Alterations in human vitreous humour following cataract extraction.

Cataract extraction is associated with the risk of posterior vitreous detachment, macular edema and retinal detachment possibly as a result of a disturbance to the vitreous body during surgery. While it is common for lens cortical fiber debris to leak into the vitreous humour during cataract extraction, the extent to which the vitreous humour is altered post-surgery is unknown. The current study examines the integrity of the vitreous humour of pseudophakic and phakic human donor eyes by comparing the proteome, the viscosity and the size distribution of macromolecules in different regions of the vitreous humour from human pseudophakic and phakic donor eyes. Major differences between the proteomes of anterior and posterior vitreous humour were observed in phakic and pseudophakic donor eyes. Seventeen spots identified as complete, modified or cleaved forms of alphaA-, alphaB-, betaA4-, betaB2, and gammaS-crystallins were present in the anterior vitreous humour of all pseudophakic eyes studied. Crystallins were not detected in the posterior vitreous humour of the pseudophakic eye or the vitreous humour of the phakic eye. Significant alterations in abundance and/or modification of transthyretin, alpha antitrypsin, and retinoic acid binding protein were observed in all locations of pseudophakic vitreous humour as compared to phakic samples. In addition, a significant decrease in the number and intensity of protein spots was observed for the posterior vitreous humour of pseudophakic eyes when compared to posterior vitreous humour of phakic eyes. Proteins which were affected include antioxidant proteins and enzymes such as carbonic anhydrase and trisephosphate isomerase. A reversal of the viscosity gradient, anterior to posterior, in the vitreous humour of pseudophakic eyes was observed concomitant with alterations in the distribution of 50 nm particles. These particles are likely primarily composed of hyaluronan. While varying degrees of vitreous degradation may have existed prior to surgery and may have contributed to the cataract formation, in no case did the phakic donor eyes exhibit the same alterations in the vitreous humour proteome, viscosity or particle sizes as did the pseudophakic donor eyes. The examination of phakic/pseudophakic donor eye pairs confirmed that the vitreous humour proteome and structural integrity were very similar in the matched phakic donor eye to eyes from donors with no history of cataract. Even though the number of samples for this study was limited, the observed changes support the hypothesis that alterations in the vitreous humour proteome occur in psuedophakic eyes with concurrent alterations in the structure of the vitreous humor. These modifications of the microenvironment of the retina may contribute to the development of retinal complications following cataract surgery.

NMR relaxation studies of syneretic response to pressure change in bovine lenses.

PURPOSE: Reversible syneretic response to pressure in bovine and rhesus monkey lenses has been demonstrated previously by invasive techniques, such as differential scanning calorimetry and thermogravimetric analysis. This study is designed to investigate whether such a response could be observed by non-invasive techniques, namely by relaxographic imaging studies, in situ, in the intact, albeit excised lens. METHODS: Excised bovine lenses were incubated in media at 37 degrees C in specialized pressure chambers for 24 hrs. Three pressures, 2, 1 and 0.03 atm, were employed. The pressure chambers were placed in the cavity of an NMR magnet. Seven sections of the lens, under 2 atm pressure, from anterior outer cortex to posterior outer cortex were imaged and the T(1) (spin-lattice) and T(2 ) (spin-spin) relaxation data on each section were collected. The pressure was then released and NMR data were collected under 1 atm. Similar arrangement was followed on lenses under initial 0.03 atm pressure. T(1) and T(2) relaxations were analyzed by fitting pixel intensity to one and two term exponential expressions. RESULTS: Analysis of the time dependence of the T(2) relaxation time indicated that the response to a change in pressure is complete within 2 hours. Both T(1) and T(2) relaxation times showed minimal values in the nuclear region and maxima at the two outer cortexes. With increasing pressure both relaxation times decrease. The effect of pressure on both relaxation times was smaller in the nucleus and more enhanced at the outer cortexes. The pre-exponential terms of the fittings of both T(1) and T( 2) relaxations indicate the amount of protons participating in the relaxation. Thus they serve as a population index. The T(2) population index had a maximum in the nucleus and minima in the two cortexes. The population index of T(1) relaxation exhibited minimal value in the nucleus and maxima at the two cortexes. The pre-exponential term of T(2) relaxation increased with increasing pressure. The pre-exponential term of T(1) relaxation did not show consistent pressure dependence. CONCLUSIONS: The positional dependence of T(2) relaxation times as well as that of its population index indicated that it represents the behavior of the bound water in the lens. The positional dependence of T(1) population index suggests that this relaxation represents the total water that has a minimal value in the nucleus. Both the relaxation times as well as the population indices indicated that as pressure increases the strength of hydrogen bonding as well as the amount of bound water increases. This also means that the free water/bound water ratio decreases with increasing pressure. Thus NMR imaging and relaxation studies confirm significant syneretic response to applied hydrostatic pressure in bovine lenses.

Syneretic response to pressure in bovine and rhesus monkey vitreous.

Excised bovine and rhesus monkey vitreous samples were subjected to different hydrostatic pressures (0.03, 1 and 2 atm) for 12 hr. At the end of the experiment the samples were frozen in a -70 degrees C dry ice-acetone bath and thereafter maintained in frozen conditions. Differential scanning calorimetry (DSC) was used to obtain freezable water (FW) content. The total water content of the samples was obtained by thermogravimetric (TGA) techniques. The non-freezable water (NFW) content was obtained as a difference between total and freezable water contents. The total water content did not show a trend in pressure dependence, neither in bovine nor in rhesus monkey vitreous samples. However, the free water/bound water ratio, i.e. FW/NFW, increased with increasing pressure in vitreous samples of both species. The trend of the average FW/NFW values as a function of hydrostatic pressure implies a simple syneretic response, i.e. the conversion of bound (non-freezable) water to free water as pressure was increased.

On the equilibrium between monomeric alpha-lactalbumin and the chaperoning complex of alpha-crystallin.

In chaperoning dithiothreitol-denatured alpha-lactabumin, alpha-crystallin forms a chaperoning complex. In order to study the kinetics of such chaperoning it needs to be established whether the formation of the chaperoning complex is a reversible or irreversible process. The chaperoning reaction was studied by dynamic light scattering as a function of concentration and weight ratio of alpha-lactalbumin/alpha-crystallin. HPLC and subsequent SDS-PAGE gel electrophoresis experiments established that the chaperoning complex formed contains both alpha-crystallin and alpha-lactalbumin. Upon rechromatographing the chaperoning complex, the presence of monomeric alpha-lactalbumin has been demonstrated in addition to the chaperoning complex itself. This and equilibrium dialysis experiments demonstrated conclusively the existence of an equilibrium between monomeric partially denatured alpha-lactalbumin and the chaperoning complex made of alpha-lactalbumin and alpha-crystallin.

The mode of chaperoning of dithiothreitol-denatured alpha-lactalbumin by alpha-crystallin.

Molecular chaperones prevent the aggregation of partially folded or misfolded forms of protein. alpha-crystallin performs such a function in the ocular lens. To gain insight into the mechanism of the anti-aggregation activity of alpha-crystallin, we performed dynamic light scattering (DLS) measurements investigating its interaction with partially denatured alpha-lactalbumin over a 24 hr period. Analyses were conducted as a function of the concentration of alpha-lactalbumin as well as the bovine alpha-crystallin/alpha-lactalbumin ratio. Additional studies of the systems were performed by HPLC and SDS gel electrophoresis. The particle distribution patterns derived from the DLS data indicated that the chaperoned complex (lactalbumin plus crystallin) is a loose fluffy globular entity. After the complex becomes saturated with lactalbumin, it appears to release the partially denatured lactalbumin which may aggregate into high molecular weight moieties. These eventually may precipitate out of solution. On longer standing, 24hr and over, the chaperoned complex as well as the lactalbumin aggregates become more compact. The chaperoned complex (alpha-crystallin plus alpha-lactalbumin) is in dynamic equilibrium both with the monomeric and the aggregated alpha-lactalbumin population.

Pressure-induced syneretic response in rhesus monkey lenses.

PURPOSE: To investigate the effect of pressure on the freezable and nonfreezable water content of the lens. METHODS: Excised rhesus monkey lenses in tissue culture media were subjected to three different hydrostatic pressures (2 atm, 1 atm, and 0.03 atm) for 24 hours. Then while still under the experimental pressure, the vessels were cooled in dry ice-acetone until the lenses were frozen. While the lenses were kept frozen, nuclear and cortical samples were dissected, enclosed in a sample pan, and weighed. Differential scanning calorimetry (DSC) measurements were performed between -30 degrees C and 30 degrees C. Total water content of each lens sample was obtained by thermogravimetric analysis at 105 degrees C. The nonfreezable water content was obtained by subtracting the freezable water content calculated from the DSC data from the total water content. RESULTS: The total water content of the lenses did not change significantly as a function of pressure applied. This was true both for cortical and for nuclear sections. The freezable water content increased as the pressure decreased both in cortex and nucleus. Similarly, the freezable water/nonfreezable water ratio also decreased with increasing pressure. CONCLUSIONS: External hydrostatic pressure would generate an influx of water into the lens. To alleviate this diluting tendency and to prevent turbidity as a result of dilution, the lens must effect an osmotic pressure change equivalent to the applied pressure. Change in the osmotic pressure is caused by changing the activity of the water (i.e., converting free water to bound water). This is a reversible and energetically the least expensive response. The release of bound water from the hydration layers of macromolecules and its conversion to free water in condensed systems are known as syneresis. In the lens decreasing pressures induce syneresis as demonstrated by the increase in freezable water content and the freezable water/nonfreezable water ratio. Such a response may be operative also in accommodating lenses.

Thermodynamic stability of bovine alpha-crystallin in its interactions with other bovine crystallins.

Light scattering measurements were performed on dilute solutions of alpha-crystallin mixed with different combinations of beta H, beta L and gamma-fractions of bovine lens crystallins. Light scattering intensities were obtained as a function of scattering angle, concentration and temperature. The temperature dependence of the second virial coefficients was used to obtain partial molar enthalpy and end entropy of solutions. The difference between the thermodynamic parameters of the crystallin mixtures and those of the weighted averages of the individual components yielded the excess enthalpy and entropy functions of the solutions. Both the excess enthalpy and entropy functions indicated that thermodynamic stability of alpha-crystallin is progressively enhanced by its interactions with gamma [symbol: see text] (beta H + gamma) [symbol: see text] (beta H + beta L + gamma) crystallins. The last two combinations showed negative values both for excess enthalpy as well for excess entropy of solutions. Other combinations demonstrated increasing positive values. This implies that the combination of all four crystallins in the vertebrate lens enables the best solvation property as well as the best packing as opposed to any other single or combinatorial arrangements of crystallins. Similar conclusions have been obtained in the past from water and other vapor sorption studies.

Do changes in the hydration of the diabetic human lens precede cataract formation?

Cataract formation in diabetic lenses has been attributed to polyol-osmotic pressure-generated influx of water. The ensuing swelling in the form of pocket and lake accumulations cause light scattering. The authors tested whether clear lenses of diabetic patients show different hydration properties than age matched normal lenses. Normal and diabetic human lenses were investigated for their nonfreezable water content by differential scanning calorimetry. The total water content of the lens sections were studied by thermogravimetric analysis. Non-cataractous diabetic lenses in all three regions showed a higher total water content than normal lenses. The nonfreezable water content, seems to increase with age in diabetic lenses and decrease with age in normal human lenses. Thus, hydration changes in human diabetic lenses precede cataract formation. While syneresis, the release of bound water into the bulk, is part of the normal aging process, it appears to occur in the younger diabetics only. In older diabetics syneresis is halted or even reversed. This may be due to the glycation of lens proteins in diabetic patients which tends to immobilize water and therefore, reverse the syneresis due to aging.

On the nature of hereditary cataract in strain 13/N guinea pigs.

PURPOSE: Congenital nuclear cataracts in strain 13/N guinea pigs are caused by a single splice-site mutation in the zeta-crystallin gene. Very little is known of the physical factors involved in lens opacification of this system. The aim of this study is to elucidate the biophysical processes causing the nuclear turbidity. METHODS: Normal, homozygous and heterozygous mutant guinea pig lenses were studied. Polarized light scattering measurements were performed on thin sections of lenses as a function of scattering angle. Scattering intensities were collected in two modes, I- and I+. The total water content of lenses was determined by thermogravimetric analysis. The nonfreezable (bound) water content was obtained by differential scanning calorimetry. The morphology of lenses was investigated by scanning electron microscopy. RESULTS: Normal lenses scatter 5- to 10-fold less light than cataractous lenses at wide angles in both modes. The intensity ratios of the two modes imply that most of the scattering comes from density fluctuations; 10-20% of the turbidity may be contributed by orientation fluctuations. The nucleus of heterozygous cataractous lenses contain less total water than normal lenses, whereas the cortex has the same hydration as the normal lens. The nonfreezable water content of the cataractous nucleus is higher than that of the normal lens. Scanning electron microscopy showed frequent truncation of the fiber cells, cavitations and occasional longitudinal splitting resulting in hollow cylinder formation in the nucleus of the cataractous lens. CONCLUSION: Mutation of zeta-crystallin in guinea pigs causes a congenital cataract. A number of supramolecular events contribute to the turbidity. The mobile water leaves the nuclear fiber cells, causing a collapse of supramolecular structures. Both the size of the aggregates and their refractive index increase by this dehydration process, contributing to the turbidity. The truncation and hollowing of fiber cells causes the orientation fluctuations that also increase turbidity.

Light scattering and morphology of cataract formation in transgenic mice containing the HIV-1 protease linked to the lens alpha A-crystallin promoter.

Two constructs of transgenic mice, TG61 and TG72, containing the HIV-1 protease linked to the lens alpha A-crystallin promoter develop cataract. The TG61 construct develop cataract in utero, while the TG72 construct exhibit frank opacities on the 24th day (homozygotes) and 26th day (hemizygotes) post natum. Polarized light scattering measurements were performed on cortical and nuclear sections of TG72 lenses from day 19 to day 26 as a function of scattering angle. The parallel components of the scattered light intensity increase gradually during opacification, the perpendicular components show very low values from day 19 to day 22 after which they increase exponentially. Analysis of the scattered light intensities yielded parameters describing the size of the protein aggregates, the size of the domains exhibiting optical anisotropy/birefringence, the difference in refractive index between (a) the protein aggregates and their surroundings and (b) the birefringent units and their surroundings. The last parameter accounts for the major portion of the increase in lens turbidity. The TG72 construct shows normal lens development on day 16. By day 21 the posterior cortex shows some disintegration, while the lens is still clear. By day 26 the lens nucleus migrates toward the posterior pole and there is a major alteration in the cortical fibers. Scanning electron microscopic studies reveal normal fiber cell organizations in control animals. In the TG72 construct the fiber cells are well organized at 16 days after birth but already develop some slight separation in the posterior cortical part of the lens. By post-natal day 21, the nucleus and the anterior cortex still exhibit well aligned fiber cell organization, but the posterior cortex shows disalignment. By day 26 in the TG72 construct, all areas of the lens show complete disintegration of the fiber cells and amorphous masses are present throughout. The light scattering parameters describing changes on the nanometer scale can be correlated with the changes in lens morphology during cataractogenesis that occur on the micrometer scale. In comparison, the light and scanning electron microscopic examinations of the postnatal TG61 construct show that the lens is severely disrupted and contains completely disintegrated fiber cell remnants at an early age.

Light scattering parameters of rat lenses with calcium-induced cataracts.

Light scattering intensities of rat lenses obtained in the I,, and I+ modes were analysed using the random density and orientation fluctuation theory. Rat lenses incubated in calcium rich media had the same density fluctuation parameters as rat lenses incubated in control (low-calcium) media. However, the correlation length of the orientation fluctuations decreased during cataract formation by 100 to 200 nm while the amplitude of the fluctuations increased. The correlation length, or the size of the optically anisotropic domains, is related to the size of the cytoskeleton, especially vimentin. Vimentin has been shown to degrade when calcium activates calpain. This has been observed in SDS-gel electrophoretic experiments in rat lenses in calcium rich media. The amplitude factor of orientation fluctuations, that is, the mean squared deviation from the average refractive index, increased between two- and seven-fold during cataractogenesis. These results indicate that calcium cataract formation at the beginning (first 72 hr incubation) has little to do with aggregation or syneresis but it is largely the result of changes in the intrinsic birefringence of the lens due to vimentin degradation.

Degradation of vimentin in frozen rat lenses.

Rat lenses stored for extended periods at -20 degrees C showed degradation and eventual disappearance of vimentin. This may have been caused by the rupture of cells and/or organelles by growing ice crystals and the subsequent release and diffusion of proteases. Besides the disappearance of vimentin there was also a decrease in the intensity of the beta B1 polypeptide after 3 months storage. The observations reported herein are generally qualitative in nature, but should serve to alert other investigators of a potential problem.

Lens hydration in transgenic mice containing HIV-1 protease linked to the lens alpha A-crystallin promoter.

Two constructs of transgenic mice, TG61 and TG72, containing HIV-1 protease linked to lens alpha A-crystallin promoter develop cataract. The TG61 construct exhibits cataractogenesis in utero, while in the TG72 construct frank opacities appear 24 days (homozygotes) and 26 days (hemizygotes) after birth. Differential scanning calorimetry and thermogravimetric analysis studies indicate that the hydration of lenses is strongly correlated with cataractogenesis. In all clear lenses (normal and precataractous) the total water content was the same, 68%, and increased upon opacification. The bound water, measured as percentage nonfreezable water of the total water, decreased upon cataract formation, indicating a syneretic process. On the other hand, the bound water expressed as grams of nonfreezable water per gram dry weight increases upon opacification. This implies that proteolysis and subsequent enhanced hydration is the primary supramolecular event in cataractogenesis and that syneresis in the lens of transgenic mice is of secondary importance.

Enthalpy and entropy of hydration of bovine crystallins.

Transparency of the lens of the eye is the result of a short range order in the packing of crystallin molecules within the fiber cells. Short range order depends on crystallin-crystallin as well as water-crystallin interactions. Light scattering measurements can provide information on the hydration of crystallins. Light scattering intensities were obtained as a function of scattering angle, concentration, and temperature on dilute solutions of beta H, beta L, and gamma fractions of bovine lens crystallins. The temperature dependence of the second virial coefficient was negative for the beta crystallin fractions and positive for the gamma fraction as well as that for alpha crystallin (Wang, X., and Bettelheim, F. A. (1989) Proteins Struct. Funct. Genet. 5, 166-169). The partial molar enthalpy values of the solutions were negative for the beta crystallin fractions, indicating a tendency for homo- and heterodimer and -oligomer association. The enthalpy values were positive for the alpha and gamma fractions. The negative values of the enthalpy of solutions differentiate the beta crystallins from the other crystallins. The partial molar entropy values of solutions of beta L and gamma fractions were identical, those of the oligomeric beta H fraction were higher, whereas those of alpha crystallin were a magnitude larger than those of the smaller crystallin molecules.

A study of collagen-hyaluronan interaction through swelling in polyacrylamide gels.

The equilibrium swelling of biopolymer loaded polyacrylamide gels can yield information on polymer-polymer interactions. Using the temperature dependence of excess equilibrium swelling, equations were derived to yield homopolymer as well as heteropolymer interactions. Hyaluronan and collagen (Type I) encapsulated in polyacrylamide gels showed increasing isothermal swelling with increase in the bipolymer concentration. The pH dependence of isothermal swelling caused by hyaluronan indicated that the swelling was largely due to repulsion of the charges of the carboxylate groups. Temperature dependent studies showed a decrease in swelling with an increase in temperature for collagen. The opposite trend was found for hyaluronan. This indicates that the interactions among collagen monomers are largely hydrophobic, while the interactions among hyaluronan molecules are mainly hydrogen bonding and repulsive ionic forces. Combined hyaluronan and collagen in polyacrylamide gels demonstrated an increase in swelling with an increase in temperature albeit much reduced compared with hyaluronan alone. Collagen interacts with hyaluronan through ionic attraction.

Calcium cataract: a model for optical anisotropy fluctuations.

Young rat lenses were incubated in organ culture media enriched with 20 mM calcium. Lenses in the calcium rich medium developed cataracts and were characterized by the absence of vimentin in the urea soluble protein fractions. Sections from the same lenses were studied by polarized light scattering. The I+/Iparallel scattering intensity ratios were higher from the lenses in calcium-rich media than from the control lenses. This indicated an increase in the optical anisotropy fluctuations during cataractogenesis. The turbidity that developed due to these fluctuations was caused partly by the disappearance of vimentin and which in turn caused the enhancement of birefringence of the lens.

Hydration of gamma-crystallins.

Four fractions of gamma-crystallins, B(II), C(IIIb), D(IIIa) and E(IVa), were isolated from bovine lenses. The hydration properties of each fraction were measured as a function of protein concentration. The freezable water content was obtained by differential scanning calorimetry and the total water content by thermogravimetric analysis. The difference yields the non-freezable (bound) water content of the crystallins. At physiological protein concentrations the order of the bound water content was C(IIIb) > D(IIIa) >> E(IVa) > B(II). This order was reversed between C and D and between B and E at low protein concentrations. This trend was evident whether the bound water was calculated as a percentage of the total water or as quantity per gram of gamma-crystallin.

The hydration of vimentin.

The hydration of vimentin isolated from the cortex of bovine lenses was studied by thermal analysis. Differential scanning calorimetry between -30, and 30 degrees C provided the freezable water content, and thermogravimetric analysis up to 105 degrees C yielded the total water content. The difference between the two gives the nonfreezable water content (bound water) as a function of concentration. The nonfreezable water content of the polymerized vimentin is higher than that of the monomeric vimentin. This behavior is exactly the opposite of that of actin. This difference is explained on the basis of different modes of supramolecular assembly in the two cytoskeletal bodies.