Optimizing the flattening filter free beam selection in RapidArc®-based stereotactic body radiotherapy for Stage I lung cancer.

OBJECTIVE: To optimize the flattening filter-free (FFF) beam selection in stereotactic body radiotherapy (SBRT) treatment for Stage I lung cancer in different fraction schemes. METHODS: Treatment plans from 12 patients suffering from Stage I lung cancer were designed using the 6XFFF and 10XFFF beams in different fraction schemes of 4 × 12, 3 × 18 and 1 × 34 Gy. Plans were evaluated mainly in terms of organs at risk (OARs) sparing, normal tissue complication probability (NTCP) estimation and treatment efficiency. RESULTS: Compared with the 10XFFF beam, 6XFFF beam showed statistically significant lower dose to all the OARs investigated. The percentage of NTCP reduction for both lung and chest wall was about 10% in the fraction schemes of 4 × 12 and 3 × 18 Gy, whereas only 7.4% and 2.6% was obtained in the 1 × 34 Gy scheme. For oesophagus, heart and spinal cord, the reduction was greater with the 6XFFF beam, but their absolute estimates were <10(-6)%. The mean beam-on time for 6XFFF and 10XFFF beams at 4 × 12, 3 × 18 and 1 × 34 Gy schemes were 2.2 ± 0.2 vs 1.5 ± 0.1, 3.3 ± 0.9 vs 2.0 ± 0.5 and 6.3 ± 0.9 vs 3.5 ± 0.4 min, respectively. CONCLUSION: The 6XFFF beam obtains better OARs sparing and lower incidence of NTCP in SBRT treatment of Stage I lung cancer, whereas the 10XFFF beam improves the treatment efficiency. To balance the OARs sparing and intrafractional variation owing to the prolonged treatment time, the authors recommend using the 6XFFF beam in the 4 × 12 and 3 × 18 Gy schemes but the 10XFFF beam in the 1 × 34 Gy scheme. ADVANCES IN KNOWLEDGE: This study optimizes the FFF beam selection in different fraction schemes in SBRT treatment of Stage I lung cancer.

Vasoactive intestinal peptide: mediator of laminin synthesis in cultured Schwann cells.

To learn more about neuropeptide-induced glial responses which accompany axon regeneration, we studied effects of VIP on laminin production by cultured Schwann cells. Schwann cells were isolated from sciatic nerves of neonatal mice, purified, and incubated for 5 days in either control medium (DMEM + 15% FCS) or control medium containing 10-7 -10-11 M VIP. At 10-7 and 10-8 M VIP, laminin levels measured by enzyme-linked immunosorbent assay were significantly higher (55% and 35%) than those in control cultures. Lower VIP concentrations (10-9 -10-11 M) produced smaller increases which were not significant. Low-affinity VIP receptors which mediated this effect were demonstrated on Schwann cells by radioligand binding studies. The increased Schwann cell synthesis of laminin induced by VIP was blocked when either a VIP antagonist or a VIP receptor antagonist was added to the VIP-containing incubation medium. In contrast to astrocytes, when Schwann cells were loaded with fura-2, VIP did not increase cytosolic Ca2+. This indicates that Schwann cells and astrocytes may have different intracellular transduction pathways; their receptor subtypes also may differ. We suggest that the VIP-induced increase in laminin synthesis which we have observed in cultured Schwann cells may also occur in vivo and might be an important component of axon-Schwann cell interactions during nerve regeneration.

Combinatorial expression of immediate early genes in single neurons.

To address the question how relatively small numbers of immediate early gene (IEG) could specifically couple a wide range of stimulus-response cascades, we examined the possibility that IEG could be expressed heterogeneously in individual neurons. Analysis of multiple IEG in single neurons revealed that many individual DRG neurons express several IEGs. The combinatorial expression of IEGs by individual DRG displays substantial heterogeneity. Analysis of mRNA species encoding AP-1 composition in single cells also revealed coordinated change of mRNAs coding for AP-1 factors after membrane depolarization. Our results indicate that differential expression of IEG in individual cells, and the possible interaction among them may represent a mechanism by which the specificity in stimulation-response coupling may be achieved by IEGs.

Analysis of multiple heterogeneous mRNAs in single cells.

We describe a two-stage amplification procedure designed to analyze multiple heterogeneous mRNA from single cells. Using an oligo(dT) primer with an attached phage promoter, the whole mRNA pool from a single cell was first transcriptionally amplified. This step brings the weak signal from one cell to a range in which it can be reliably picked up by the polymerase chain reaction (PCR) procedure. The cDNA was then divided for separate PCR amplification to obtain an unambiguous signal for each gene product. The linear amplification of phage transcription increased the convenience and reliability of detecting multiple messengers in the second stage. The procedure is extremely sensitive because it combines the amplification generated by both phage transcription and PCR. Using a pAW109 artificial RNA, we demonstrated that this procedure detects 10 copies of pAW109 RNA per original sample with 90% confidence and 50 copies per sample with > 95% confidence. This procedure of multiple mRNA analysis allows "phenotyping" of any cell for its mRNA composition. Examples involving several immediate early genes and subunits of the gamma-aminobutyric acidA receptor genes are given. The method should greatly facilitate the analysis of combinatorial expression of various regulatory or channel molecules in their native environments. The procedure should also provide a direct and efficient way of decoding the developmental instruction coded through combinatorial transcriptional regulation.

Effects of nerve segment supernatants on cultured Schwann cell proliferation and laminin production.

Mouse sciatic nerves were transected and 3 hr to 16 days later proximal segments were removed and homogenized. Supernatants of these segments or of normal sciatic nerves were added to Schwann cells maintained in Dulbecco's modified Eagle's medium (DMEM) + 15% fetal calf serum (FCS). After 6 days, Schwann cells were solubilized and the protein content was measured using a Bio-Rad (Melville, NY) protein assay. Samples containing the same amounts of protein were then applied to microtiter plates and the laminin content was determined by enzyme-linked immunosorbent assay (ELISA). Lysates of cultures treated with 24 hr proximal segment supernatants contained significantly higher levels of laminin than those prepared from other intervals, from distal segments, or from control nerves. Increased surface and cytoplasmic anti-laminin immunoreactivity also was found in Schwann cells treated with 24 hr supernatants. To identify the source(s) of this effect, proximal segments removed 24 hr after transection were bisected; supernatants were prepared from each half and tested. Significant increases in laminin production were produced by supernatants from both halves. When supernatants from proximal and distal halves were compared, the latter produced significantly higher laminin levels. Electron microscopic examination of both halves showed that distal halves contained sprouting neurites and growth cones ensheathed by Schwann cells which had a basal lamina and resembled those seen during development and regeneration. Proximal halves appeared normal. Schwann cell proliferation also was compared in supernatant-treated cultures by using a bromodeoxy-uridine (BrdU) ELISA. The 24 hr and 2 day supernatants increased Schwann cell proliferation significantly; 12 hr, 4 day, and 8 day supernatants produced smaller increases. Our observations suggest that axons undergoing early regenerative changes are one of several possible sources of substance(s) in our proximal segment supernatants which increased Schwann cell proliferation and laminin production.

Effects of electrical stimulation on GAP-43 expression in mouse sensory neurons.

Effects of electrical activity on GAP-43 expression were tested in mouse dorsal root ganglion (DRG) neurons subjected to electrical stimulation in culture. Patterned electrical stimulation was provided through extracellular electrodes placed in multicompartment cell culture chambers. Stimulation was delivered at 10 Hz, in 0.5 s bursts every 2 s for up to 3 days. Expression of GAP-43 was assessed by immunocytochemistry, two ELISA methods, and Northern blot analysis within three experimental protocols: (1) prior to synaptogenesis, (2) after synaptogenesis with spinal cord neurons, and (3) within the context of activity-dependent synaptic competition, in which synapses from active and inactive DRG neurons converge on the same postsynaptic neurons. None of the stimulation treatments produced a measurable change in GAP-43 or RNA message for the protein, although this electrical stimulus induces persistent changes in synaptic strength, and alters neurite outgrowth in these cultures. The decline in GAP-43 levels between 1 and 3 weeks in culture, which has been reported in other studies, was readily detectable by our measurements. We conclude that regulation of GAP-43 expression is not required for activity-dependent regulation of growth cone motility, synaptogenesis and synapse elimination, or changes in synaptic strength. Instead, post-translational modification, such as phosphorylation, may be the primary means of regulating any GAP-43 functions associated with these activity-dependent processes.

Heterogeneous calcium currents and transmitter release in cultured mouse spinal cord and dorsal root ganglion neurons.

1. Calcium currents and transmitter release were studied in cocultures of fetal mouse neurons from the ventral half of the spinal cord (VH neurons) and from dorsal root ganglion (DRG neurons). The effects of BayK 8644 and omega-conotoxin on calcium currents and transmitter release were compared. 2. The presence of low voltage-activated (LVA) calcium current in both VH and DRG neurons is variable. Some cells exhibit only high voltage-activated (HVA) currents, whereas others show both HVA and LVA currents. 3. BayK 8644 did not affect LVA currents but strongly augmented both steady and transient components of the HVA calcium conductance. 4. omega-Conotoxin GVIA reduces both transient and steady components of the HVA but does not abolish either component even after 3 h of application. 5. Calcium currents that were resistant to omega-contoxin were augmented by BayK 8644. 6. Synaptic transmission between pairs of spinal cord neurons from the ventral half of the spinal cord (VH-VH connections) or between dorsal root ganglion neurons and VH neurons (DRG-VH connections) were studied with two-cell recording and stimulation techniques. 7. In approximately 70% of VH-VH connections and 50% of DRG-VH connections, BayK 8644 or its active optical isomer failed to affect transmitter output. Substantial augmentation of the remainder of the connections could be reliably produced by the dihydropyridines. Raised calcium in the extracellular medium produced augmentation of synaptic connections in all cases. BayK 8644 produced substantial, consistent augmentation of voltage-sensitive calcium channels in both VH and DRG neurons. 8. The toxin, omega-conotoxin, produced no consistent effect on excitatory or inhibitory postsynaptic potentials (EPSPs or IPSPs) elicited in VH neurons by stimulation of nearby VH neurons. VH EPSPs elicited by stimulation of nearby DRG neurons were reduced to approximately 50% of control values after 10 min of omega-conotoxin perfusion. Spontaneous and evoked synaptic activity could be recorded in VH neurons as long as 2 h after cultures were incubated in 0.5 microM omega-conotoxin. omega-Conotoxin produced a modest reduction in HVA currents in both VH and DRG neurons. 9. BayK 8644 did not produce consistent augmentation of transmission at the frog neuromuscular junction. omega-Conotoxin produced total blockade of transmission in this preparation. 10. We conclude that neither sustained nor inactivating high-threshold voltage-sensitive (HVA) calcium channels sensitive to BayK 8644 or omega-conotoxin such as those measured in the neuronal cell bodies are responsible for action-potential-evoked transmitter release from the majority of VH neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of cholecystokinin octapeptide on genetically determined seizure susceptibility.

Utilizing audiogenic seizure-prone P77PMC rats, the effects of cholecystokinin octapeptide (CCK-8) on genetic seizure susceptibility were studied in vivo, in cerebral cortical synaptosomes, and in cortical neuronal cell cultures. The results showed that CCK-8 could decrease seizure susceptibility, and that the K(+)-stimulated release of GABA in cerebral cortex synaptosomes from seizure-prone animals was depressed. The presence of exogenous CCK-8 (10(-7) M) together with elevated K+ (25 mM) causes a higher increased magnitude in GABA release from synaptosomes (enhanced by 100%) and cell cultures (17 days in vitro, increased by 177%) derived from seizure-prone rats than the controls (increased by 42%, in synaptosomes; and 107% in cell cultures). These preliminary results raise the possibility that the developmental abnormalities in modulation effect of CCK-8 on GABA release in central nervous system may play a role causing greater seizure susceptibility in genetic seizure-prone rats. The analysis of the brain tissue level and gene-expression of CCK-8 will be the important step of further investigation.