Ifeanyi Iwuchukwu

The Novel Methods for Analysis of Exosomes Released from Endothelial Cells and Endothelial Progenitor Cells

Exosomes (EXs) are cell-derived vesicles that mediate cell-cell communication and could serve as biomarkers. Here we described novel methods for purification and phenotyping of EXs released from endothelial cells (ECs) and endothelial progenitor cells (EPCs) by combining microbeads and fluorescence quantum dots (Q-dots®) techniques. EXs from the culture medium of ECs and EPCs were isolated and detected with cell-specific antibody conjugated microbeads and second antibody conjugated Q-dots by using nanoparticle tracking analysis (NTA) system. The sensitivities of the cell origin markers for ECs (CD105, CD144) and EPCs (CD34, KDR) were evaluated. The sensitivity and specificity were determined by using positive and negative markers for EXs (CD63), platelets (CD41), erythrocytes (CD235a), and microvesicles (Annexin V). Moreover, the methods were further validated in particle-free plasma and patient samples. Results showed that anti-CD105/anti-CD144 and anti-CD34/anti-KDR had the highest sensitivity and specificity for isolating and detecting EC-EXs and EPC-EXs, respectively. The methods had the overall recovery rate of over 70% and were able to detect the dynamical changes of circulating EC-EXs and EPC-EXs in acute ischemic stroke. In conclusion, we have developed sensitive and specific microbeads/Q-dots fluorescence NTA methods for EC-EX and EPC-EX isolation and detection, which will facilitate the functional study and biomarker discovery.

Propofol–ketamine combination therapy for effective control of super-refractory status epilepticus ☆

Retrospective analysis was conducted of patients with SRSE who were treated simultaneously with propofol and ketamine. Sixty-seven patients were identified from 2012 to 2015, and outcomes documented were resolution and mortality. The duration of combined ketamine and propofol use ranged from 1 to 28 days (mean - 3.6 days). Infusion rates ranged up to 145 and 175 mcg/kg/min. Vasopressors were used in 53 patients (79%), and were given within the first 5 days of the ICU admission in 48 (91%) patients. The overall SRSE resolution rate was 91%, and the overall mortality including patients with anoxic brain injury was 39%. Of the 13 patients with SRSE as a result of anoxic brain injury, SRSE was controlled in 5 (56%). The primary determinant of mortality was family withdrawing care related to the presence of severe medical/neurological diseases.

Analyses of Endothelial Cells and Endothelial Progenitor Cells Released Microvesicles by Using Microbead and Q-Dot Based Nanoparticle Tracking Analysis

Accurate analysis of specific microvesicles (MVs) from biofluids is critical and challenging. Here we described novel methods to purify and detect MVs shed from endothelial cells (ECs) and endothelial progenitor cells (EPCs) by combining microbeads with fluorescence quantum dots (Q-dots) coupled nanoparticle tracking analysis (NTA). In the in vitro screening systems, we demonstrated that 1) anti-CD105 (EC marker) and anti-CD34 (EPC marker) conjugated-microbeads had the highest sensitivity and specificity for isolating respective MVs, which were confirmed with negative controls, CD41 and CD235a; 2) anti-CD144 (EC marker) and anti-KDR (EPC marker) conjugated-Q-dots exhibited the best sensitivity and specificity for their respective MV NTA detection, which were confirmed with positive control, anti-Annexin V (MV universal marker). The methods were further validated by their ability to efficiently recover the known amount of EC-MVs and EPC-MVs from particle-depleted plasma, and to detect the dynamical changes of plasma MVs in ischemic stroke patients, as compared with traditional flow cytometry. These novel methods provide ideal approaches for functional analysis and biomarker discovery of ECs- and EPCs- derived MVs.

Transgenic expression of HuR increases vasogenic edema and impedes functional recovery in rodent ischemic stroke

BACKGROUND AND PURPOSE Ischemic stroke produces significant morbidity and mortality, and acute interventions are limited by short therapeutic windows. Novel approaches to neuroprotection and neurorepair are necessary. HuR is an RNA-binding protein (RBP) which modulates RNA stability and translational efficiency of genes linked to ischemic stroke injury. METHODS Using a transgenic (Tg) mouse model, we examined the impact of ectopic HuR expression in astrocytes on acute injury evolution after transient middle cerebral artery occlusion (tMCAO). RESULTS HuR transgene expression was detected in astrocytes in perilesional regions and contralaterally. HuR Tg mice did not improve neurologically 72h after injury, whereas littermate controls did. In Tg mice, increased cerebral vascular permeability and edema were observed. Infarct volume was not affected by the presence of the transgene. CONCLUSIONS Ectopic expression of HuR in astrocytes worsens outcome after transient ischemic stroke in mice in part by increasing vasogenic cerebral edema. These findings suggest that HuR could be a therapeutic target in cerebral ischemia/reperfusion.

Description of a novel telemedicine-enabled comprehensive system of care: drip and ship plus drip and keep within a system of stroke care delivery.

United States (US) and worldwide telestroke programs frequently focus only on emergency room hyper-acute stroke management. This article describes a comprehensive, telemedicine-enabled, stroke care delivery system that combines “drip and ship” and “drip and keep” models with a comprehensive stroke center primary hub at Ochsner Medical Center in New Orleans, advanced stroke-capable regional hubs, and geographically-aligned, “stroke-ready” spokes. The primary hub provides vascular neurology expertise via telemedicine and monitors care for patients remaining at regional hubs and spokes using a multidisciplinary team approach. By 2014, primary hub telestroke consults grew to ≈1000/year with 16 min average door to consult initiation and 20 min to completion, and 29% of ischemic stroke patients received recombinant tissue-type plasminogen activator (rtPA), increasing 275%. Most patients remained in hospitals close to home, but neurointensive care and interventional procedures were common reasons for primary hub transfer. Given the time sensitivity and expert consultation needed for complex acute stroke care delivery paradigms, telestroke programs are effective for fulfilling unmet care needs. Combining drip and ship and drip and keep management allows more patients to stay “local,” limiting primary hub transfer unless more advanced services are required. Post admission telestroke management at spokes increases personnel efficiency and can positively impact stroke outcomes.

Characterization of the transient middle cerebral artery occlusion model of ischemic stroke in a HuR transgenic mouse line

This set of experiments characterizes a model of transient cerebral ischemic stroke in a transgenic (Tg) mouse line in which the glial fibrillary acidic protein (GFAP) promoter is utilized to drive expression of a human RNA-binding protein, HuR. Additionally, the effect of cerebral ischemia on the expression of endogenous Hu proteins is presented.

Management of Sodium Abnormalities in the Neurosurgical Intensive Care Unit

Patients with neurological injury commonly acquire changes in their sodium levels, both as a consequence of their disease process and iatrogenically. Hyper- or hyponatremia in the neurosurgical intensive care unit can easily be broken down to neurological causes, medical causes, and iatrogenic causes. Promptly diagnosing conditions in which sodium levels rapidly change are especially important in this patient population because of the effect on cerebral volume. When sodium levels change quickly, large shifts in water occur over the cellular membrane, which can lead to neuronal and cell swelling or shrinkage. Inappropriate or delayed diagnosis and treatment may lead to severe morbidity or even death. The rate of correction of these derangements depends on how rapidly the onset occurred. In this review, we will discuss hypernatremia and hyponatremia and their retrospective etiologies, diagnoses, treatments, and complications.

Macroglossia Associated with Brainstem Injury

BackgroundMacroglossia has been reported in patients undergoing posterior fossa neurosurgical procedures and is thought to be as a result of venous engorgement from intubation or mechanical positioning during these prolonged procedures.MethodsWe report three patients who developed macroglossia and dysautonomia of central neurogenic origin following brainstem injury.ResultsThe three patients developed macroglossia and dysautonomia with wide hemodynamic fluctuations in the setting of posterior fossa injury of the lower brainstem structures, necessitating tracheostomy placement. Macroglossia was managed with dexamethasone and there was complete resolution of dysautonomia while treated with beta-blockers and gabapentin.ConclusionsNeurointensivists should be aware of macroglossia with dysautonomia complicating brainstem injury, which may have perilous consequences in the setting of cerebral edema or intracranial hypertension.