorrhagic brain lesions, where murine models allow major mechanistic discoveries, ushering genetic manipulations and preclinical assessment of therapies. Histology for lesion counting and morphometry is essential yet tedious and time consuming. We herein describe the application and validations of X-ray micro-computed tomography (micro-CT), a non-destructive technique allowing three-dimensional CCM lesion count and volumetric measurements, in transgenic murine brains. NEW METHOD We hereby describe a new contrast soaking technique not previously applied to murine models of CCM disease. Volumetric segmentation and image processing paradigm allowed for histologic correlations and quantitative validations not previously reported with the micro-CT technique in brain vascular disease. RESULTS Twenty-two hyper-dense areas on micro-CT images, identified as CCM lesions, were matched by histology. The inter-rater reliability analysis showed strong consistency in the CCM lesion identification and staging (K=0.89, p<0.0001) between the two techniques. Micro-CT revealed a 29% greater CCM lesion detection efficiency, and 80% improved time efficiency. COMPARISON WITH EXISTING METHOD Serial integrated lesional area by histology showed a strong positive correlation with micro-CT estimated volume (r(2)=0.84, p<0.0001). CONCLUSIONS Micro-CT allows high throughput assessment of lesion count and volume in pre-clinical murine models of CCM. This approach complements histology with improved accuracy and efficiency, and can be applied for lesion burden assessment in other brain diseases.

B-Cell Depletion Reduces the Maturation of Cerebral Cavernous Malformations in Murine Models

Cerebral cavernous malformations (CCMs) are relatively common vascular malformations, characterized by increased Rho kinase (ROCK) activity, vascular hyper-permeability and the presence of blood degradation products including non-heme iron. Previous studies revealed robust inflammatory cell infiltration, selective synthesis of IgG, in situ antigen driven B-cell clonal expansion, and deposition of immune complexes and complement proteins within CCM lesions. We aimed to evaluate the impact of suppressing the immune response on the formation and maturation of CCM lesions, as well as lesional iron deposition and ROCK activity. Two murine models of heterozygous Ccm3 (Pdcd10), which spontaneously develop CCM lesions with severe and milder phenotypes, were either untreated or received anti-mouse BR3 to deplete B cells. Brains from anti-mouse BR3-treated mice exhibited significantly fewer mature CCM lesions and smaller lesions compared to untreated mice. B cell depletion halted the progression of lesions into mature stage 2 lesions but did not prevent their genesis. Non-heme iron deposition and ROCK activity was decreased in lesions of B cell depleted mice. This represents the first report of the therapeutic benefit of B-cell depletion in the development and progression of CCMs, and provides a proof of principle that B cells play a critical role in CCM lesion genesis and maturation. These findings add biologics to the list of potential therapeutic agents for CCM disease. Future studies would characterize the putative antigenic trigger and further define the mechanism of immune response in the lesions.

Thrombospondin1 (TSP1) replacement prevents cerebral cavernous malformations

KRIT1 mutations are the most common cause of cerebral cavernous malformation (CCM). Acute Krit1 gene inactivation in mouse brain microvascular endothelial cells (BMECs) changes expression of multiple genes involved in vascular development. These changes include suppression of Thbs1, which encodes thrombospondin1 (TSP1) and has been ascribed to KLF2- and KLF4-mediated repression of Thbs1. In vitro reconstitution of TSP1 with either full-length TSP1 or 3TSR, an anti-angiogenic TSP1 fragment, suppresses heightened vascular endothelial growth factor signaling and preserves BMEC tight junctions. Furthermore, administration of 3TSR prevents the development of lesions in a mouse model of CCM1 (Krit1ECKO) as judged by histology and quantitative micro-computed tomography. Conversely, reduced TSP1 expression contributes to the pathogenesis of CCM, because inactivation of one or two copies of Thbs1 exacerbated CCM formation. Thus, loss of Krit1 function disables an angiogenic checkpoint to enable CCM formation. These results suggest that 3TSR, or other angiogenesis inhibitors, can be repurposed for TSP1 replacement therapy for CCMs.

CSF inflammatory response after intraventricular hemorrhage

Objective: To investigate the temporal pattern and relevant associations of CSF inflammatory measures after intraventricular hemorrhage (IVH). Methods: We analyzed prospectively collected CSF cell counts and protein and glucose levels from participants in the Clot Lysis Evaluation of Accelerated Resolution of IVH phase III (CLEAR III) trial. Corrected leukocyte count and cell index were calculated to adjust for CSF leukocytes attributable to circulating blood. Data were chronologically plotted. CSF inflammatory measures (daily, mean, median, maximum, and cases with highest quartile response) were correlated with initial IVH volume, IVH clearance rate, thrombolytic treatment, bacterial infection, and adjudicated clinical outcome at 30 and 180 days. Results: A total of 11,376 data points of CSF results from 464 trial participants were analyzed. Measures of CSF inflammatory response evolved during the resolution of IVH. This was significantly more pronounced with initial IVH volume exceeding 20 mL. Intraventricular alteplase was associated with a significantly augmented inflammatory response compared to saline, even after correcting for initial IVH volume. There was an association but nonpredictive correlation of CSF inflammation measures with culture-positive CSF bacterial infection. None of the CSF inflammatory measures, including cases with upper quartile inflammatory response, was associated with a significant detrimental effect on 30 or 180 days functional outcome or mortality after multivariate adjustment for measures of disease severity. Conclusions: Aseptic CSF inflammation after IVH is primarily dependent on the volume of initial bleed. Thrombolysis intensifies the inflammatory response, with no apparent detrimental effect on clinical outcome. Clinicaltrials.gov identifier: NCT00784134.

Demographic Risk Factors for Vascular Lesions as Etiology of Intraventricular Hemorrhage in Prospectively Screened Cases

Background: Spontaneous intraventricular hemorrhage (IVH) is associated with high rates of morbidity and mortality despite critical care and other advances. An important step in clinical management is to confirm/rule out an underlying vascular lesion, which influences further treatment, potential for further bleeding, and prognosis. Our aim is to compare demographic and clinical characteristics between IVH patients with and without an underlying vascular lesion, and among cohorts with different vascular lesions. Methods: We analyzed prospectively collected data of IVH patients screened for eligibility as part of the Clot Lysis: Evaluation Accelerated Resolution of IVH Phase III (CLEAR III) clinical trial. The trial adopted a structured screening process to systematically exclude patients with an underlying vascular lesion as the etiology of IVH. We collected age, sex, ethnicity, and primary diagnosis on these cases and vascular lesions were categorized prospectively as aneurysm, vascular malformation (arteriovenous malformation, dural arteriovenous fistula, and cavernoma), Moyamoya disease, or other vascular lesion. We excluded cases <18 or >80 years of age. Baseline characteristics were compared between the CLEAR group (IVH screened without vascular lesion) and the group of IVH patients screened and excluded from CLEAR because of an identified vascular lesion. We further analyzed the differential demographic and clinical characteristics among subcohorts with different vascular lesions. Results: A total of 10,538 consecutive IVH cases were prospectively screened for the trial between 2011 and 2015. Out of these, 496 cases (4.7%) screened negative for underlying vascular lesion, met the inclusion criteria, and were enrolled in the trial (no vascular etiology group); and 1,205 cases (11.4%) were concurrently screened and excluded from the trial because of a demonstrated underlying vascular lesion (vascular etiology group). Cases with vascular lesion were less likely to be >45 years of age (OR 0.28, 95% CI 0.20-0.40), African-American (OR 0.23, 95% CI 0.18-0.31), or male gender (OR 0.48, 95% CI 0.38-0.60), and more likely to present with primary IVH (OR 1.85, 95% CI 1.37-2.51) compared to those with no vascular etiology (p < 0.001). Other demographic factors were associated with specific vascular lesion etiologies. A combination of demographic features increases the association with the absence of vascular lesion, but not with absolute reliability (OR 0.1, 95% CI 0.06-0.17, p < 0.001). Conclusion: An underlying vascular lesion as etiology of IVH cannot be excluded solely by demographic parameters in any patient. Some form of vascular imaging is necessary in screening patients before contemplating interventions like intraventricular fibrinolysis, where safety may be impacted by the presence of vascular lesion.

A Report of 2 Cases of Brainstem Hemorrhage After Suboccipital Craniectomy for Chiari Decompression

BACKGROUND AND IMPORTANCE Decompression surgery for Chiari malformation is known to have very low procedure-related complications. There has been no report of post-Chiari malformation decompression surgery development of brainstem hemorrhage. We report 2 post-Chiari decompression surgery brainstem hemorrhage cases with 2-yr follow-up. CLINICAL PRESENTATION Two cases were reviewed in which patients underwent uncomplicated suboccipital craniectomy with expansive autologous pericranium duraplasty for Chiari decompression. Postoperatively, both patients awoke with hemibody sensory and motor deficits. Immediate postoperative magnetic resonance imaging revealed a small hemorrhage within the dorsal medulla in both cases. Follow-up imaging shows resolution along with near complete clinical recovery of deficits. CONCLUSION These cases demonstrate a rare postdecompression surgery-related complication in Chiari malformation. We hypothesize that these hemorrhages may occur from the rapid drainage of cerebrospinal fluid resulting in a loss of positive pressure, allowing a low-pressure hemorrhage to occur. Given that these hemorrhages are of low pressure, recovery is excellent.