Rhesus Monkey Lenses Research Articles

Accommodative changes in lens diameter in rhesus monkeys.

Some debate surrounds the accommodative mechanism in primates, particularly whether the lens equatorial diameter increases or decreases during accommodation. This study has been undertaken to measure the relationship between changes in lens diameter and refraction during accommodation in rhesus monkeys. Photorefraction was used to measure accommodation, and goniovideography was used to measure accommodative changes in lens diameter in the iridectomized eyes of two rhesus monkeys. Accommodation was stimulated through the full amplitude available to each eye by stimulation of the Edinger-Westphal nucleus of the brain. Dynamic measurement of refractive changes followed by dynamic measurements of changes in lens diameter for the same stimulus current amplitudes allow the relationship between refraction and lens diameter to be determined. Lens diameter decreased relatively linearly during accommodation by 0.055 mm/diopter (D), resulting in an overall decrease in lens diameter of approximately 7% of the unaccommodated lens diameter for approximately 12 D of accommodation. The rhesus monkey lens diameter decreases systematically with the refractive change during accommodation in accordance with the Helmholtz accommodative mechanism and in contrast to the accommodative mechanism originally proposed by Tscherning.

Tempol-H inhibits opacification of lenses in organ culture.

Cataract is the world's leading cause of blindness and a disease for which no efficacious medical therapy is available. To screen potential anti-cataract agents, a lens organ culture model system was used. Opacification of lenses maintained in culture was induced by specific insults including H(2)O(2) or the cataractogenic sugar xylose. Potential anti-cataract agents were added to the culture medium and their ability to inhibit opacification and certain biochemical changes associated with the opacification were assessed. Among the compounds tested, Tempol-H, the hydroxylamine of the nitroxide Tempol, gave the most promising results. It significantly inhibited opacification of rat lenses in an H(2)O(2)-induced cataract system as well as opacification of rhesus monkey lenses induced by xylose. Tempol-H inhibited the loss of glutathione, the leakage of protein, and decreases in the ability of cultured lenses to accumulate (3)H-choline from the medium, all of which were associated with the development of lens opacification. The antioxidative activity of Tempol-H and its ability to re-dox cycle make it an attractive candidate as a therapeutic agent for the prevention of aging-related cataract.

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°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.

Betaine-homocysteine Methyltransferase Is a Developmentally Regulated Enzyme Crystallin in Rhesus Monkey Lens

We describe herein the characterization of a major 45-kDa protein from the soluble betaH-crystallin fraction of rhesus monkey (Macaca mulatta) lens. Based on partial peptide sequence, immunoreactivity, and enzymatic activity, this protein has been identified as betaine-homocysteine S-methyltransferase (BHMT: EC 2.1.1.5), an enzyme that catalyzes the methylation of homocysteine using either betaine or thetins as methyl donors. This protein was found to be expressed abundantly in the nuclear region of the monkey lens, reaching approximately 10% of the total nuclear protein, but was barely detectable in the epithelium and cortex regions of the lens. Because the nucleus represents the early embryonic and fetal stages of lens development, we infer that BHMT expression in the lens of the eye is developmentally regulated. By virtue of its high abundance, BHMT can be considered an enzyme crystallin (psi-crystallin). This is the first enzyme crystallin to be found in primate lenses.

Glycosphingolipids in cultured lens epithelial cells from dog and rhesus monkey.

Vertebrate lens tissues contain several species of acidic and neutral glycosphingolipids in relatively high amounts. However, the epithelia with capsule from dog and rhesus monkey lenses had a simpler composition and lower content of glycosphingolipids than whole lenses. Gangliosides and neutral glycosphingolipids in monolayer cultures of lens epithelial cells were also different from those in whole lenses. Although alpha-galactosyl (Gal alpha 1-3Gal-R) or Lewis(x) (Gal beta 1-4[Fuc alpha 1-3]GlcNAc-R) epitopes were found in glycosphingolipids from whole lenses, they were not detected in those from monolayer cultures of dog and rhesus monkey lens cells. In addition, significant changes in ganglio-series gangliosides were induced in monolayer cultures of both cells, where GM3 and GD3 were predominant. Immunofluorescence study revealed a characteristic distribution of cell surface gangliosides in confluent monolayers. These findings suggest that glycosphingolipid synthesis in lens epithelia is intrinsically different from that in cortical and nuclear fibres, and that the expression of Lewis(x) and alpha-galactosyl epitopes in glycosphingolipids appears to be associated with the differentiation of epithelial cells to fibres.

Photolysis of intact young human, baboon and rhesus monkey lenses.

Intact young human, baboon and rhesus monkey lenses were subjected to near-UV irradiation under identical conditions and fluorophore buildup was continuously monitored for several hours. The compositional changes occurring in the lenses were monitored by analyzing the ethanol extracts of the irradiated and control lenses using high-performance liquid chromatography and thin-layer chromatography (TLC). The chromatograms of the supernatant detected at 365 nm as well as the TLC scans showed the presence of 3-hydroxykynurenine glucoside (3-HKG) and two other kynurenine-type compounds. The 3-HKG and one of the compounds were found in all three species, while the remaining one was structurally different in the lower primates. A loss of 3-HKG as a result of irradiation was apparent in all the lenses and correlated with the buildup of the blue fluorophore, suggesting that the latter may be a photoproduct(s) of 3-HKG. The kinetic analysis of baboon and human lenses showed a slowdown in the fluorophore buildup as irradiation times increased. This was probably due to the competitive absorptions of 3-HKG and other chromophores present. Rhesus monkey lenses did not exhibit this slowdown.

The Integrity of Mammalian Lenses in Organ Culture

A method to quantitate protein leakage was developed to assess the integrity of mammalian lenses in organ culture. Protein leakage into the culture medium was found to be a strong indicator of lens integrity. Rat lens incubation medium that contained more than 14 μg protein per lens after 1 hr was indicative of lens damage. Damaged lenses almost always exhibited lens opacities within 24 hr. However, on occasion, a lens predicted by this method to be damaged would maintain clarity at the 24 hr timepoint. In these instances, damage was confirmed by the inability of the lens to accumulate [3H]choline and 86RbCl from the medium at levels equivalent to undamaged lenses. When the protein concentration of the incubation medium was greater than 14 μg per lens, there was a concomitant impairment of lens transport function. This indicates that quantitation of protein leakage is a better predictive measure of lens viability than is lens clarity.Rat lenses were oxidatively stressed by exposure to a bolus of hydrogen peroxide (final concentration, 1 mM). Within 24 hr, the lenses exhibited cortical opacities and the protein concentration of the medium was significantly increased over the controls. Western and slot blot analyses of the media revealed the presence of tens crystallins, βB2-crystallin was the major component leaking from damaged lenses. The incubation media from control lenses exhibited no protein leakage. Comparative studies with Rhesus monkey lenses yielded similar data.

Thermal inactivation study of glutathione peroxidase and glutathione reductase activities in lenses of primates and non-primates

Heat lability studies of glutathione peroxidase and glutathione reductase activities were conducted on rabbit, sheep, rat, human, galago, cat and rhesus monkey lens supernatants. These species represent five mammalian orders. Incubation periods were 10.0 minutes in duration, with temperatures ranging from 25-100 degrees C (depending on which enzyme was being investigated). Results obtained for glutathione peroxidase activity demonstrated nearly identical heat lability profiles for human and rhesus monkey lenses. Both species were extremely labile to heat, losing activity at 30 degrees C and becoming totally inactive at temperatures of 50 degrees C (rhesus monkey) and 55 degrees C (human). Their profiles were very dissimilar to those of the other five species investigated, providing evidence for the existence of an evolutionary break. Glutathione reductase activity was extremely stable under conditions of highly elevated temperature for all seven species investigated. The human lens enzyme, the most stable of the species, maintained nearly 100% of its original activity up to 65 degrees C. Lenticular glutathione reductase activity did not reach zero levels in any of the seven species until a temperature of at least 80 degrees C was attained.

Phosphorylcholine and phosphorylethanolamine in human and rhesus monkey lenses

Phosphorylcholine (P-choline) and phosphorylethanolamine (P-ethanolamine) are important precursors of phospholipids. The metabolism and concentration of P-choline has been shown to change in animal models of cataract, especially in oxidatively or osmotically stressed rat lenses. The concentrations of P-choline and P-ethanolamine were determined in monkey lenses and in normal and cataractous human lenses, and the rate of synthesis of P-choline was determined in human and monkey lenses. The concentration of P-choline in 53 clear human lenses was 0.94 mM (+/- 0.31 S.D.) and was relatively unaffected by age, eye bank storage, or freezing. There was a 70% decrease in P-choline in brown cataracts but no significant change from normal in non-brown cataracts. The concentration of P-ethanolamine in human lenses was 0.45 mM (+/- 0.26 S.D.), and it appeared to decrease during frozen storage of lenses and in cataracts. The concentrations of P-choline and P-ethanolamine in 12 rhesus monkey lenses were 1.51 mM (+/- 0.27 S.D.) and 0.75 mM (+/- 0.14 S.D.), respectively. The rate of synthesis of P-choline in monkey lenses incubated with [3H]choline was 8 nmol hr-1 g-1 wet weight in 1 mM choline. Adult human lenses incubated in 1 mM choline synthesized P-choline at a rate of 23 nmol hr-1 g-1 (+/- 6 S.D.). This limited capacity for P-choline synthesis in primate lenses may contribute to the lower P-choline concentration relative to rat lenses, which contain 11 mM P-choline and can synthesize P-choline at an apparent maximum rate of 130 nmol hr-1 g-1.

Slit-lamp studies of the rhesus monkey eye: III. The zones of discontinuity

The rhesus monkey lens exhibits two zones of discontinuity, one anteriorly and one posteriorly. They are first discernible by slit-lamp photography as performed in this study at around age 7 years in iridectomized eyes, and become more distinct with increasing age. Their thickness and distance from each other along the polar axis are independent of lens age, but their distance from the lens surface increases with increasing age. Upon accommodation, the distance between the two zones increases while all other distances along the polar axis remain unchanged, indicating that, as in the human, overall alterations in rhesus lenticular shape and thickness with accommodation primarily reflect changes in the shape of the central region. The curvature of each zone becomes sharper in a linear fashion with accommodation, with the slope of the relationship being similar to those for lens surfaces.

Glutathione peroxidase, glutathione reductase and glutathione-S-transferase activities in the rhesus monkey lens as a function of age.

The activities of glutathione peroxidase, glutathione reductase and glutathione-S-transferase were determined in lenses from rhesus monkeys (Macaca mulatta) as a function of age. The ages ranged from 137 day old embryos to a 34 year old. Glutathione peroxidase activity (units/g lens) occurred at a very low level in lenses of fetuses and neonates, but increased dramatically with age, peaking in the adult of about 12 to 20 years of age and declining thereafter. Glutathione reductase activity (units/g lens) decreased throughout juvenile life, leveling off when adulthood was reached (at least 6 years of age). Glutathione-S-transferase activity showed considerable age-related variation. Calculations show that glutathione reductase is rate-limiting in the glutathione redox pathway.

Analysis of the proteins in the Rhesus monkey lens

The proteins of the Rhesus monkey (Macaca mulatta) lens were studied and characterized. In general, there is a similarity of the monkey lens to the human lens particularly in the alpha- and beta-crystallins and the membrane proteins, although the amount of beta 1-crystallin may be greater in the monkey. There is immunological cross-reactivity in the crystallins between monkey and human lenses including the monkey low molecular weight proteins and the human low molecular weight proteins. The monkey lens low molecular weight material showed three differently sized proteins by gel exclusion chromatography. In this respect, the monkey lens proteins are similar to the human. However, the monkey lens low molecular weight proteins differ from the human low molecular weight proteins in charge as well as molecular weight determined by SDS-PAGE. One of the monkey low molecular weight proteins is more prevalent in the nucleus than in the cortex suggesting that this protein may be more important to the embryonic lens that to the adult lens.

Intraocular Irrigating Solutions and Lens Clarity

By in vitro incubation of rhesus monkey lenses at 37degreesC, we determined that solutions superior to intraocular irrigating solutions in common clinical use could be prepared by aspetically adding commerically available solutions of dextrose 5% in water and sodium bicarbonate to either Ringer's injection or Ringer's lactate solutions. While not as effective as glutathione-bicarbonate-Ringer's solution (G-B-R) in maintaining lens clarity, these mixtures can be prepared easily in the operating room (in contrast to G-B-r) and may improve surgical results in procedures requiring prolonged intraocular irrigation.