Prognostic awareness and communication preferences among caregivers of patients with malignant glioma.

OBJECTIVE: Malignant glioma (MG) is a devastating neuro-oncologic disease with almost invariably poor prognosis, yet many families facing malignant glioma have poor prognostic awareness (PA), or the awareness of the patient's incurable disease and shortened life expectancy. Accurate PA is associated with favorable medical outcomes at end-of-life for patients and psychosocial outcomes for informal caregivers (ICs) through bereavement. To date, however, no study has specifically examined PA among MG ICs and the information they receive that shapes their awareness. METHODS: Thirty-two ICs of patients with malignant glioma completed a semi-structured assessment of their awareness of the incurability and life expectancy of their loved one's illness, and to understand their sources of prognostic information and preferences for communication of prognostic information. RESULTS: Twenty-two (69%) ICs had full PA-awareness of the incurability of malignant glioma and accurate estimates of their loved ones' life expectancy. Twenty-three (72%) felt that prognostic information was extremely or very important to possess, and 16 (50%) desired more prognostic information. The majority of ICs received prognostic information from physicians and the Internet. Qualitative analyses revealed that many ICs had difficulty navigating medical encounters in which they concurrently wanted to elicit prognostic information from physicians and protect patients from such information. CONCLUSIONS: Accurate and timely PA is necessary for ICs to serve as critical members of health care teams. Interventions are needed to foster ICs' skills in navigating prognostic communication with patients and health care providers and thereby improve their ability to advocate for their loved one's wishes.

Acute exposure of toluene transiently potentiates p42/44 mitogen-activated protein kinase (MAPK) activity in cultured rat cortical astrocytes.

It has been shown that the inhalation of toluene in rats can cause neuronal apoptosis in the central nervous system. However, the cellular and molecular effects of toluene directly on astrocytes are relatively unknown. We used primary cultures of astrocytes isolated from the neonatal rat cortex as a model to study the toluene effects on cell outcome and associated signal transduction pathways using immunostaining and Western blotting. We observed that acute toluene exposure significantly induced caspase-dependent cell apoptosis and transiently stimulated the activation of p42/44 mitogen-activated protein kinase (MAPK) in the primary astrocytes. Interestingly, the inhibition of the p42/44 MAPK signaling cascade by PD98059 in conjunction with the toluene treatment evoked more cellular apoptosis than toluene alone, suggesting that the toluene-induced transient MAPK activation may play a role in promoting cell survival during the toluene exposure.

The use of GABA(A) receptors expressed in neural precursor cells for cell-based assays.

GABA(A) receptors are known targets for certain classes of environmental neurotoxins and pharmaceutical compounds. Since few neural cell lines express functional GABA(A) receptors, the capacity to rapidly screen for compounds that affect GABA(A) receptor function is presently limited. Previous work has demonstrated that rat neural precursor cells express functional GABA(A) receptors that can be monitored via Ca(2+) imaging. This study examined GABA(A) receptor subunit expression to determine whether GABA(A) receptor function and its interactions with neurotoxins is preserved after passaging. Neural precursor cells isolated from embryonic day 13 rat brain were expanded in serum-free medium containing basic fibroblast growth factor and passaged three times. Reverse transcription-polymerase chain reaction analysis demonstrated early expression of abundant mRNAs encoding various GABA(A) receptor subunits. Ca(2+) imaging showed that the highly proliferating precursor cells in passaged cultures maintained expression of functional GABA(A) receptors. In addition, we showed that trimethylolpropane phosphate, a neurotoxin generated during partial pyrolysis of a synthetic ester turbine engine lubricant, potently inhibited muscimol (GABA(A) receptor agonist) but not depolarization-induced cytosolic Ca(2+) increase. The findings of this study suggest that neural precursor cells may be well suited for the evaluation of certain environmental neurotoxins with convulsant activity. The potential use of neural precursor cells in high-throughput screens for compounds acting on GABA(A) receptors is discussed.

Design and demonstration of an automated cell-based biosensor.

Cell-based biosensors have the capacity to respond to a wide range of analytes in a physiologically relevant manner and appear well-suited for toxicity monitoring of both known and unknown analytes. One means of acquiring cellular functional information for biosensor applications involves extracellular recording from excitable cells, which can generate noninvasive and long-term measurements. Previous work from our laboratory described a prototype portable system capable of high signal-to-noise extracellular recordings, in spite of deficiencies in thermal control, fluidics handling, and absence of data acquisition (DAQ) capability. The present work describes a cell-based biosensor system that incorporates low noise amplifier and filter boards, a two-stage thermal control system with integrated fluidics and a flexible graphical user interface for DAQ and control implemented on a personal computer. Wherever possible, commercial off-the-shelf components have been utilized for system design and fabrication. The system exhibits input-referred noise levels of 5-10 microV(RMS), such that extracellular potentials exceeding 50-60 microV can be readily resolved. In addition, the biosensor system is capable of automated temperature and fluidics control. Flow rates can range from 0-2.5 ml/min, while the cell recording chamber temperature is maintained within a range of 36-37 degrees C. To demonstrate the capability of this system to resolve small extracellular potentials, recordings from embryonic chick cardiac myocytes have been performed.

Ethanol blocks cytosolic Ca2+ responses triggered by activation of GABA(A) receptor/Cl- channels in cultured proliferating rat neuroepithelial cells.

GABA(A) receptor/Cl- channels and voltage-gated Ca2+ channels are believed to be important sites of ethanol action in the CNS. Acute exposure of ethanol potentiates GABA(A) receptor/Cl- channel activity and inhibits voltage-gated Ca2+ channels in a number of preparations, mostly post-mitotic neurons. The effects of ethanol on these channels in primary cultures of undifferentiated neural precursor cells remain unknown. To address this issue, we examined the effects of ethanol on GABA(A) agonist-activated elevation of cytosolic Ca2+ in an in vitro model of the cortical neuroepithelium derived from rat basic fibroblast growth factor-expanded neural precursor cells. We found a potent inhibition of GABA(A)-activated elevation of cytosolic Ca2+ by ethanol in actively proliferating cells. Since we had recently demonstrated that GABA(A) receptor activation depolarizes these cells and elevates their cytosolic Ca2+, we tested whether the effects of ethanol involved both GABA(A) receptors and voltage-gated Ca2+ channels. Both extracellular K+- and muscimol-induced cytosolic Ca2+ elevations were abolished by nitrendipine, indicating that both depolarizing stimuli triggered Ca2+ influx through L-type voltage-gated Ca2+ channels. Exposure of proliferating cells to different concentrations of ethanol revealed that the drug was more potent in blocking muscimol-induced compared to K+-evoked cytosolic Ca2+ elevations. These results raise the possibility that ethanol blocks GABAergic stimulation of cytosolic Ca2+ levels in proliferating precursors primarily by interacting with GABA(A) receptor/Cl- channels and secondarily with voltage-gated Ca2+ channels.

Detection of physiologically active compounds using cell-based biosensors.

Cell-based biosensors are portable devices that contain living biological cells that monitor physiological changes induced by exposure to environmental perturbations such as toxicants, pathogens or other agents. Methods of detecting physiological changes include extracellular electrical recordings, optical measurements, and, in the future, functional genomics and proteomics. Several technical developments are occurring that will increase the feasibility of cell-based biosensors for field applications; these developments include stem cell and 3D culture technologies. Possible scenarios for the use of cell-based biosensors include broad-range detectors of unknown threat agents and functional assessment of identified agents.

Survival and neurite outgrowth of rat cortical neurons in three-dimensional agarose and collagen gel matrices.

To better understand interactions between neurons and extracellular matrix equivalents, embryonic day-18 rat cortical neurons were immobilized and maintained in culture for up to 24 days in agarose and type I collagen gels. Using live/dead staining, neuronal cultures in low density collagen gel lasted at least 3 weeks. At 14 days, over 50% of immobilized cells in collagen gel were found viable while in low density agarose gel no cells survived. In situ cell death detection showed that most, if not all, dead cells in either of the gels underwent apoptosis. The collagen-trapped neurons exhibited normal neuronal polarity and developed long neurites, estimated at over 500 microm. The results suggest that collagen, because it is a major extracellular matrix constituent, suppresses apoptosis and provides a suitable substrate for neuronal survival and differentiation.

Primary neural precursor cell expansion, differentiation and cytosolic Ca(2+) response in three-dimensional collagen gel.

To investigate the ability to culture neural precursor cells in a three-dimensional (3D) collagen gel, neuroepithelial cells were isolated from embryonic day 13 rat cortex, dispersed within type I collagen and maintained for up to 30 days in vitro. Cultured in Neuorobasal medium supplemented with B27 containing basic fibroblast growth factor, the collagen-entrapped precursor cells actively expanded and formed clone-like clusters. Many cells in the center of the cluster were proliferating as revealed by 5-bromo-2'-deoxyuridine uptake. Some cells began to migrate away from the center at 5 days and were labeled by either neuronal marker neuron-specific beta-tubulin (TuJ1) or astrocytic marker glial fibrillary acidic protein. The differentiated neurons (TuJ1(+)) exhibited characteristic cytosolic Ca(2+) oscillations in response to excitatory neurotransmitter glutamate. These findings suggest the suitability of the 3D culture system for the proliferation and differentiation of neural precursor cells.

Immobilization of neural cells in three-dimensional matrices for biosensor applications.

To overcome logistical difficulties with current designs of cell- or tissue-based biosensors which have individual cells or tissue slices immobilized on membranes or microelectrode arrays, we have proposed a system that uses three-dimensional cultures of neural cells immobilized in hydrogel matrices. In this design, immobilized cells would be maintained in a reservoir and then transferred to a detector platform when needed for analysis. The development of such a system relies upon a renewable supply of cells and the ability to culture cells for long periods of time in three-dimensions while maintaining their physiological function. To investigate the ability to culture neural cells in 3D matrices, embryonic rat cortical neurons and astrocytes were immobilized by matrix entrapment in a novel sugar poly(acrylate) hydrogel and collagen gels. The sugar poly(acrylate) hydrogel does not appear to support neural cell growth as a result of a lack of cell adherence, small pore size and, possibly, harshness of synthesis conditions. In contrast, collagen gels support the growth of cortical neurons, astrocytes, as well as neural progenitor cells. Evidence is also presented from immunocytochemistry and patch-clamp measurements which shows that neural progenitor cells proliferate in culture and can be induced to differentiate into neural cell types. Thus, they potentially represent a renewable cell source.

Synaptic connectivity in hippocampal neuronal networks cultured on micropatterned surfaces.

Embryonic rat hippocampal neurons were grown on patterned silane surface in order to organize synapse formations in a controlled manner. The surface patterns were composed of trimethoxysilylpropyl-diethylenetriamine (DETA) lines separated by tridecafluoro-1,1,2,2-tetrahydrooctyl-1-dimethylchlorosilane (13F) spaces. Pre- and post-synaptic specializations were identified by immunostaining for synapsin I and microtubule-associated protein-2 (MAP-2). Functional synaptic connections were examined by recording simultaneously from pairs of neurons using the whole-cell configuration of the patch-clamp technique. Spontaneous and evoked synaptic currents were recorded in neurons cultured for 2-14 days. The formation of functional connections was accompanied by the appearance of spontaneous synaptic currents (SSCs), which could be detected after approximately 3 days in culture in the absence of evoked synaptic currents (ESCs). ESCs were detected only after approximately 7 days in culture, mostly in the form of unidirectional synaptic connections. Other forms of synaptic connectivity, such as bidirectional and autaptic connections, were also identified. Both transient GABAergic and glutamatergic signals mediated the transmissions between communicating cells. These results demonstrate the combination of various types of synaptic connections forming simple and complex networks in neurons cultured on line (DETA)-space (13F) patterns. Finally, precisely synchronized SSCs were recorded in neuron pairs cultured on pattern indicating the existence of a fast-acting feedback mechanism mediated by pre-synaptic GABA(A) receptors.

Acetylcholine stimulates cortical precursor cell proliferation in vitro via muscarinic receptor activation and MAP kinase phosphorylation.

Increasing evidence has shown that some neurotransmitters act as growth-regulatory signals during brain development. Here we report a role for the classical neurotransmitter acetylcholine (ACh) to stimulate proliferation of neural stem cells and stem cell-derived progenitor cells during neural cell lineage progression in vitro. Neuroepithelial cells in the ventricular zone of the embryonic rat cortex were found to express the m2 subtype of the muscarinic receptor. Neural precursor cells dissociated from the embryonic rat cortical neuroepithelium were expanded in culture with basic fibroblast growth factor (bFGF). reverse transcriptase-polymerase chain reaction (RT-PCR) revealed the presence of m2, m3 and m4 muscarinic receptor subtype transcripts, while immunocytochemistry demonstrated m2 protein. ACh and carbachol induced an increase in cytosolic Ca2+ and membrane currents in proliferating (BrdU+) cells, both of which were abolished by atropine. Exposure of bFGF-deprived precursor cells to muscarinic agonists not only increased both cell number and DNA synthesis, but also enhanced differentiation of neurons. These effects were blocked by atropine, indicating the involvement of muscarinic ACh receptors. The growth-stimulating effects were also antagonized by a panel of inhibitors of second messengers, including 1,2-bis-(O-aminophenoxy)-ethane-N,N,N', N'-tetraacetic acid (BAPTA-AM) to chelate cytosolic Ca2+, EGTA to complex extracellular Ca2+, pertussis toxin, which uncouples certain G-proteins, the protein kinase C inhibitor H7 and the mitogen-activated protein kinase (MAPK) inhibitor PD98059. Muscarinic agonists activated MAPK, which was significantly inhibited by atropine and the same panel of inhibitors. Thus, muscarinic receptors expressed by neural precursors transduce a growth-regulatory signal during neurogenesis via pathways involving pertussis toxin-sensitive G-proteins, Ca2+ signalling, protein kinase C activation, MAPK phosphorylation and DNA synthesis.

Investigation of in vitro toxicity of jet fuels JP-8 and Jet A.

The in vitro cytotoxicity and electrophysiological toxicity of Jet Propulsion-8 (JP-8 jet fuel) on four cell types: H4IIE liver cell line, NIH Swiss 3T3 cell line, neuroblastoma x glioma NG108-15 cells, and embryonic hippocampal neurons were investigated. H4IIE cells exposed to Jet A (a commercial fuel) and JP-8 demonstrated identical toxicity with an IC50 of 12.6 +/- 0.4 micrograms/ml for the two fuels. Comparison of H4IIE and NIH/3T3 toxicity to JP-8 revealed that NIH/3T3 cells were more sensitive to JP-8 than H4IIE cells, with an IC50 8.5 +/- 0.1 micrograms/ml. JP-8 exposure for the hippocampal neurons proved to be highly toxic (IC50 of < 2 micrograms/ml), while in contrast, the NG108-15 cells were much less sensitive. Electrophysiological examination of NG108-15 cells showed that administration of JP-8 at 1 microgram/ml did not alter significantly any of the electrophysiological properties. However, exposure to JP-8 at 10 micrograms/ml during a current stimulus of +46 pA decreased the amplitude of the action potential to 83 +/- 7% (n = 4), the rate of rise, dV/dtMAX to 50 +/- 8% (n = 4), and the spiking rate to 25 +/- 11% (n = 4) of the corresponding control levels. These results demonstrate JP-8 induced cytotoxic varies among cell types. The possible mechanisms underlying these observations are presented.

Kir 4.1 channel expression in neuroblastomaxglioma hybrid NG108-15 cell line.

To study a possible involvement of inwardly rectifying K+ 4.1 (Kir 4. 1) channels in neural cell development, RT-PCR, immunocytochemistry and whole-cell patch-clamp techniques were used to assess expression of Kir 4.1 channels in proliferating and differentiated NG108-15 cells. RT-PCR revealed co-expression of Kir 4.1 and rat ether-a-go-go-related gene (R-ERG) mRNAs in both proliferating and differentiated cells. The relative Kir 4.1 mRNA concentration increased markedly as cells progressed from undifferentiated to differentiated cells. Kir 4.1-immunoreactivity was barely detectable in undifferentiated cells, but clearly detected in differentiated cells, indicating that Kir 4.1 gene and protein expressions are developmentally regulated. However, corresponding Kir 4.1 current could not be detected in differentiated cells using whole-cell patch-clamp recording. The 'silent' channel/receptor, often found in tumor cells, may carry genetic defects, which prevent functional expression of the channel. NG108-15 may serve as unique model for studying the relationship between the expression of an ion channel gene and the electrophysiological phenotype it encodes.

A role for inwardly rectifying K+ channels in differentiation of NG108-15 neuroblastoma x glioma cells.

The whole-cell patch-clamp technique was used to assess the current carried by inwardly rectifying K+ channels (K(ir)) and the resting membrane potential (RMP) during long-term culture of NG108-15 cells. Culture of this cell line in serum-free medium triggers differentiation of a type I, neuron-like cell type followed by an eventual predominance of a type II, proliferative cell type. NG108-15 K(ir) currents, which strongly resemble currents carried by human ether-a-go-go related gene (HERG) K+ channels, exhibited significantly smaller current density for the more depolarized undifferentiated cells in growth media (GM) and type II cells compared to the neuron-like type I cells. Detailed examination of the transition from undifferentiated GM cells to type I cells revealed a shift in the voltage dependence of K(ir) activation which paralleled the more hyperpolarized RMP, neurite outgrowth, and biochemical differentiation characteristic of type I cells. Reverse-transcription polymerase chain reaction experiments using primers for the rat variant of HERG, RERG, revealed a a nearly twofold increase in RERG mRNA as cells differentiate from GM to type I, a finding entirely consistent with the increased K(ir) current density derived from patch-clamp recordings. Administration of CsCl(5 mM) blocked K(ir) currents and depolarized the RMP of type I cells. Furthermore, culture of NG108-15 cells in serum-free medium but with CsCl added significantly prevented neurite extension, an effect which was entirely reversible upon subsequent removal of CsCl. In contrast, other K+ channel inhibitors (4-aminopyridine and tetraethylammonium), at concentrations without marked effects on K(ir), failed to affect neurite extension. These results suggest an important role of the K(ir) channels in determining the RMP and triggering morphological differentiation of the cell line.

Cardiac transplantation for pediatric patients. With inoperable congenital heart disease.

Recent studies have reported the expanding use of transplantation as the definitive option for pediatric patients with inoperable congenital heart disease. This study compares perioperative risk factors and outcomes in pediatric patients who received heart transplants for congenital heart disease with those in pediatric patients who received heart transplants for cardiomyopathy. Retrospective data collected on 40 consecutive pediatric patients undergoing cardiac transplantation from 1 January 1990 through 31 January 1995 provided the following results: 26 patients with cardiomyopathy (mean age, 7.6 years) and 14 patients with congenital heart disease (mean age, 7.2 years) underwent heart transplantation. Between groups, no significant difference was detected in waiting time for a donor heart (cardiomyopathy = 85 days, range = 2 to 409; congenital heart disease = 126 days, range = 9 to 396; P = NS); in donor/recipient weight ratio (1.27 +/- 0.34 vs 1.27 +/- 0.28, P = NS); or in ischemic times (209 +/- 92 minutes vs 248 +/- 70 minutes, P = NS). Cardiopulmonary bypass times accounted for the only significant difference (73 +/- 21 minutes vs 102 +/- 29 minutes, P = 0.003). No significant difference was found in the number of infection episodes, total days hospitalized, rejection episodes, or incidence of transplant coronary artery disease. Forty-month actuarial survival was 88% +/- 6% and 92% +/- 7% for cardiomyopathy and congenital heart disease transplant recipients, respectively (P = NS). We conclude that post-transplantation morbidity and mortality in patients with previous congenital heart disease are not significantly different from morbidity and mortality in patients with cardiomyopathy. Transplantation should be considered an acceptable therapeutic option for patients with congenital heart disease when surgical repair of the native heart is not possible.

Intravascular stents in congenital heart disease: short- and long-term results from a large single-center experience.

OBJECTIVES: This report describes the results of the Food and Drug Administration's phase 1 and 2 clinical trials of intravascular stents at Texas Children's Hospital. BACKGROUND: Since the late 1980s, intravascular stent implantation for the treatment of arterial and venous stenoses in congenital heart disease has been highly successful. METHODS: Stents were placed in postoperative pulmonary artery (PA) stenoses, congenital PA stenoses or stenoses of systemic veins/venous anastomoses. Prospective collection of data according to protocol was done before intervention, after stent implantation and at follow-up catheterization. RESULTS: At stent implantation, pressure gradients decreased significantly in all three groups (mean +/- SD): from 46 +/- 25 to 10 +/- 13 mm Hg in postoperative PA stenoses (p < 0.001); from 71 +/- 45 to 15 +/- 21 mm Hg in congenital PA stenoses (p < 0.001); and from 7 +/- 6 to 1 +/- 2 mm Hg in stenoses of systemic veins/venous anastomoses stenoses (p < 0.001). Vessel diameters markedly increased: from 6 +/- 3 to 12 +/- 3 mm in postoperative PA stenoses (p < 0.001); from 3 + 1 to 9 + 1 mm in congenital PA stenoses (p < 0.001); and from 3 +/- 4 to 12 +/- 4 mm in stenoses of systemic veins/venous anastomoses (p < 0.001). In the postoperative and congenital PA stenoses groups, right ventricular pressure decreased (right ventricular pressure indexed to femoral artery pressure ratio): from 0.63 +/- 0.2 to 0.41 +/- 0.02 (p < 0.001) and from 0.71 +/- 0.3 to 0.55 +/- 0.35 (p = 0.04), respectively. Perfusion to a single affected lung increased from 31 +/- 17% to 46 +/- 14% (p < 0.001). On recatheterization (mean 14 months), results varied minimally. Repeat angioplasty of residual stent stenoses was safe and effective. Complications included four early patients with stent migration, three with stent thrombosis and two deaths. There were no late complications. Significant restenosis occurred in only three patients. CONCLUSIONS: Intravascular stents for the treatment of vascular stenoses in congenital heart disease provide excellent immediate and long-term results.

Cardinal clinical signs in the differentiation of heart murmurs in children.

OBJECTIVE: To determine the diagnostic accuracy of clinical assessment of heart murmurs in children and specific clinical features that are predictive of cardiac disease. DESIGN: Concurrent case series with pretest-posttest assessment. SETTING: Pediatric cardiology outpatient clinic. PARTICIPANTS: Five full-time academic pediatric cardiologists. MEASURES: For each of 222 consecutive patients who were seen for first-time evaluation of a heart murmur, the clinical findings and diagnostic impressions were recorded after clinical assessment. The results of electrocardiograms and echocardiograms were then reviewed, and changes in diagnostic impressions were recorded and compared with the original impressions. RESULTS: The prevalence of cardiac disease was 33%. Clinical assessment differentiated those patients with pathologic murmurs with a sensitivity of 92%, specificity of 94%, positive predictive value of 88%, and negative predictive value of 96%. If diagnostic uncertainty was considered an indication for echocardiography, then sensitivity and specificity increased to 97% and 98%, respectively. Missed disease included only trivial or minor lesions. Clinical features that were independently predictive of the presence of disease included murmurs that were pansystolic (odds ratio [OR], 54.0), grade 3 or more in intensity (OR, 4.84), heard best at the left upper sternal border (OR, 4.24) and harsh in quality (OR, 2.37), and the presence of an abnormal second heart sound (OR, 4.09) and an early or midsystolic click (OR, 8.35). CONCLUSIONS: Clinical assessment by a pediatric cardiologist is sufficient to distinguish pathologic from innocent heart murmurs. A genetic approach by using specific clinical features that are independently associated with disease may have some practical utility to noncardiologists.

An evaluation of parental concerns and misperceptions about heart murmurs.

This study sought to determine the extent of anxiety and misperceptions about heart murmurs for consenting parents of 182 children referred for first-time pediatric cardiology assessment (including echocardiography) of a heart murmur. From questionnaires completed before assessment, 22% of parents indicated that they were extremely concerned, and only 16% could define a heart murmur as a sound made by the heart. From 1-month follow-up questionnaires obtained from parents of children without heart disease at assessment, 10% continued to believe that their child had a heart problem. Cardiology assessment may not provide complete reassurance to all families and additional interventions may be necessary.