Dimitris G. Placantonakis

High-Throughput Screening Assay for the Identification of Compounds Regulating Self-Renewal and Differentiation in Human Embryonic Stem Cells

High-throughput screening (HTS) of chemical libraries has become a critical tool in basic biology and drug discovery. However, its implementation and the adaptation of high-content assays to human embryonic stem cells (hESCs) have been hampered by multiple technical challenges. Here we present a strategy to adapt hESCs to HTS conditions, resulting in an assay suitable for the discovery of small molecules that drive hESC self-renewal or differentiation. Use of this new assay has led to the identification of several marketed drugs and natural compounds promoting short-term hESC maintenance and compounds directing early lineage choice during differentiation. Global gene expression analysis upon drug treatment defines known and novel pathways correlated to hESC self-renewal and differentiation. Our results demonstrate feasibility of hESC-based HTS and enhance the repertoire of chemical compounds for manipulating hESC fate. The availability of high-content assays should accelerate progress in basic and translational hESC biology.

Safety of low-dose intrathecal fluorescein in endoscopic cranial base surgery.

OBJECTIVE Intraoperative identification of cerebrospinal fluid (CSF) leakage is critical in successful closure after endoscopic cranial base surgery. Intrathecal injection of fluorescein is quite useful in identifying CSF leaks. However, complications have been reported with various doses and the technique has fallen out of favor. We explored the safety of low-dose intrathecal fluorescein administered to patients undergoing endoscopic cranial base surgery. METHODS A retrospective chart review and postoperative patient survey were performed. The nature and incidence of complications and subjective complaints were recorded in 54 patients who underwent endoscopic, endonasal approaches to the anterior cranial base and received intrathecal fluorescein after premedication with dexamethasone and diphenhydramine. RESULTS Intraoperative CSF leak was identified with fluorescein in 46.3% of the patients and helped determine the reconstruction technique. Postoperative CSF leak occurred in 9.3% of the patients and resolved with lumbar drainage. There were no seizures. Most side effects were nonspecific, transient, and likely not caused by fluorescein including malaise (57.4%), headache (51.9%), dizziness (31.5%), or nausea/vomiting (24.1%). Three patients (5.6%) experienced persistent subjective lower extremity weakness (n = 2) and numbness (n = 2) postoperatively; however, two of them had undergone lumbar drainage. CONCLUSION Low-dose injection of intrathecal fluorescein after premedication with steroid and antihistamine agents is generally safe. Most symptoms are nonspecific and transient, likely caused by the surgery or lumbar drainage. However, fluorescein should be administered with some caution because it may be responsible for occasional lower extremity weakness and numbness.

Is autism due to brain desynchronization

The hypothesis is presented that a disruption in brain synchronization contributes to autism by destroying the coherence of brain rhythms and slowing overall cognitive processing speed. Particular focus is on the inferior olive, a precerebellar structure that is reliably disrupted in autism and which normally generates a coherent 5–13 Hz rhythmic output. New electrophysiological data reveal that the continuity of the rhythmical oscillation in membrane potential generated by inferior olive neurons requires the formation of neuronal assemblies by the connexin36 protein that mediates electrical synapses and promotes neuronal synchrony. An experiment with classical eyeblink conditioning is presented to demonstrate that the inferior olive is necessary to learn about sequences of stimuli presented at intervals in the range of 250–500 ms, but not at 700 ms, revealing that a disruption of the inferior olive slows stimulus processing speed on the time scale that is lost in autistic children. A model is presented in which the voltage oscillation generated by populations of electrically synchronized inferior olivary neurons permits the utilization of sequences of stimuli given at, or faster than, 2 per second. It is expected that the disturbance in inferior olive structure in autism disrupts the ability of inferior olive neurons to become electrically synchronized and to generate coherent rhythmic output, thereby impairing the ability to use rapid sequences of cues for the development of normal language skill. Future directions to test the hypothesis are presented.

BAC Transgenesis in Human Embryonic Stem Cells as a Novel Tool to Define the Human Neural Lineage

Human embryonic stem cells (hESCs) have enormous potential for applications in basic biology and regenerative medicine. However, harnessing the potential of hESCs toward generating homogeneous populations of specialized cells remains challenging. Here we describe a novel technology for the genetic identification of defined hESC‐derived neural cell types using bacterial artificial chromosome (BAC) transgenesis. We generated hESC lines stably expressing Hes5::GFP, Dll1::GFP, and HB9::GFP BACs that yield green fluorescent protein (GFP)+ neural stem cells, neuroblasts, and motor neurons, respectively. Faithful reporter expression was confirmed by cell fate analysis and appropriate transgene regulation. Prospective isolation of HB9::GFP+ cells yielded purified human motor neurons with proper marker expression and electrophysiological activity. Global mRNA and microRNA analyses of Hes5::GFP+ and HB9::GFP+ populations revealed highly specific expression signatures, suggesting that BAC transgenesis will be a powerful tool for establishing expression libraries that define the human neural lineage and for accessing defined cell types in applications of human disease. STEM CELLS 2009;27:521–532

Intrathecal Fluorescein in Endoscopic Skull Base Surgery

OBJECTIVES: Reconstruction following endoscopic skull base surgery requires a high degree of success to avoid the morbidity of postoperative cerebrospinal fluid (CSF) leak. The impact on outcomes of CSF visualization with intrathecal fluorescein, however, is unknown. STUDY DESIGN: A retrospective review of patients undergoing endoscopic skull base surgery with intrathecal fluorescein. A possible correlation between intraoperative fluorescein identification and postoperative CSF leak was analyzed. RESULTS: 61 patients underwent surgery for a variety of lesions including pituitary adenoma (55.7%), encephalocele (14.8%), and meningioma (9.8%). Seven (19.4%) of the 37 patients with intraoperative fluorescein leak experienced postoperative CSF leak compared to 0 of the 24 patients who did not have intraoperative fluorescein leak (P = 0.02). All cases of CSF leak resolved with lumbar drainage alone. CONCLUSIONS: The lack of intraoperative fluorescein leak-age correlates strongly with a low risk for postoperative CSF leak. This can be used to stratify the extent of skull base reconstruction required during endoscopic skull base surgeries.

Continuous Electrical Oscillations Emerge from a Coupled Network: A Study of the Inferior Olive using Lentiviral Knockdown of Connexin36

Do continuous subthreshold oscillations in membrane potential within an electrically coupled network depend on gap junctional coupling? For the inferior olive (IO), modeling and developmental studies suggested that the answer is yes, although physiological studies of connexin36 knock-out mice lacking electrical coupling suggested that the answer is no. Here we addressed the question differently by using a lentivirus-based vector to express, in the IO of adult rats, a single amino acid mutation of connexin36 that disrupts the intracellular trafficking of wild-type connexin36 and blocks gap junctional coupling. Confocal microscopy of green fluorescence protein-labeled dendrites revealed that the mutant connexin36 prevented wild-type connexin36 from being expressed in dendritic spines of IO neurons. Intracellular recordings from lentivirally transduced IO networks revealed that robust and continuous subthreshold oscillations require gap junctional coupling of IO neuron somata within 40 μm of one another. Topological studies indicated that the minimal coupled network for supporting such oscillations may be confined to the dendritic arbor of a single IO neuron. Occasionally, genetically uncoupled IO neurons showed transient oscillations; however, these were not sustained longer than 3 s and were 69% slower and 71% smaller than the oscillations of normal IO neurons, a finding replicated with carbenoxolone, a pharmacological antagonist of gap junctions. The experiments provided the first direct evidence that gap junctional coupling between neurons, specifically mediated by connexin36, allows a continuous network oscillation to emerge from a population of weak and episodic single-cell oscillators. The findings are discussed in the context of the importance of gap junctions for cerebellar rhythms involved in movement.

Transsphenoidal Resection of Sellar Tumors Using High-Field Intraoperative Magnetic Resonance Imaging

There has been increasing experience in the utilization of intraoperative magnetic resonance imaging (iMRI) for intracranial surgery. Despite this trend, only a few U.S centers have examined the use of this technology for transsphenoidal resection of tumors of the sella. We present the largest series in North America examining the role of iMRI for pituitary adenoma resection. We retrospectively reviewed our institutional experience of 59-patients who underwent transsphenoidal procedures for sellar and suprasellar tumors with iMRI guidance. Of these, 52 patients had a histological diagnosis of pituitary adenoma. The technical results of this subgroup were examined. A 1.5-T iMRI was integrated with the BrainLAB (Feldkirchen, Germany) neuronavigation system. The majority (94%) of tumors in our series were macroadenomas. Seventeen percent of tumors were confined to the sella, 49% had suprasellar extensions without involvement of the cavernous sinus, 34% had frank cavernous sinus invasion. All patients underwent at least one iMRI, and 19% required one or more additional sets of intraoperative imaging. In 58% of patients, iMRI led to the surgeon attempting more resection. A gross total resection was obtained in 67% of the patients with planned total resections. There was one case of permanent postoperative diabetes insipidus and no other instances of new hormone replacement. In summary, iMRI was most useful for tumors of the sella with and without suprasellar extension where the information from the iMRI extended the complete resection rate from 40 to 72% and 55 to 88%, respectively. As one would expect, it did not substantially increase the rate of resection of tumors with cavernous sinus invasion. Overall, iMRI was particularly useful in guiding resection safely, aiding in clinical decision making, and allowing identification and preservation of the pituitary stalk and normal pituitary gland. Limitations of the iMRI include a need for additional personnel and training as well as additional operative time, which diminishes over time as personnel learn to optimize workflow efficiency. Additional costs are mitigated in part by using the iMRI as an immediate postoperative scan. Other data emerging from our experience suggest that preservation of normal gland and thus avoidance of hypopituitarism may be improved by iMRI use, but longer follow-up periods are required to test this conclusion. iMRI can detect unsuspected complications sooner than routine postoperative imaging, potentially leading to improved outcomes. However, larger studies are needed.

Neurosurgical management of medically intractable epilepsy associated with hypomelanosis of Ito.

To the Editor: Hypomelanosis of Ito (HI) is a rare neurocutaneous disorder described by Ito in 1952 (1). HI is first seen with hypopigmentation along the lines of Blaschko, whereas extracutaneous anomalies are encountered in 94% of cases, with central nervous system involvement seen in most cases (2). Neurologic manifestations include mental retardation (65%), seizures (53%), autism (12%), and psychiatric symptoms (2,3). The etiology of the seizure disorder associated with HI involves several neuronal migration abnormalities including hemimegalencephaly, pachygyria, cortical dysplasia, gray matter heterotopias, white matter pathology, abnormal neuronal morphology, and hamartomas of the gray– white matter interface (4–8). The therapeutic efficacy of surgical resection in the treatment of intractable epilepsy in HI has not been reported, although the use of neurosurgical intervention for epilepsy associated with hemimegalencephaly has been documented (9). Here we present two patients with HI who had medically intractable epilepsy. Both patients underwent resection of epileptogenic cortex with marked improvement in their seizure disorder.

Localization in Epilepsy

Pharmacologic therapy represents the first line of treatment of epilepsy and is effective in most patients. However, about 20% to 30% of cases develop intractable seizures that cannot be controlled by medication alone. In such cases, surgical intervention is considered for therapeutic, often curative purposes. Dynamic spatiotemporal variability in the epileptic focus renders seizure localization a challenge to the clinician. Many diagnostic modalities have been developed to identify different aspects of the epileptic focus. Older techniques are being increasingly supplemented by a variety of anatomic and functional imaging modalities that can help clarify discrepancies. Invasive electroencephalography remains the gold standard for identifying epileptic foci and guiding the surgeon to successful resections.