Matthew C. Garrett

BEAMing and Droplet Digital PCR Analysis of Mutant IDH1 mRNA in Glioma Patient Serum and Cerebrospinal Fluid Extracellular Vesicles

Development of biofluid-based molecular diagnostic tests for cancer is an important step towards tumor characterization and real-time monitoring in a minimally invasive fashion. Extracellular vesicles (EVs) are released from tumor cells into body fluids and can provide a powerful platform for tumor biomarkers because they carry tumor proteins and nucleic acids. Detecting rare point mutations in the background of wild-type sequences in biofluids such as blood and cerebrospinal fluid (CSF) remains a major challenge. Techniques such as BEAMing (beads, emulsion, amplification, magnetics) PCR and droplet digital PCR (ddPCR) are substantially more sensitive than many other assays for mutant sequence detection. Here, we describe a novel approach that combines biofluid EV RNA and BEAMing RT-PCR (EV-BEAMing), as well droplet digital PCR to interrogate mutations from glioma tumors. EVs from CSF of patients with glioma were shown to contain mutant IDH1 transcripts, and we were able to reliably detect and quantify mutant and wild-type IDH1 RNA transcripts in CSF of patients with gliomas. EV-BEAMing and EV-ddPCR represent a valuable new strategy for cancer diagnostics, which can be applied to a variety of biofluids and neoplasms.

Clinical features, surgical treatment, and long-term outcome in adult patients with moyamoya disease. Clinical article.

Object To report the clinical features, surgical treatment, and long-term outcomes of adults with moyamoya phenomenon treated at a single institution in the United States.

A mouse model of intracerebral hemorrhage using autologous blood infusion

The development of controllable and reproducible animal models of intracerebral hemorrhage (ICH) is essential for the systematic study of the pathophysiology and treatment of hemorrhagic stroke. In recent years, we have used a modified version of a murine ICH model to inject blood into mouse basal ganglia. According to our protocol, autologous blood is stereotactically infused in two stages into the right striatum to mimic the natural events of hemorrhagic stroke. Following ICH induction, animals demonstrate reproducible hematomas, brain edema formation and marked neurological deficits. Our technique has proven to be a reliable and reproducible means of creating ICH in mice in a number of acute and chronic studies. We believe that our model will serve as an ideal paradigm for investigating the complex pathophysiology of hemorrhagic stroke. The protocol for establishing this model takes about 2 h.

Adjuvant Embolization With N-Butyl Cyanoacrylate in the Treatment of Cerebral Arteriovenous Malformations Outcomes, Complications, and Predictors of Neurologic Deficits

Background and Purpose— The purpose of this study was to assess the frequency, severity, and predictors of neurological deficits after adjuvant embolization for cerebral arteriovenous malformations. Methods— From 1997 to 2006, 202 of 275 patients with arteriovenous malformation received embolization before microsurgery (n=176) or radiosurgery (n=26). Patients were examined before and after endovascular embolization and at clinical follow-up (mean, 43.4±34.6 months). Outcome was classified according to the modified Rankin Scale. New neurological deficits after embolization were defined as minimal (no change in overall modified Rankin Scale), moderate (modified Rankin Scale ≤2), or significant (modified Rankin Scale >2). Results— Two hundred two patients were treated in 377 embolization procedures. There were a total of 29 new clinical deficits after embolization (8% of procedures; 14% of patients), of which 19 were moderate or significant. Postembolization deficits resolved in a significant number of patients over time (P<0.0001). Five patients had persistent neurological deficits due to embolization (1.3% of procedures; 2.5% of patients). In multivariate analysis, the following variables significantly predicted new neurological deficit after embolization: complex arteriovenous malformation with treatment plan specifying more than one embolization procedure (OR, 2.7; 95% CI, 1.4 to 8.6), diameter <3 cm (OR, 3.2; 95% CI, 1.2 to 9.1), diameter >6 cm (OR, 6.2; 95% CI, 1.0 to 57.0), deep venous drainage (OR, 2.7; 95% CI, 1.1 to 6.9), or eloquent location (OR, 2.4; 95% CI, 1.0 to 5.7). These variables were weighted and used to compute an arteriovenous malformation Embolization Prognostic Risk Score for each patient. A score of 0 predicted no new deficits, a score of 1 predicted a new deficit rate of 6%, a score of 2 predicted a new deficit rate of 15%, a score of 3 predicted a new deficit rate of 21%, and a score of 4 predicted a new deficit rate of 50% (P<0.0001). Conclusions— Small and large size, eloquent location, deep venous drainage, and complex vascular anatomy requiring multiple embolization procedures are risk factors for the development of immediate postembolization neurological deficits. Nevertheless, a significant number of patients with treatment-related neurological deficits improve over time. The low incidence of permanent neurological deficits underscores the usefulness of this technique in carefully selected patients.

A Comprehensive Review of Radiosurgery for Cerebral Arteriovenous Malformations: Outcomes, Predictive Factors, and Grading Scales

The management of cerebral arteriovenous malformations (AVMs) continues to present a challenge to neurosurgeons. The natural history of this condition, as well as the morbidity and mortality of therapeutic interventions, remains incompletely elucidated. Predictive factors and grading scales in AVM management allow risk-benefit analysis of treatment options and comparison of outcomes. Stereotactic radiosurgery is one of the established treatment modalities for AVMs and is generally used to treat lesions that are high risk for surgical resection. Radiosurgery aims to obliterate AVMs and thus prevent hemorrhage or seizure without any new or worsening of existing symptoms. Lesion characteristics and postsurgical complications differ markedly in patientstreated by radiosurgery versus microsurgery. Radiosurgery-based grading systems account for factors that have been associated with various aspects of radiosurgical outcomes including obliteration, hemorrhage, and postoperative complications, particularly those induced by radiation. The purpose of this paper is to describe the most current predictive factors and grading systems for radiosurgical treatment of cerebral AVMs.

The efficacy of direct extracranial-intracranial bypass in the treatment of symptomatic hemodynamic failure secondary to athero-occlusive disease: A systematic review

OBJECTIVE The 1985 International Extracranial-Intracranial (EC-IC) Bypass Trial failed to show a benefit following surgery in patients with varying degrees of angiographic ICA stenosis. More recent studies using modern technology to identify appropriate candidates, however, have generated promising findings. As a result, controversy exists regarding the role of this technique in the treatment of symptomatic athero-occlusive disease. To this end, we performed a systematic review and quantitative analysis of the literature to determine if a subset of patients with symptomatic hemodynamic failure secondary to athero-occlusive disease may benefit from direct EC-IC bypass. METHODS We performed a MEDLINE (1985-2007) database search using the following keywords, singly and in combination: EC-IC bypass, hemodynamic failure and misery perfusion. Additional studies were identified manually by scrutinizing references from identified manuscripts, major neurosurgical journals and texts, and personal files. Our literature search divided studies into three categories: natural history of patients with stage I hemodynamic failure (16 studies, 2320 patients), natural history of patients with stage II hemodynamic failure (3 studies 163 patients), and outcomes of patients with hemodynamic failure treated by EC-IC bypass (23 studies 506 patients). RESULTS Patients with severe stage I and stage II hemodynamic failure are at higher risk of cerebral infarction than those with mild disease (p=.014, OR 1.17-4.08 and p=0.10, OR 0.89-3.63, respectively). Additionally, patients with severe hemodynamic failure respond better to surgery than those with mild disease (p=0.03, OR 0.16-0.92). CONCLUSIONS Patients with severe hemodynamic failure secondary to athero-occlusive disease appear to benefit from direct EC-IC bypass surgery. As a result, the conclusions of the 1985 International EC-IC Bypass Trial may not be applicable to this subset of patients. A randomized clinical trial involving this patient population is warranted.

Clinical application of preconditioning and postconditioning to achieve neuroprotection.

Ischemic conditioning is a form of endogenous protection induced by transient, subcritical ischemia in a tissue. Organs with high sensitivity to ischemia, such as the heart, the brain, and spinal cord, represent the most critical and potentially promising targets for potential therapeutic applications of ischemic conditioning. Numerous preclinical investigations have systematically studied the molecular pathways and potential benefits of both pre- and postconditioning with promising results. The purpose of this review is to summarize the present knowledge on cerebral pre- and postconditioning, with an emphasis in the clinical application of these forms of neuroprotection. A systematic MEDLINE search for the terms preconditioning and postconditioning was performed. Publications related to the nervous system and to human applications were selected and analyzed. Pre- and postconditioning appear to provide similar levels of neuroprotection. The preconditioning window of benefit can be subdivided into early and late effects, depending on whether the effect appears immediately after the sublethal stress or with a delay of days. In general, early effects have been associated posttranslational modification of critical proteins (membrane receptors, mitochondrial respiratory chain) while late effects are the result of gene up- or downregulation. Transient ischemic attacks appear to represent a form of clinically relevant preconditioning by inducing ischemic tolerance in the brain and reducing the severity of subsequent strokes. Remote forms of ischemic pre- and postconditioning have been more commonly used in clinical studies, as the remote application reduces the risk of injuring the target tissue for which protection is pursued. Limb transient ischemia is the preferred method of induction of remote conditioning with evidence supporting its safety. Clinical studies in a variety of populations at risk of central nervous damage including carotid disease, cervical myelopathy, and subarachnoid hemorrhage have shown improvement in surrogate markers of injury. Promising preclinical and early clinical studies noting improvement in surrogate markers of central nervous injury after the use of remote pre- and postconditioning treatments demand follow-up systematic investigations to address effectiveness. Challenges in the application of these techniques to pressing clinical cerebrovascular disease ought to be overcome through careful, well-designed, translational investigations.

C3a receptor antagonist attenuates brain injury after intracerebral hemorrhage.

Neuroprotective therapy targeting the complement cascade may reduce injury associated with intracerebral hemorrhage (ICH). We investigated the role of C3a-receptor antagonist (C3aRA) after ICH in mice. Autologous whole blood was infused into the right striatum of mice that were treated with C3aRA or vehicle, using both a pre- and postinjury dosing regimen. Hematoma volume, brain water content, and inflammatory cell profile were assessed at 72 h post-ICH. Neurologic dysfunction was assessed by evaluating both spatial memory and sensorimotor capacity. Animals pretreated with C3aRA showed significantly improved neurologic function, brain water content, and granulocyte infiltration relative to vehicle-treated animals when assessed at 72 h. There was no significant difference in hemorrhagic/nonhemorrhagic ratio of microglial activation among all groups. Hematoma volumes were also not significantly different between C3aRA-treated and vehicle-treated animals. Administration of C3aRA beginning 6 h postinjury afforded significant amelioration of neurologic dysfunction as well as a reduction in brain water content. Treatment with C3aRA improved neurologic outcome while reducing inflammatory cell infiltration and brain edema formation after experimental ICH in mice. Results of this study suggest that the C3a receptor may be a promising target for therapeutic intervention in hemorrhagic stroke.

Treatment guidelines for cerebral arteriovenous malformation microsurgery

The goal of cerebral arteriovenous malformation (AVM) treatment is to eliminate intracerebral hemorrhage risk and to preserve or maximize neurological functions of the patient. Interventional planning must determine the modality or combination of modalities with the greatest success rate according to patient characteristics, AVM architecture, and the capabilities of the treatment option to fulfill the goals of treatment. Although there is a lack of data from randomized trials to guide AVM management, microsurgery is a mainstay of therapy in patients receiving definitive intervention. In this paper, we review current guidelines for surgical planning, risk-benefit analysis, and prediction of outcome in AVM patients.

Synergistic neuroprotective effects of C3a and C5a receptor blockade following intracerebral hemorrhage.

BACKGROUND Intracerebral hemorrhage (ICH) is associated with neurological injury that may be ameliorated by a neuroprotective strategy targeting the complement cascade. We investigated the role of C5a-receptor antagonist (C5aRA) solely and in combination with C3a-receptor antagonist (C3aRA) following ICH in mice. METHODS Adult male C57BL/6J mice were randomized to receive vehicle, C5aRA alone or C3aRA and C5aRA 6 and 12 h after ICH, and every 12 h thereafter. A double injection technique was used to infuse 30 microL of autologous whole blood into the right striatum. A final group of mice received a sham procedure consisting only of needle insertion followed by vehicle injections. Brain water content and flow cytometry analysis for leukocyte and microglia infiltration and activation in both hemispheres were measured on day 3 post ICH. Neurological dysfunction was assessed using a Morris water-maze (MWM), a 28-point scale, and a corner test at 6, 12, 24, 48 and 72 h after ICH induction. RESULTS Neurological deficits were present and comparable in all three cohorts 6 h after ICH. Animals treated with C5aRA and animals treated with combined C3aRA/C5aRA demonstrated significant improvements in neurological function assessed by both the corner turn test and a 28-point neurological scale at 24, 48 and 72 h relative to vehicle-treated animals. Similarly, C5aRA and C3aRA/C5aRA-treated mice demonstrated better spatial memory retention in the Morris water-maze test compared with vehicle-treated animals (C3aRA/C5aRA: 23.4+/-2.0 s p< or =0.0001 versus vehicle: 10.0+/-1.7 s). Relative to vehicle-treated mice, the brain water content in C3aRA/C5aRA-treated mice was significantly decreased in the ipsilateral cortex and ipsilateral striatum (ipsilateral cortex: C3aRA/C5aRA: 0.755403+/-0.008 versus 0.773327+/-0.003 p=0.01 striatum: 0.752273+/-0.007 versus 0.771163+/-0.0036 p=0.02). C5aRA-treated mice and C3aRA/C5aRA-treated mice had a decreased ratio of granulocytes (CD45(+)/CD11b(+)/Ly-6G(+)) in the hemorrhagic versus non-hemorrhagic hemispheres relative to vehicle-treated animals (C5aRA: 1.78+/-0.36 p=0.02 C3aRA/C5aRA: 1.59+/-0.22 p=0.005 versus vehicle: 3.01). CONCLUSIONS While administration of C5aRA alone provided neuroprotection, combined C3aRA/C5aRA therapy led to synergistic improvements in neurofunctional outcome while reducing inflammatory cell infiltration and brain edema. The results of this study indicate that simultaneous blockade of the C3a and C5a receptors represents a promising neuroprotective strategy in hemorrhagic stroke.