Cellular energetics measured by phosphorous nuclear magnetic resonance spectroscopy are not correlated with chronic nutrient deficiency in multicellular tumor spheroids.

We have measured the 31P nuclear magnetic resonance spectra of EMT6/Ro multicellular tumor spheroids over a wide range of sizes under constant nutrient conditions which matched those used for culturing the spheroids. The amount of nucleotide triphosphate per cell decreased with spheroid growth, roughly in proportion to the decrease in cell volume. There was no correlation between the intracellular pH, the nucleotide triphosphate:Pi ratio, or the phosphocreatine:Pi ratio and either the spheroid cellularity, the mean cell volume, the S-phase fraction, the clonogenic capacity, or the amount of central necrosis. The phosphoryethanolamine:phosphorylcholine ratio also increased with increasing spheroid size. There was a negative correlation between the phosphoryethanolamine:phosphorylcholine ratio and the S-phase cell fraction or the mean cell volume; this ratio was positively correlated with the extent of central necrosis. The membrane degradation components glycerophosphorylcholine and glycerophosphorylethanolamine showed no significant changes with increasing spheroid size. These results imply that spheroid necrotic areas induced by chronic nutrient deficiencies are "invisible" to 31P nuclear magnetic resonance and that the development of cellular quiescence in spheroids is not caused by a decrease in the steady-state level of high-energy phosphates or a reduced intracellular pH. Together, these data support a model in which cells maintain normal steady-state levels of high energy phosphates until they are very close to necrotic cell death. This implies that the deterioration of 31P nuclear magnetic resonance spectra of tumors with increasing size is not caused by chronic nutrient deficiencies resulting from cells outgrowing the capillary supply, but rather is more related to transient nutrient deprivation phenomena.

PTTL method applied to UV radiation detection during refractive surgery using excimer laser.

The method of phototransferred thermoluminescence (PTTL), using CaSO(4):Dy pellets produced at IPEN as sensitive material, was used to detect the spread laser radiation inside the surgery room during refractive surgical procedures using ArF excimer lasers. The purpose of this work was to study the viability of performing the ultraviolet radiation (UVR) exposure detection of patients and the hospital's surgical staff during a refractive surgery. The CaSO(4):Dy pellets were positioned at different distances from the laser source inside the surgery room: patient's ( congruent with 0.15 m), surgeon's ( congruent with 0.5 m) and nurse's ( congruent with 1.0 m) foreheads, lateral ( congruent with 1.5 m) and back ( congruent with 4.0 m) walls. The measurements of PTTL were carried out at two different conditions: five surgeries, each one taking approximately 10 min, and during a period of 4 h (cumulative), when several operations were performed. The detectors positioned as far as 4.0 m from the UV laser source were sensitised, making the UVR detection feasible at large source-detector distances. The absorbed energy was detected in the range from 40 microJ to 30 mJ during a surgery. This result indicates that the method studied can be used to detect the spread UVR.

Radiobiology of ultrasoft X rays. I. Cultured hamster cells (V79).

Ultrasoft X rays (approximately less than keV) provide a useful probe for the study of the physical parameters associated with the induction of biological lesions because the spatial scale of their energy depositions is of nanometer dimensions, comparable to that of critical structures within the cell. We report on cell-killing experiments using cultured hamster cells (V79) exposed to carbon K (0.28 keV), aluminum K (1.5 keV), copper K (8.0 keV), and 250 kVp X rays, under oxic and hypoxic conditions, and as a function of cell-cycle phase. Our principal results are: RBE increases with decreasing X-ray energy; OER decreases with decreasing X-ray energy; and cell-cycle response is similar for all X-ray energies. Our RBE results confirm earlier observations using ultrasoft X rays on mammalian cells. The shapes of fitted curves through the data for each energy are statistically indistinguishable from one another, implying that the enhanced effectiveness is purely dose modifying. The results reported herein generally support the view that single-track effects of radiation are predominantly due to very local energy depositions on the nanometer scale, which are principally responsible for observed radiobiological effects.

Calibration of trephine blade depth.

PURPOSE: To assess the accuracy and precision of the Barron radial vacuum trephine as an instrument for corneal lamellar dissection. METHODS: We describe a practical method to calibrate individual trephines prior to lamellar corneal procedures using the calibration microscope designed for diamond knives. RESULTS: We observed that one turn (360 degrees) of the trephine produced a mean blade displacement of 0.25 mm. However, individual trephines demonstrated a significant range of variability. CONCLUSION: For highly accurate trephination, it is useful to calibrate each individual trephine using the calibration microscope.

Factors affecting corneal strip stroma-to-stroma adhesion.

BACKGROUND: Successful laser in situ keratomileusis depends on strong adhesion between the dissected anterior corneal flap and the underlying stromal bed, without suturing. This study attempts to generate new information about the mechanism of this adhesion. METHODS: Strips of fresh bovine corneal stroma were dissected and split apart. The split strips were then made to adhere to each other with varying amounts of combined heat and drying. A relationship was developed between heat applied and hydration of strips. After each heat and drying treatment, the strips were pulled apart and the force needed to accomplish this task was measured. RESULTS: There was a tight linear correlation between increase in temperature and decrease in water content (R2 = 0.7414) and between decrease in water content and increase in the adhesive force (R2 = 0.3355). CONCLUSION: This model suggests that drying increases stromal-stromal adhesion. We speculate this is due to the increased concentration of surface molecules, which have high ionic charge densities and ionic bonding. A higher concentration of these molecules produces stroma-to-stroma adhesion via ionic bonding.

A clinical follow up of PRK and LASIK in eyes with preoperative abnormal corneal topographies.

AIM: To assess the safety and predictability of photorefractive keratotomy (PRK) and laser in situ keratomileusis (LASIK) based on preoperative corneal topography. METHODS: A non-randomised comparative study was carried out on 84 eyes that presented with topographic abnormalities before undergoing PRK (n = 44) or LASIK (n = 40) procedures. 84 spherical equivalent paired normal eyes served as the control group. Either PRK or LASIK procedures were performed on 168 eyes using the Summit apex plus excimer laser. Topographic abnormalities, including apex displacement (AD), increased asphericity (AS), meridional irregularity (MI), increased inferior-superior asymmetry (IS), increased curvature (CU), and combined features (CO), were assessed preoperatively using the EyeSys analysis system. Safety and predictability of the two procedures were defined as a postoperative visual acuity of 20/40 or better and the loss of one or more lines of spectacle corrected visual acuity (SCVA). RESULTS: All patients were followed for 6 months. There was a significant loss of best corrected visual acuity in the PRK-AD (p<0.001), PRK-CO (p<0.05), and LASIK-AS (p<0.001) patients. The number of eyes within plus or minus 1.0D of the surgical plan postoperatively was similar in all groups. CONCLUSION: These data suggest that although predictability was similar, PRK and LASIK performed in corneas with topographic abnormalities might cause loss of vision.

HIV-antibody detection in vitreous humor and serum by enzyme immunosorbent assay and particle agglutination test.

PURPOSE: To compare enzyme immunosorbent assay (EIA) and the particle agglutination (PA) test for the detection of antibodies to the human immunodeficiency virus (HIV) in cadaveric vitreous humor and serum. METHODS: Seventeen cadavers presenting pathologic signs of acquired immunodeficiency syndrome (AIDS) at autopsy and 33 without AIDS signs were evaluated. Consistency (k) and significance (p) between the diagnostic tests were established. RESULTS: The right and left eyes of each of 48 bilaterally tested cadavers presented identical diagnostic results in all laboratory tests. All tests were positive for antibodies in the AIDS cadavers except for two EIA-negative vitreous results. Three autopsy-negative cadavers showed by both EIA and PA testing positive results in serum and negative in vitreous. Two of them showed a serum-positive Western blot. CONCLUSION: Serum EIA anti-HIV antibody detection test is the current gold standard for evaluating corneal donor material. Neither the PA test nor EIA should be used for vitreous testing because of their low sensitivity.

Effect of differences in binocularity on perceived absolute distance.

1. Using a 0.9 x 4.0 m visual alley, perceived absolute distance was studied in four groups of subjects: binocular, cross-eyed, monocular, and induced-monocular individuals. 2. A power function between the different physical and perceived distances was adjusted and the relative and absolute errors made by the observers were calculated. Despite a tendency to overestimate distance in the monocular group, no significant differences were detected among groups. 3. The data suggest that, under natural-cue conditions, binocularity is not a determining factor for the perception of absolute distance.

Advanced techniques: developing quality drawings.

Just like writing, developing ideas for illustrating your articles is a skill you can learn. With creativity, planning, and a cooperative relationship with a graphic artist you can include drawings that enhance your reader's appreciation of every publication you write.

Evaluation of the impact of intracorneal ring segments implantation on the quality of life of patients with keratoconus using the NEI-RQL (National Eye Institute Refractive Error Quality of life) instrument.

BACKGROUND/AIMS: A prospective study evaluating the impact of intracorneal ring segment (ICRS) implantation on the quality of life of patients with keratoconus using the NEI-RQL (National Eye Institute Refractive Error Quality of Life) instrument. METHODS: Sixty-nine eyes of 42 keratoconus patients were implanted with the Keraring. All patients self-administered the NEI-RQL before and 4(1/2) to 8 months after surgery. The results were evaluated and analysed, along with best-corrected visual acuity (BCVA), refraction, contrast sensitivity, corneal topography and aberrometry preoperatively and 3 months postoperatively. RESULTS: There was a significant improvement in all NEI-RQL scales and the overall scale. "Clarity of vision," "far vision," "near vision," "activity limitations," "appearance" and "satisfaction with correction" had the greatest improvement, ranging from 25 to 50 preoperatively to 80 to 90 after surgery. Best spectacle-corrected visual acuity and binocular BCVA improved significantly, and there was a significant decrease in keratometric values, from 50.22 dioptres preoperatively to 46.27 dioptres postoperatively. Contrast sensitivity function improved postoperatively, especially at the frequency of 6 cpd. Low-order root mean square (RMS) improved after surgery, but high-order RMS did not. CONCLUSION: ICRS implantation not only improves the quantitative metrics such as visual acuity and refraction, but also has a positive impact on patients' quality of life.

Regrowth kinetics of cells from different regions of multicellular spheroids of four cell lines.

A basic understanding of the recruitment of quiescent tumor cells into the cell cycle would be an important contribution to tumor biology and therapy. As a first step in pursuing this goal, we have investigated the regrowth kinetics of cells from different regions in multicellular spheroids of rodent and human origin. Cells were isolated from four different depths within the spheroids using a selective dissociation technique. The outer cells were proliferating and resumed growth after replating with a 0-8-hour lag period, similar to cells from exponentially growing monolayers. With increasing depth of origin, the lag periods prior to regrowth increased to 2-3 times the monolayer doubling time; cells from plateau-phase monolayers showed a lag period of 1-1.5 times the doubling period. After resuming growth, all cells of a given cell line grew with the same doubling time and achieved the same confluency level. The inner spheroid cells and cells from plateau-phase monolayers had reduced clonogenic efficiencies. The inner cells were initially 1.5-3 times smaller than the outer cells, but began to increase in volume within 4 hours of replating. The fractions of S-phase cells were greatly decreased with increasing depth of origin in the spheroids; there were long delays prior to S-phase recovery after plating, to a maximum of 1-1.5 times the normal doubling time. These results suggest that those quiescent cells from spheroids and monolayers which are able to reenter the cell cycle are predominantly in the G1-phase. However, quiescent cells from the innermost spheroid region require approximately twice as long to enter normal cell cycle traverse as cells from plateau-phase monolayers. The selective dissociation method can isolate very pure populations of proliferating and quiescent cells in a rapid and nonperturbing manner; this system will be valuable in further characterizing quiescent cells from spheroids.

Automated selective dissociation of cells from different regions of multicellular spheroids.

In this report we describe a new apparatus which has been developed for the automated selective dissociation of multicellular spheroids into fractions of viable cells from different locations in the spheroid. This device is based on the exposure of spheroids to a 0.25% solution of trypsin under carefully controlled conditions, such that the cells are released from the outer spheroid surface in successive layers. Study of the spheroid size, number of cells per spheroid, and sections through the spheroid with increasing exposure to trypsin demonstrate the effectiveness of this technique. The technique has been successfully used on spheroids from five different cell lines over a wide range of spheroid diameters. We also present data detailing the effect of varying the dissociation temperature, the mixing speed, the trypsin concentration, and the number of spheroids being dissociated. The new apparatus has several advantages over previous selective dissociation methods and other techniques for isolating cells from different regions in spheroids, including: a) precise control over dissociation conditions, improving reproducibility; b) short time to recover cell fractions; c) ability to isolate large numbers of cells from many different spheroid locations; d) use of common, inexpensive laboratory equipment; and e) easy adaptability to new cell lines or various spheroid sizes. Applications of this method are demonstrated, including the measurement of nutrient consumption rates, regrowth kinetics, and radiation survivals of cells from different spheroid regions.

Partial purification of a protein growth inhibitor from multicellular spheroids.

We report the partial purification of growth inhibitors extracted from human and mouse multicellular tumor spheroids with extensive necrosis. Sephadex G-100 column chromatography of spheroid extracts separated inhibitory fractions which eluted just after the void volume of the column. Identical chromatography of monolayer cell extracts showed no inhibitory activity. High-performance liquid chromatography of spheroid extracts separated single active peaks at apparent molecular weights of 80-89 kD. These extracts were extremely heat labile, and activity was destroyed by moderate trypsin treatment. The isolation of similar growth inhibitors from spheroids of two cell lines suggests that inhibition is important in tumor cell growth control in a three-dimensional situation.

A simple electronic volume cell sorter for clonogenicity assays.

A single-parameter electronic volume flow cell sorter that can be easily and inexpensively constructed using existing technology is described. The instrument is designed for ease and flexibility of operation, including such features as a large open area for recovering sorted cells into a variety of dishes or vessels; a remote, electrically activated fluidics system; a mechanism for heating or cooling samples during sorting; a simple arrangement for monitoring and adjusting the sorting control parameters; and an interface to a standard IBM personal computer for data acquisition, analysis, and control of the sorting windows. Several researchers in our laboratory now routinely use this sorter for plating precise numbers of cells directly into culture dishes in an aseptic manner for clonogenicity assays. The instrument can sort cells at rates of up to approximately 2,000 per second with greater than 80% sorting efficiency and no cytotoxicity. An advantage of this system is that the sorting windows can be set to exclude acellular debris and include either the entire cell volume distribution or a subset thereof. Applications of the instrument are detailed, including 1) precise cell plating for low-dose survival studies, 2) separation of cells into age compartments, and 3) rapid inoculation of single cells into multiwell dishes for cloning studies. Advantages of this technology for cell survival studies are detailed, along with some limitations to its applicability.

A system for viably maintaining a stirred suspension of multicellular spheroids during NMR spectroscopy.

We have developed a system for the perfusion of a stirred suspension of multicellular spheroids during nuclear magnetic resonance spectroscopy. Measurement of the medium temperature, pH, oxygen tension, and glucose and lactate concentrations demonstrated that the macroenvironmental conditions around the spheroids during perfusion matched those in standard spinner culture flasks. Spheroids cultured in the NMR perfusion chamber for up to 48 h were virtually identical to spheroids cultured under standard conditions in terms of volume and cell number growth, the extent of central necrosis, cellular clonogenicity, and proliferative status. To avoid problems in interpreting the NMR spectra, we have used a medium containing 10% of the normal inorganic phosphate concentration; comparative growth and NMR studies showed that this medium had no effect on the results reported. 31P NMR spectroscopic analysis demonstrated that the mean pH, nucleotide triphosphate (NTP) to inorganic phosphate (Pi) ratio, the total amount of NTP, and the total energy charge were essentially constant over 8 h of analysis. Stopping the stirring of the spheroid culture during analysis resulted in depletion of the nucleotide phosphate pool in 30 min, with an accumulation of Pi and a shift to a more acid intracellular pH. This effect could be reversed if stirring was resumed within 30 min. Stopping the perfusion while maintaining stirring resulted in a deterioration of the 31P spectra until no high energy phosphates remained at 120 min and the pH fell to approximately 6. This effect was also partially reversible after 30 min of reperfusion, with recovery to a normal 31P spectrum requiring 10 h. The combination of the spheroid model system with 31P NMR spectroscopic analysis will provide a powerful tool for investigating basic questions about the regulation of tumor cell energy metabolism and viability.

Myopic angle-supported intraocular lenses: two-year follow-up.

OBJECTIVE: To evaluate clinical and refractive results of myopic angle-supported intraocular lenses (IOLs). DESIGN: Prospective, noncomparative case series. PARTICIPANTS: Twenty-one eyes of 12 patients (7 female) with a mean age of 29.5 years and a follow-up of 24 months. The prospective study included highly myopic eyes (more than -11.00 diopters [D]) with spectacle-corrected visual acuity better than 20/200. METHODS: Twenty-one eyes underwent implantation of a single-piece IOL with Z-shaped haptics for angle support (each haptic with two footplates) and an optical zone of 4.5 mm (NuVita, Bausch & Lomb Surgical, Irvine, CA). The dioptric power of the IOL was calculated considering refraction, keratometry, and anterior chamber depth (specific nomogram), and its diameter was determined by adding 0.5 mm to the corneal diameter. MAIN OUTCOME MEASURES: We evaluated the following clinical and refractive data: visual acuity without and with correction (VAsc and VAcc), spherical equivalent (SE) obtained under cycloplegia, slit-lamp biomicroscopy, keratometry, applanation tonometry, endothelial cell count, ultrasound pachymetry, gonioscopy, indirect ophthalmoscopy, subjective complaints, and descriptions of complications. RESULTS: Mean postoperative VAsc was 20/74. Mean preoperative VAcc (20/50) increased to 20/30; 65% gained at least two lines of VAcc, and no eye had a decrease in VAcc. Preoperative SE (-18.95 D) evolved to -2. 06 D, stabilizing after one month. Iris retraction (pupil ovalization) more than 0.5 mm was noted in eight eyes (40%). Mean keratometry, corneal astigmatism, and ultrasound pachymetry were stable during the study (P > 0.01). Significant endothelial cell loss was demonstrated in the second year. Gonioscopy showed 70 (87. 5%) footplates ideally positioned with no iris depression. Reports of glare and haloes in dark environment were considered light in 80% and not referred in 20%, and spectacles were used for residual refraction in 75%. Intraocular lens exchange was needed in one eye because of undersizing, and the IOL was removed in one eye because of chronic inflammatory reaction associated with ocular hypertension (this patient was excluded from the statistical analysis). CONCLUSIONS: This report demonstrates good efficacy for correction of high myopia by the phakic IOL used during the two years of follow-up. Long-term complications (safety) such as iris retraction and endothelial cell loss remain a concern.

Effect of differences in binocularity on perceived absolute distance

Using a 0.9 x 4.0m visual alley, perceived absolute distance was studied in four groups of subjects: binocular, cross-eyed, monocular, and induced-monocular individuals. A power function between the different physical and perceived distances was adjusted and the relative and absolute errors mad by the observers were calculated. Despite a tendency to overestimate distance in the monocular group, no significant differences were detected among groups. The data suggest that, under natural-cue conditions, binocularity is not a determining factor for the perception of absolute distance

Desenvolvimento de um instrumento computadorizado para medida do poder refrativo da córnea (videoceratógrafo) / Development of a computerized instrument (videokeratograph)for the measurement of the corneal refractive power

Objetivo: Resumir o desenvolvimento de um Videoceratógrafo (ou menos precisamente, Topógrafo de Córnea) projetado e fabricado no Brasil. Métodos: Discos de Plácido pintados num anteparo em forma de cone säo refletidos pela córnea. As imagens passam por um sistema óptico de aumento e säo focalizadas num CCD ('' Charge Coupled Device ''; nada mais do que uma abreviaçäo para câmeras fabricadas com a tecnologia de semicondutores) atrás do cone. O sinal do CCD é enviado para uma placa de captura de imagens ('' frame grabber '')intalada em um PC-IBM compatível. Por meio de algoritmos de processamento de imagens, extraem-se das imagens digitalizadas distâncias de borda dos Discos de Plácido. Estes valores säo inseridos em algoritmos com modelos matemáticos de curvatura da córnea, resultando no cálculo da dioptria de aproximadamente 5.760 pontos. Resultados: Assim como em outros aparelhos importados, imprime-se na tela do computador um mapa colorido plano com código de cores relativo aos valores de dioptria. Para um conjunto de 9 esferas de raios: 5.50; 5.75; 6.00; 6.25; 6.75; 7.00; 7.25; 7.50; 7.75 mm obteve-se um desvio médio de 0.1 para dioptria e 0.02 mm para o raio. Foram analizados computacionalmente 5.760 pontos para cada esfera. Em uma amostra de 20 córneas obteve-se desvio médio de 0.2 dioptrias com relaçäo ao topógrafo EyeSys. Conclusöes: Podemos afirmar que o instrumento contruído obtém resultados equivalentes àqueles do intrumento importado.