Three-dimensional localization of fluorescent masses deeply embedded in tissue.

In this paper we present preliminary results obtained for tissue-like phantoms and ex vivo tissue slabs using a prototype system for CW infrared fluorescence imaging of fluorescent markers. Besides the design of the experimental system itself, we have developed a theoretical model of photon migration under experimental conditions and associated 3D reconstruction algorithms to estimate the distribution of fluorescent markers. Application of the developed algorithms in the analysis of 2D intensity distributions of the fluorescent light demonstrated their ability to reconstruct positions of the markers (including their depths) with good accuracy (error < or = 10%) both for the Intralipid phantoms and ex vivo tissue slabs.

Brownian flocculation of polymer colloids in the presence of a secondary minimum.

Most analyses of Brownian flocculation apply to conditions where London-van der Waals attractive forces cause particles to be strongly bound in a deep interparticle potential well. In this paper, results are reported that show the interaction between primary- and secondary-minimum flocculation when the interparticle potential curve reflects both attractive and electrostatic repulsive forces. The process is highly time-dependent because of transfer of particles from secondary- to primary-minimum flocculation. Essential features of the analysis are corroborated by experiments with 0.80-microm polystyrene spheres suspended in aqueous solutions of NaCl over a range of ionic strengths. In all cases, experiments were restricted to the initial stage of coagulation, where singlets and doublets predominate.

Elucidating the pathophysiology of syringomyelia.

OBJECT: Syringomyelia causes progressive myelopathy. Most patients with syringomyelia have a Chiari I malformation of the cerebellar tonsils. Determination of the pathophysiological mechanisms underlying the progression of syringomyelia associated with the Chiari I malformation should improve strategies to halt progression of myelopathy. METHODS: The authors prospectively studied 20 adult patients with both Chiari I malformation and symptomatic syringomyelia. Testing before surgery included the following: clinical examination; evaluation of anatomy by using T1-weighted magnetic resonance (MR) imaging; evaluation of the syrinx and cerebrospinal fluid (CSF) velocity and flow by using phase-contrast cine MR imaging; and evaluation of lumbar and cervical subarachnoid pressure at rest, during the Valsalva maneuver, during jugular compression, and following removal of CSF (CSF compliance measurement). During surgery, cardiac-gated ultrasonography and pressure measurements were obtained from the intracranial, cervical subarachnoid, and lumbar intrathecal spaces and syrinx. Six months after surgery, clinical examinations, MR imaging studies, and CSF pressure recordings were repeated. Clinical examinations and MR imaging studies were repeated annually. For comparison, 18 healthy volunteers underwent T1-weighted MR imaging, cine MR imaging, and cervical and lumbar subarachnoid pressure testing. Compared with healthy volunteers, before surgery, the patients had decreased anteroposterior diameters of the ventral and dorsal CSF spaces at the foramen magnum. In patients, CSF velocity at the foramen magnum was increased, but CSF flow was reduced. Transmission of intracranial pressure across the foramen magnum to the spinal subarachnoid space in response to jugular compression was partially obstructed. Spinal CSF compliance was reduced, whereas cervical subarachnoid pressure and pulse pressure were increased. Syrinx fluid flowed inferiorly during systole and superiorly during diastole on cine MR imaging. At surgery, the cerebellar tonsils abruptly descended during systole and ascended during diastole, and the upper pole of the syrinx contracted in a manner synchronous with tonsillar descent and with the peak systolic cervical subarachnoid pressure wave. Following surgery, the diameter of the CSF passages at the foramen magnum increased compared with preoperative values, and the maximum flow rate of CSF across the foramen magnum during systole increased. Transmission of pressure across the foramen magnum to the spinal subarachnoid space in response to jugular compression was normal and cervical subarachnoid mean pressure and pulse pressure decreased to normal. The maximum syrinx diameter decreased on MR imaging in all patients. Cine MR imaging documented reduced velocity and flow of the syrinx fluid. Clinical symptoms and signs improved or remained stable in all patients, and the tonsils resumed a normal shape. CONCLUSIONS: The progression of syringomyelia associated with Chiari I malformation is produced by the action of the cerebellar tonsils, which partially occlude the subarachnoid space at the foramen magnum and act as a piston on the partially enclosed spinal subarachnoid space. This creates enlarged cervical subarachnoid pressure waves that compress the spinal cord from without, not from within, and propagate syrinx fluid caudally with each heartbeat, which leads to syrinx progression. The disappearance of the abnormal shape and position of the tonsils after simple decompressive extraarachnoidal surgery suggests that the Chiari I malformation of the cerebellar tonsils is acquired, not congenital. Surgery limited to suboccipital craniectomy, C-I laminectomy, and duraplasty eliminates this mechanism and eliminates syringomyelia and its progression without the risk of more invasive procedures.

Down-regulation of nitric oxide production by ibuprofen in human volunteers.

Ibuprofen has been shown in vitro to modulate production of nitric oxide (NO), a mediator of sepsis-induced hypotension. We sought to determine whether ibuprofen alters NO production and, thereby, vascular tone, in normal and endotoxin-challenged volunteers. Techniques for detecting NO were validated in 17 subjects infused with sodium nitroprusside, a NO donor. Then, endotoxin (4 ng/kg) or saline (vehicle alone) was administered in a single-blinded, crossover design to 12 other subjects randomized to receive either ibuprofen (2400 mg p.o.) or a placebo. Endotoxin decreased mean arterial pressure (MAP; P =.002) and increased alveolar NO flow rates (P =.04) and urinary excretion of nitrite and nitrate (P =.07). In both endotoxemic and normal subjects, ibuprofen blunted the small fall in MAP associated with bed rest (P =.005) and decreased alveolar NO flow rates (P =.03) and urinary excretion of nitrite and nitrate (P =.02). However, ibuprofen had no effect on the decrease in MAP caused by endotoxin, although it blocked NO production to the point of disrupting the normal relationship between increases in exhaled NO flow rate and decreases in MAP (P =.002). These are the first in vivo data to demonstrate that ibuprofen down-regulates NO in humans. Ibuprofen impaired the NO response to bed rest, producing a small rise in blood pressure. Although ibuprofen also interfered with the ability of endotoxin to induce NO production, it had no effect on the fall in blood pressure, suggesting that the hemodynamic response to endotoxin is not completely dependent on NO under these conditions.

Quenching revisited: low level direct current inhibits amygdala-kindled seizures.

We have reported that low frequency stimulation (1 Hz for 15 min), applied after kindling stimulation of the amygdala, inhibited the development and expression of amygdala-kindled seizures, an effect we termed quenching. Subsequently, we discovered that this effect could only be achieved when certain stimulators were used that also emitted a low-level direct current (DC). The studies reported here indicate that DC, applied once daily for 15 min at intensities of 5-15 microA, produced an intensity-related attenuation of kindling development and an increase in the afterdischarge threshold. This effect persisted in some animals for at least 1 month after discontinuation of the DC. In fully kindled animals, a robust increase in seizure threshold and persistent seizure inhibition were also observed using 10 microA of DC administered for 14 days. These results clarify and extend our original findings of a quenching effect; however, the mechanisms by which low level DC induces quenching require further elucidation.

A head motion measurement system suitable for emission computed tomography.

Subject motion during brain imaging studies can adversely affect the images through loss of resolution and other artifacts related to movement. We have developed and tested a device to measure head motion externally in real-time during emission computed tomographic (ECT) brain imaging studies, to be used eventually to correct ECT data for that motion. The system is based on optical triangulation of three miniature lights affixed to the patient's head and viewed by two position-sensitive detectors. The computer-controlled device converts the three sets of lamp positions into rotational and translational coordinates every 0.7 seconds. When compared against a mechanical test fixture, the optical system was found to be linear and accurate with minimal crosstalk between the coordinates. In a study of two subjects, comparing the angular motions measured by the optical device and a commercially available electromagnetic motion detector, the two systems agreed well, with an root mean square (rms) difference of less than 0.6 degree for all rotations.

A simple method for measurement of cell-substrate attachment forces: application to HIV-1 Tat.

In order to understand the importance of cell attachment to HIV-1 Tat, we quantified the strength of cell attachment to immobilized Tat in microtiter plate wells by the application of buoyant force. By replacing the attachment medium with dense medium, and subjecting the attached cells in the microtiter plates to centrifugal force in the conventional upright position, weakly binding and strongly binding cells could be discriminated (and separated) by varying the centrifugal speed. The strength of attachment of HT1080 cells to Tat was compared with that of the well-known extracellular matrix (ECM) proteins fibronectin and vitronectin. We observed that all three proteins mediated significant attachment of HT1080 cells both at 4 degrees C and 37 degrees C. However, unlike the ECM proteins, Tat was unable to engage in higher strength binding when the temperature was raised to 37 degrees C. The relatively weak binding of HT1080 cells to Tat (in the order of 3.0 mudynes/picomole of coated Tat) and lack of strengthening of binding to Tat at physiologic temperature suggests that this protein does not mimic adhesion molecule function. We anticipate that the methodology developed and described here will be useful in a wide variety of cell-matrix and cell-cell interaction studies.

Experimental human endotoxemia increases cardiac regularity: results from a prospective, randomized, crossover trial.

OBJECTIVE: To determine whether human endotoxemia is associated with a loss of the physiologic beat-to-beat variability of heart rate. DESIGN: Prospective, randomized, crossover, single-blind study. SETTING: Clinical research center in a federal, nonuniversity hospital. SUBJECTS: Healthy volunteers. INTERVENTIONS: Intravenous administration of reference (Escherichia coli) endotoxin or saline placebo, with or without previous administration of oral ibuprofen. MEASUREMENTS AND MAIN RESULTS: Electrocardiograms were continuously recorded and digitized using series of 1000 beat epochs of R-R intervals over 8 hrs. Analyses included measures in the time domain (standard deviation), frequency domain (power spectra), and a measure of regularity (approximate entropy). Endotoxin administration was associated with loss of variability by all measures. This loss of variability remained significant even with administration of ibuprofen, which blocked the development of fever and endotoxin-related symptoms. CONCLUSIONS: Infusion of endotoxin into human volunteers causes loss of heart rate variability, as measured by standard deviation and power spectra, as well as an increase in heart rate regularity, as measured by approximate entropy. Changes in approximate entropy occurred earlier than changes in other heart rate variability measures and may be a useful means of detecting early sepsis. This reduction in regularity is consistent with a model in which the pathogenesis of multiple organ system dysfunction syndrome involves the physiologic uncoupling of vital organ systems.

Aequorin targeted to the endoplasmic reticulum reveals heterogeneity in luminal Ca++ concentration and reports agonist- or IP3-induced release of Ca++.

A chimeric protein (ERaeq) comprised of the invariant chain (Ii) of class II major histocompatability complex (MHC-II) and aequorin was localized in the endoplasmic reticulum (ER) of transfected human embryonal kidney 293 cells. The targeted aequorin resided in the lumen of the ER membrane system, including the nuclear cistern, and following addition of the chromophore coelenterazine underwent Ca(++)-activated chemiluminescence. The majority of chemiluminescence produced by coelenterazine treatment of ERaeq-expressing 293 cells was consumed rapidly (within 2-4 min) upon re-addition of Ca++ to coelenterazine-loaded cells, a finding consistent with very high Ca++ concentrations (approximately 10(-5)-10(-3) M Ca++ ion) inside the ER. However, following the initial rapid consumption of ERaeq chemiluminescence, the activity that remained (10-30% of total sample luminescence of permeabilized cells or 50-70% of total sample luminescence of intact cells) was found to produce a stable baseline corresponding to a Ca++ ion concentration < or = 1-2 microM. The stable baseline of luminescence observed following rapid consumption of the majority of the sample's activity was not derived from re-binding of fresh chromophore to spent photoprotein, suggesting that a minority fraction of the ER membrane system within which the ERaeq chimera was distributed contained a relatively low Ca++ concentration. Addition of IP3 to digitonin-permeabilized cells, or agonist treatment of intact cells decreased this residual signal. Luminescence recordings from cells expressing an ER-targeted aequorin with relatively high affinity for Ca++ thus reveal heterogeneity in luminal ER Ca++ concentration and permit observation of receptor- and IP3-activated release of Ca++ from the ER membrane system.