James W. Y. Chen

In vitro characterization of human dental pulp cells: various isolation methods and culturing environments

Our purpose was to characterize human dental pulp cells isolated by various methods and to examine the behavior of cells grown under various conditions for the purpose of pulp/dentin tissue engineering and regeneration. We compared the growth of human pulp cells isolated by either enzyme digestion or the outgrowth method. Expression of dentin sialophosphoprotein, Cbfa1, and two types of collagen (I and III) in these cells was examined by Western blot or reverse transcription/polymerase chain reaction. Growth of pulp cells on dentin and in collagen gel was also characterized. We found that different isolation methods give rise to different populations or lineages of pulp cells during in vitro passage based on their collagen gene expression patterns. Cells isolated by enzymedigestion had a higher proliferation rate than those isolated by outgrowth. Pulp cells did not proliferate or grew minimally on chemically and mechanically treated dentin surface and appeared to establish an odontoblast-like morphology with a cytoplasmic process extending into a dentinal tubule as revealed by scanning electron microscopy. The contraction of the collagen matrix caused by pulp cells was dramatic: down to 34% on day 14. Our data indicate that (1) the choice of the pulp cell isolation method may affect the distribution of the obtained cell populations, (2) a treated dentin surface might still promote odontoblast differentiation, and (3) a collagen matrix may not be a suitable scaffold for pulp tissue regeneration because of the marked contraction caused by pulp cells in the matrix. The present study thus provides important information and a basis for further investigations pre-requisite to establishing pulp tissue engineering/regeneration protocols.

Primary Glioblastomas Express Mesenchymal Stem-Like Properties

Glioblastoma is the most common and aggressive primary brain cancer. Recent isolation and characterization of brain tumor-initiating cells supports the concept that transformed neural stem cells may seed glioblastoma. We previously identified a wide array of mesenchymal tissue transcripts overexpressed in a broad set of primary glioblastoma (de novo) tumors but not in secondary glioblastoma (derived from lower-grade) tumors, low-grade astrocytomas, or normal brain tissues. Here, we extend this observation and show that a subset of primary glioblastoma tumors and their derived tumor lines express cellular and molecular markers that are associated with mesenchymal stem cells (MSC) and that glioblastoma cell cultures can be induced to differentiate into multiple mesenchymal lineage-like cell types. These findings suggest either that a subset of primary glioblastomas derive from transformed stem cells containing MSC-like properties and retain partial phenotypic aspects of a MSC nature in tumors or that glioblastomas activate a series of genes that result in mesenchymal properties of the cancer cells to effect sustained tumor growth and malignant progression. (Mol Cancer Res 2006;4(9):607–19)

Functional signal- and paradigm-dependent linear relationships between synaptic activity and hemodynamic responses in rat somatosensory cortex

Linear relationships between synaptic activity and hemodynamic responses are critically dependent on functional signal etiology and paradigm. To investigate these relationships, we simultaneously measured local field potentials (FPs) and optical intrinsic signals in rat somatosensory cortex while delivering a small number of electrical pulses to the hindpaw with varied stimulus intensity, number, and interstimulus interval. We used 570 and 610 nm optical signals to estimate cerebral blood volume (CBV) and oxygenation, respectively. The spatiotemporal evolution patterns and trial-by-trial correlation analyses revealed that CBV-related optical signals have higher fidelity to summed evoked FPs (ΣFPs) than oxygenation-derived signals. CBV-related signals even correlated with minute ΣFP fluctuations within trials of the same stimulus condition. Furthermore, hemodynamic signals (CBV and late oxygenation signals) increased linearly with ΣFP while varying stimulus number, but they exhibited a threshold and steeper gradient while varying stimulus intensity, suggesting insufficiency of the homogeneity property of linear systems and the importance of spatiotemporal coherence of neuronal population activity in hemodynamic response formation. These stimulus paradigm-dependent linear and nonlinear relationships demonstrate that simple subtraction-based analyses of hemodynamic signals produced by complex stimulus paradigms may not reflect a difference in ΣFPs between paradigms. Functional signal- and paradigm-dependent linearity have potentially profound implications for the interpretation of perfusion-based functional signals.

Mechanistic and pharmacologic aspects of status epilepticus and its treatment with new antiepileptic drugs

We review recent advances in our understanding and treatment of status epilepticus (SE). Repeated seizures cause an internalization of γ‐aminobutyric acid (GABA)A receptors, together with a movement of N‐methyl‐d‐aspartate (NMDA) receptors to the synapse. As a result, the response of experimental SE to treatment with GABAergic drugs (but not with NMDA antagonists) fades with increasing seizure duration. Prehospital treatment, which acts before these changes are established, is finding increased acceptance, and solid evidence of its efficacy is available, particularly in children. Rational polypharmacy aims at multiple receptors or ion channels to increase inhibition and simultaneously reduce excitation. Combining GABAA agonists with NMDA antagonists and with agents acting at other sites is successful in treating experimental SE, and in reducing SE‐induced brain damage and epileptogenesis. The relevance of these experimental data to clinical SE is actively debated. Valproate and levetiracetam have recently become available for intravenous use, and the use of ketamine and of other agents (topiramate, felbamate, etc.) have seen renewed interest. A rapidly increasing but largely anecdotal body of literature reports success in seizure control at the price of relatively few complications with the clinical use of those agents in refractory SE.

Advances in the pathophysiology of status epilepticus

Status epilepticus (SE) describes an enduring epileptic state during which seizures are unremitting and tend to be self‐perpetuating. We describe the clinical phases of generalized convulsive SE, impending SE, established SE, and subtle SE. We discuss the physiological and biochemical cascades which characterize self‐sustaining SE (SSSE) in animal models. At the transition from single seizures to SSSE, GABAA (gamma‐aminobutyric acid) receptors move from the synaptic membrane to the cytoplasm, where they are functionally inactive. This reduces the number of GABAA receptors available for binding GABA or GABAergic drugs, and may in part explain the development of time‐dependent pharmacoresistance to benzodiazepines and the tendency of seizures to become self‐sustaining. At the same time, ‘spare’ subunits of AMPA (alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid) and NMDA (N‐methyl‐D‐aspartic acid) receptors move from subsynaptic sites to the synaptic membrane, causing further hyperexcitability and possibly explaining the preserved sensitivity to NMDA blockers late in the course of SE. Maladaptive changes in neuropeptide expression occur on a slower time course, with depletion of the inhibitory peptides dynorphin, galanin, somatostatin and neuropeptide Y, and with an increased expression of the proconvulsant tachykinins, substance P and neurokinin B. Finally, SE‐induced neuronal injury and epileptogenesis are briefly discussed.

Characterization of optical intrinsic signals and blood volume during cortical spreading depression.

Cortical spreading depression (CSD) was imaged in vivo in a rodent model with optical intrinsic signals (OIS). This is the first study to identify a triphasic OIS response and to characterize the rate and timing of the response. The initial OIS phase had a highly uniform wavefront, which spread at a rate characteristic of CSD, 3.5 mm/min. Later phases were more diffuse and inhomogeneous. Blood volume changes, measured with intravascular fluorescent dye, correlated in time and location with the later phases of OIS reponse. This suggests that the inhomogeneity of the late OIS response may be due to complex residual hemodynamic contributions, as opposed to underlying cortical circuitry.

Optical intrinsic signal imaging in a rodent seizure model

Article abstract The authors studied seizure activity with optical intrinsic signal (OIS) imaging in a rat seizure model. OIS, which measures vascular and metabolic effects associated with neuronal activity, showed significant cortical reflectance changes from penicillin-induced seizures, and correlated well with EEG epileptiform discharges. Furthermore, OIS changes often preceded initial EEG spikes. These observations suggest that OIS is well coupled with seizure activity, and may provide sensitive cues for seizure detection.

Source localization and time delay estimation using constrained least-squares and best-path smoothing

The problem of source localization from arrival time delay estimates requires a computationally costly iterative solution of a set of nonlinear equations. Most known methods assume that the propagation speed is known. In this paper, we provide several effective source localization and propagation velocity estimation methods which only use measurements of the relative arrival time delays between sensors. The formulae for source localization and propagation speed estimation are derived based on least squares, total least squares, bounded data uncertainty, and constrained least squares methods. Statistical performance of these methods are compared via computer simulation. In addition, in order to avoid time delay ambiguity problems and obtain smoother time delays, two time delay smoothing methods based on the forward backward algorithm and the Viterbi algorithm are also proposed. Field experiment results based on these techniques are also presented.

Posttraumatic Epilepsy in Operation Enduring Freedom/Operation Iraqi Freedom Veterans

Penetrating traumatic brain injury (TBI) is a well-established risk factor for post-traumatic epilepsy (PTE). However, many veterans in Operation Enduring Freedom (OEF) and Operation Iraqi Freedom (OIF) who suffer from TBI do so from blast injury, and its consequences are not fully known. Two neurologists performed a chart review to describe patterns of injury and health care among all 16 OEF/OIF veterans at the VA Greater Los Angeles Healthcare System who were assigned an outpatient diagnosis of both epilepsy and TBI in 2008-2009. All Veterans were male, and the mean age was 30 years. Blast exposure was the most common mechanism of TBI (81%). Although all Veterans were assigned a diagnosis code of seizures, the diagnosis of PTE was clinically confirmed in only 3 veterans. On the other hand, the diagnosis of post-traumatic stress disorder was confirmed in 81% of the sample and a diagnosis of nonepileptic seizures was suspected in 44% of the sample. Researchers who study PTE among the OEF/OIF population using administrative data also should perform chart reviews to account for the prevalence of psychogenic nonepileptic seizures.

Treatment of Post-Traumatic Epilepsy

Opinion statementPost-traumatic epilepsy (PTE) due to traumatic brain injury is a diagnosis with multifactorial causes, diverse clinical presentations, and an evolving concept of management. Due to sports injuries, work-related injuries, vehicular accidents, and wartime combat, there is rising demand to understand the epidemiology, pathophysiology, diagnosis, prognosis, and treatment of PTE. PTE could occur at any time after injury and up to decades post-injury. The frontal and temporal lobes are the most commonly affected regions, and the resulting epilepsy syndrome is typically localization related. PTE should be actively considered as a diagnosis in any patient with a history of head trauma and episodic neurologic compromise regardless of how temporally remote the trauma occurred. The standard work-up includes a thorough history, neurological examination, neuroimaging, and electroencephalogram. Psychogenic nonepileptic seizures have a high comorbidity with seizures and need to be carefully excluded. PTE can spontaneously remit. For patients who do not go into remission, treatment for confirmed PTE includes antiepileptics, vagal nerve stimulator, and, when appropriate, surgical resection of an epileptogenic lesion. Lifestyle modification and counseling are critical for patients with PTE and should be routinely included in clinical management. The published evidence on the efficacy of various treatment modalities specific to PTE consists largely of retrospective studies and case reports. Despite a unique pathogenesis, the majority of current care parameters for PTE parallel those of standard care for localization-related epilepsy. The potential and need for rigorous clinical research in PTE continue to be in great demand.