Viscosities encountered during the cryopreservation of dimethyl sulphoxide systems.

This study determined the viscous conditions experienced by cells in the unfrozen freeze concentrated channels between ice crystals in slow cooling protocols. This was examined for both the binary Me2SO-water and the ternary Me2SO-NaCl-water systems. Viscosity increases from 6.9 ± 0.1 mPa s at -14.4 ± 0.3 °C to 958 ± 27 mPa s at -64.3 ± 0.4 °C in the binary system, and up to 55387 ± 1068 mPa s at -75 ± 0.5 °C in the ternary (10% Me2SO, 0.9% NaCl by weight) solution were seen. This increase in viscosity limits molecular diffusion, reducing adsorption onto the crystal plane. These viscosities are significantly lower than observed in glycerol based systems and so cells in freeze concentrated channels cooled to between -60 °C and -75 °C will reside in a thick fluid not a near-solid state as is often assumed. In addition, the viscosities experienced during cooling of various Me2SO based vitrification solutions is determined to below -70 °C, as is the impact which additional solutes exert on viscosity. These data show that additional solutes in a cryopreservation system cause disproportionate increases in viscosity. This in turn impacts diffusion rates and mixing abilities of high concentrations of cryoprotectants, and have applications to understanding the fundamental cooling responses of cells to Me2SO based cryopreservation solutions.

Calcium effects on frog retinal cyclic guanosine 3', 5'-monophosphate levels and their light-initiated rate of decay.

When retinal sections were isolated from dark-adapted bullfrogs and placed in normal ringer's solution, they contained 40.7 +/- 0.2 pmol cGMP/mg protein (mean +/- SEM, 30 samples). When isolated, dark-adapted retinal sections were removed from normal ringer's solution and placed in calcium-deficient ringer's solution with 3 mM EGTA, there was about a threefold rise in cyclic GMP (cGMP) levels by 1.5 min and about a 10-fold rise by 5 min. The cGMP level remained high with no detectable decrease for at least 40 min (the longest time measured). When isolated, dark- adapted retinal sections were removed from normal ringer's solution and placed in ringer's solution which contained high- calcium (20 mM CaCl(2)), there was a slow but significant decrease in cGMP levels. After 20 min in high-calcium ringer's solution the cGMP level was 0.58 +/- 0.07 (mean +/- SEM, eight samples) of the cGMP level in normal ringer's solution incubated for the same time. The rate at which 10-fold elevated cGMP levels in low calcium decreased upon illumination was examined using quick-freezing techniques on the retinal sections. The elevated cGMP level in retinal sections incubated in low-calcium decreased upon illumination was examined using quick-freezing techniques on the retinal sections. The elevated cGMP level in retinal sections incubated in low-calcium ringer's solution was found to decay about 15-fold faster than cGMP levels in retinal sections incubated in normal ringer's solution. The CGMP level in low calcium was significantly different (P=0.005) after 1 s illumination, whereas the cGMP level in normal calcium was not significantly different.

Light-initiated changes of cyclic guanosine monophosphate levels in the frog retina measured with quick-freezing techniques.

Although there is good agreement that light reduces the amount of cyclic GMP (cGMP) in the retina, the exact time-course of this decrease is not well established. Bullfrog retinal sections were isolated under infrared light and quick-frozen with liquid nitrogen-cooled, metal hammers after exposure to various intensities of continuous illumination. This quick-freezing should stop the degradation of cGMP within 50-100 ms. The frozen retinal sections were then slowly warmed up in the presence of perchloric acid to denature enzymes involved in cGmp metabolism. cGMP was determined by radioimmunoassay and comparison was made between light- and dark-adapted retinal sections from the same animal. The average cGMP concentration was 44.3 +/- 0.7 pmol cGMP/mg protein or 170.9 +/- 3.2 pmol cGMP/retina. After 1 s of illumination no significant change in cGMP concentration was found even with the brightest light used (approximately 7 x 10(7) rhodopsins bleached/second per rod. At this intensity the first significant decrease in cGMP from dark-adapted levels was detected 3-5 s after the initiation of illumination; cGMP decayed to 70-75% of the dark-adapted value after approximately 30 s. With lower intensity illumination the cGMP levels recovered to dark-adapted levels after the initial decrease even though the bleaching light remained on.

A form of congenital stationary night blindness with apparent defect of rod phototransduction.

We report findings obtained from an individual with an unusual form of congenital stationary night blindness (CSNB). Although the rhodopsin density difference of this subject was normal, there was no evidence of rod-mediated visual function. Dark-adapted thresholds were cone-mediated, and dark-adapted electroretinograms (ERGs) represented activity of the cone system exclusively. ERG a- and b-waves obtained under light-adapted conditions were normal. The absence of a rod a-wave but the presence of normal rhodopsin density, in combination with normal cone function, indicates that this form of CSNB likely involves a defect of phototransduction that is limited to the rods. In addition, light-adapted b-wave responses to high luminance flashes were larger than dark-adapted responses, whereas a-wave amplitudes were reduced by light adaptation. These ERG results address proposed mechanisms by which light adaptation might enhance cone system responses.

Foveal cone pigment density difference and reflectance in retinitis pigmentosa.

Cone pigment density difference refers to a change in light absorption by cones before and after bleaching of their visual pigments. With a television ophthalmoscope image processor, we measured the foveal cone pigment density difference in patients with retinitis pigmentosa (RP), good central vision, and no clinically apparent foveal lesion. Foveal reflectance was obtained at 12 different wavelengths of illumination. Compared with similar-aged normal subjects, most patients with dominantly inherited RP had normal or minimally reduced cone pigment density difference within the central fovea, relatively lower than normal density difference at the foveal margin, and increased foveal reflectance. Compared with these normal subjects, patients with recessively inherited RP had significantly reduced cone pigment density difference within the central fovea, relatively more normal density difference at the foveal margin, and normal foveal reflectance.

Human macular pigment assessed by imaging fundus reflectometry.

A computerized, television-based, imaging fundus reflectometer was used to obtain estimates of the spatial distribution of macular pigment (xanthophylls) from seven normal subjects. Digitized images of the bleached macula of each subject were acquired at illuminating wavelengths from 462 to 697 nm. An analysis of spectral reflectances indicated that differences in short-wavelength reflectance between the foveal center and parafovea were influenced by spatial variations in melanin and oxyhemoglobin absorption as well as by the distribution of macular pigment. To provide an estimate of the spatial distribution of macular pigment alone, we have corrected fundus images obtained at 462 nm for the effect of melanin and oxyhemoglobin absorption. The spatial variation in macular pigment double density across the horizontal and vertical meridians of the retina was well described by Gaussian functions. The peak double densities for the individual subjects ranged from 0.22 to 0.45 and the standard deviations of the Gaussian functions averaged approx. 1 degree.

Determination of human cone pigment density difference spectra in spatially resolved regions of the fovea.

A new method was developed to measure spectrally and spatially resolved cone pigment optical density difference in normal human subjects. Using digitized television images of human retinas scanned before and after bleaching the cone pigments with a bright light, unique high-resolution images of cone pigment density difference were produced. The spectral peak density difference within the fovea was found to be at 560 nm. These measurements demonstrate a decrease in cone pigment optical density difference with increased distance from the subject's central fixation point in the fovea. The cone pigment density difference was asymmetrically distributed in the fovea with higher amounts on the nasal side.

Macular pigment and reduced foveal short-wavelength sensitivity in retinitis pigmentosa.

Some patients with retinitis pigmentosa (RP) show a reduced foveal short-wavelength sensitivity that cannot be attributed to a reduction in the sensitivity of the short-wavelength cone system. To determine whether an increased amount of macular pigment (xanthophyll) might account for this finding, we derived estimates of the two-way optical density of the macular pigment of five such RP patients as well as of five normals. The spectral reflectance of the foveal region of each subject was obtained from digitized images of the bleached fundus provided by a television-based reflectometer. The density spectra of the macular pigment, melanin, and oxygenated hemoglobin were fit by a least-squares procedure to the log of the ratio of parafoveal to foveal spectral reflectance in order to obtain a quantitative estimate of the contribution of each of these ocular pigments to foveal short-wavelength reflectance. By this analysis, the two-way densities of the macular pigment, melanin, and oxyhemoglobin of the RP patients were not significantly different from those of the normals. Therefore, the reduced foveal short-wavelength sensitivity of these patients was not due to an increased amount of macular pigment, but may result instead from morphological abnormalities in the foveal cones such that a normal amount of macular pigment screens the cones more effectively.

Foveal cone pigment density difference in the aging human eye.

Using fundus reflectometry, we have measured a decrease in the density difference of the foveal cone visual pigments with age in human subjects. This decrease is consistent with a loss of visual pigment in the retina with age. Fundus reflectance and normalized density difference spectra data are presented for these subjects. A decrease in cone pigment with age would be consistent with both anatomic studies, which indicate a loss and displacement of photoreceptors with age, and psychophysical studies, which demonstrate loss of photoreceptor function with age.

Visual pigments in the human macula assessed by imaging fundus reflectometry.

Multispectral imaging fundus reflectometry and multiple linear regression fitting routines were used to simultaneously assess the spatial distributions of cone visual pigment and rhodopsin in the human macula. As expected from anatomic studies, the cone visual pigment distribution showed a peak in the central fovea and was elliptical, with the broader axis along the horizontal meridian. The rhodopsin distribution showed a minimum in the fovea and the rhodopsin density increased with eccentricity. Both visual pigment distributions showed striking variability among individuals. These data provide visual pigment distributions in the relatively unexplored parafoveal region.