Acceleration of age-induced proteolysis in the guinea pig lens nucleus by in vivo exposure to hyperbaric oxygen: A mass spectrometry analysis.

Hyperbaric oxygen (HBO) treatment of animals or ocular lenses in culture recapitulates many molecular changes observed in human age-related nuclear cataract. The guinea pig HBO model has been one of the best examples of such treatment leading to dose-dependent development of lens nuclear opacities. In this study, complimentary mass spectrometry methods were employed to examine protein truncation after HBO treatment of aged guinea pigs. Quantitative liquid chromatography-mass spectrometry (LC-MS) analysis of the membrane fraction of guinea pig lenses showed statistically significant increases in aquaporin-0 (AQP0) C-terminal truncation, consistent with previous reports of accelerated loss of membrane and cytoskeletal proteins. In addition, imaging mass spectrometry (IMS) analysis spatially mapped the acceleration of age-related αA-crystallin truncation in the lens nucleus. The truncation sites in αA-crystallin closely match those observed in human lenses with age. Taken together, our results suggest that HBO accelerates the normal lens aging process and leads to nuclear cataract.

[The rate of development of myopia during hyperbaric oxygen therapy].

BACKGROUND: Hyperbaric oxygenation (HBO) is a common treatment both for emergency medicine as well as for chronic treatments. One of the most common indications for treatment is a non-healing wound due to diabetes. It is known that prolonged HBO treatments cause a myopic change in refraction. From the literature we know that the myopic shift is usually temporary, reversing back to basic refraction within a few weeks of cessation of the HBO course. There is controversy in the literature regarding the cause of refraction change, but research with animals reinforces the view that the source is the lens nucleus. PURPOSE: To measure the refractive change following 30 HBO treatments, and to quantify the rate of change. METHODS: A prospective study that included 44 eyes of diabetic patients treated with HBO in Rambam and Elisha Hyperbaric Center. Follow-up examinations were carried out every 10 treatments for spherical equivalents, sphere components, cylinder powers and axes (SPHER, SE, CYLINDER and AXIS respectively). The measurement device was an autorefractometer. A single examiner performed all the measurements; the examiner was blinded to the previous results. For each parameter, a regression equation was calculated after plotting the myopic change over time. RESULTS AND CONCLUSIONS: A positive correlation was found between HBO treatments and a myopic shift in the refraction. The correlation was statistically significant for: SPHER, SE. (0.58D, 0.61D) respectively. The change accrued from the first examination and remained throughout the follow-ups at a steady rate. Both eyes behaved similarly. The authors did not find a correlation between the myopic shift and gender, age or basic refraction. This information is vital both to clinicians, to understand the physiologic changes occurring during chronic HBO treatments, and especially to our patients, who are about to receive HBO treatments.

Phospholipid composition of the rat lens is independent of diet.

Dietary fatty acids are known to influence the phospholipid composition of many tissues in the body, with lipid turnover occurring rapidly. The aim of this study was to investigate whether changes in the fatty acid composition of the diet can affect the phospholipid composition of the lens. Male Sprague-Dawley rats were fed three diets with distinct profiles in both essential and non-essential fatty acids. After 8 weeks, lenses and skeletal muscle were removed, and the lenses sectioned into nuclear and cortical regions. In these experiments, the lens cortex was synthesised during the course of the variable lipid diet. Phospholipids were then identified by electrospray ionisation tandem mass spectrometry, and quantified via the use of internal standards. The phospholipid compositions of the nuclear and cortical regions of the lens differed slightly between the two regions, but comparison of the equivalent regions across the diet groups showed remarkable similarity. In contrast, the phospholipid composition of skeletal muscle (medial gastrocnemius) in these rats varied significantly. This study provides the first direct evidence to show that the phospholipid composition of the lens is tightly regulated and thus appears to be independent of diet. As phospholipids determine membrane fluidity and influence the activity and function of integral membrane proteins, regulation of their composition may be important for the function of the lens.

Aggregation of lens crystallins in an in vivo hyperbaric oxygen guinea pig model of nuclear cataract: dynamic light-scattering and HPLC analysis.

PURPOSE: The role of oxygen in the formation of lens high-molecular-weight (HMW) protein aggregates during the development of human nuclear cataract is not well understood. The purpose of this study was to investigate lens crystallin aggregate formation in hyperbaric oxygen (HBO)-treated guinea pigs by using in vivo and in vitro METHODS: methods. Guinea pigs were treated three times weekly for 7 months with HBO, and lens crystallin aggregation was investigated in vivo with the use of dynamic light-scattering (DLS) and in vitro by HPLC analysis of water-insoluble (WI) proteins. DLS measurements were made every 0.1 mm across the 4.5- to 5.0-mm optical axis of the guinea pig lens. RESULTS: The average apparent diameter of proteins in the nucleus (the central region) of lenses of HBO-treated animals was nearly twice that of the control animals (P < 0.001). Size distribution analysis conducted at one selected point in the nucleus and cortex (the outer periphery of the lens) after dividing the proteins into small-diameter and large-diameter groups, showed in the O2-treated nucleus a threefold increase in intensity (P < 0.001) and a doubling in apparent size (P = 0.03) of large-diameter aggregate proteins, compared with the same control group. No significant changes in apparent protein diameter were detected in the O2-treated cortex, compared with the control. The average diameter of protein aggregates at the single selected location in the O2-treated nucleus was estimated to be 150 nm, a size capable of scattering light and similar to the size of aggregates found in human nuclear cataracts. HPLC analysis indicated that one half of the experimental nuclear WI protein fraction (that had been dissolved in guanidine) consisted of disulfide cross-linked 150- to 1000-kDa aggregates, not present in the control. HPLC-isolated aggregates contained alphaA-, beta-, gamma-, and zeta-crystallins, but not alphaB-crystallin, which is devoid of -SH groups and thus does not participate in disulfide cross-linking. All zeta-crystallin present in the nuclear WI fraction appeared to be there as a result of disulfide cross-linking. CONCLUSIONS: The results indicate that molecular oxygen in vivo can induce the cross-linking of guinea pig lens nuclear crystallins into large disulfide-bonded aggregates capable of scattering light. A similar process may be involved in the formation of human nuclear cataract.

Impact of aging and hyperbaric oxygen in vivo on guinea pig lens lipids and nuclear light scatter.

PURPOSE: To measure lipid compositional and structural changes in lenses as a result of hyperbaric oxygen (HBO) treatment in vivo. HBO treatment in vivo has been shown to produce increased lens nuclear light scattering. METHODS: Guinea pigs, approximately 650 days old at death, were given 30 and 50 HBO treatments over 10- and 17-week periods, respectively, and the lenses were sectioned into equatorial, cortical, and nuclear regions. Lipid oxidation, composition, and structure were measured using infrared spectroscopy. Phospholipid composition was measured using (31)P-NMR spectroscopy. Data were compared with those obtained from lenses of 29- and 644-day-old untreated guinea pigs. RESULTS: The percentage of sphingolipid approximately doubled with increasing age (29-544 days old). Concomitant with an increase in sphingolipid was an increase in hydrocarbon chain saturation. The extent of normal lens lipid hydrocarbon chain order increased with age from the equatorial and cortical regions to the nucleus. These order data support the hypothesis that the degree of lipid hydrocarbon order is determined by the amount of lipid saturation, as regulated by the content of saturated sphingolipid. Products of lipid oxidation (including lipid hydroxyl, hydroperoxyl, and aldehydes) and lipid disorder increased only in the nuclear region of lenses after 30 HBO treatments, compared with control lenses. Enhanced oxidation correlated with the observed loss of transparency in the central region. HBO treatment in vivo appeared to accelerate age-related changes in lens lipid oxidation, particularly in the nucleus, which possesses less antioxidant capability. CONCLUSIONS: Oxidation could account for the lipid compositional changes that are observed to occur in the lens with age and cataract. Increased lipid oxidation and hydrocarbon chain disorder correlate with increased lens nuclear opacity in the in vivo HBO model.

Hyperbaric oxygen in vivo accelerates the loss of cytoskeletal proteins and MIP26 in guinea pig lens nucleus.

Previous studies have shown that treatment of guinea pigs with hyperbaric oxygen (HBO) produces certain changes in the lens nuclei of the animals which are typical of those occurring during aging. These include an increase in nuclear light scattering (NLS), elevation in levels of oxidized thiols, loss of water-soluble protein and damage to nuclear membranes. The present study investigated the effect of HBO-treatment in vivo on lens cytoskeletal proteins and MIP26 which are also known to undergo alteration with age. Young (2-month-old) and old (18-month-old) guinea pigs were treated 15 and 30 times with HBO (3 times per week with 2.5 atmospheres of 100% oxygen for 2.5 hr periods). SDS-PAGE and Western blotting showed that HBO-treatment of the older animals accelerated the age-related loss of five nuclear cytoskeletal proteins including actin, vimentin, ankyrin, alpha-actinin and tubulin, compared to levels present in age-matched controls (effects on spectrin and the beaded filaments were not investigated in this study). Treatment of the young animals with HBO produced losses which were primarily associated with concentrations of the nuclear alpha- and beta-tubulins; these cytoskeletal proteins were observed to be most sensitive to the induced oxidative stress, and were affected earliest in the study. Disulfide-crosslinking, rather than proteolysis, appeared to be the main cause of the HBO-induced cytoskeletal protein loss (elevated levels of calcium, which might have induced proteolysis, were not found in the experimental nuclei). Loss of MIP26 was observed only in the older guinea pigs treated 30 times with HBO; both disulfide-crosslinking and degradation to MIP22 were associated with the disappearance. Thus, nuclear MIP26 was susceptible to oxidative stress, but less so than the cytoskeletal proteins, particularly the tubulins. No cortical effects on either MIP26 or the cytoskeletal proteins were observed under any of the treatment protocols. No direct link was observed between an HBO-induced increase in NLS (observed in both the young and old animals using slit-lamp biomicroscopy) and losses of either MIP26 or the cytoskeletal proteins. The appearance of HBO-induced nuclear opacity without any change in the levels of nuclear sodium, potassium or calcium is similar to that observed previously for human senile pure nuclear cataracts. The results provide additional evidence that molecular oxygen can enter the nucleus of the lens and promote age-related events. The observed effects on MIP26 and the cytoskeletal proteins are indicative of an increased level of lens nuclear oxidative stress in the HBO model, possibly a precursor to nuclear cataract.

Nuclear light scattering, disulfide formation and membrane damage in lenses of older guinea pigs treated with hyperbaric oxygen.

Nuclear cataract, a major cause of loss of lens transparency in the aging human, has long been thought to be associated with oxidative damage, particularly at the site of the nuclear plasma membrane. However, few animal models have been available to study the mechanism of the opacity. Hyperbaric oxygen (HBO) has been shown to produce increased nuclear light scattering (NLS) and nuclear cataract in lenses of mice and human patients. In the present study, older guinea pigs (Initially 17-18 months of age) were treated with 2.5 atmospheres of 100% O2 for 2-2.5-hr periods, three times per week, for up to 100 times. Examination by slit-lamp biomicroscopy showed that exposure to HBO led to increased NLS in the lenses of the animals after as few as 19 treatments, compared to lenses of age-matched untreated and hyperbaric air-treated controls. The degree of NLS and enlargement of the lens nucleus continued to increase until 65 O2-treatments, and then remained constant until the end of the study. Exposure to O2 for 2.5 instead of 2 hr accelerated the increase in NLS; however, distinct nuclear cataract was not observed in the animals during the period of investigation. A number of morphological changes in the experimental lens nuclei, as analysed by transmission electron microscopy, were similar to those recently reported for human immature nuclear cataracts (Costello, Oliver and Cobo, 1992). O2-induced damage to membranes probably acted as scattering centers and caused the observed increased NLS. A general state of oxidative stress existed in the lens nucleus of the O2-treated animals, prior to the first appearance of increased NLS, as evidenced by increased levels of protein-thiol mixed disulfides and protein disulfide. The levels of mixed disulfides in the experimental nucleus were remarkably high, nearly equal to the normal level of nuclear GSH. The level of GSH in the normal guinea pig lens decreased with age in the nucleus but not in the cortex; at 30 months of age the nuclear level of GSH was only 4% of the cortical value. HBO-induced changes in the lens nucleus included loss of soluble protein, increase in urea-insoluble protein and slight decreases in levels of GSH and ascorbate; however, there was no accumulation of oxidized glutathione. Intermolecular protein disulfide in the experimental nucleus consisted mainly of gamma-crystallin, but crosslinked alpha-, beta- and zeta-crystallins were also present.(ABSTRACT TRUNCATED AT 400 WORDS)

Disulfide cross-linking of urea-insoluble proteins in rabbit lenses treated with hyperbaric oxygen.

In vivo exposure of human patients and experimental animals to hyperbaric O2 has been shown by other investigators to lead to opacification of the lens especially in the nuclear region. In the present study, cultured rabbit lenses were treated with hyperbaric O2 in order to investigate possible formation of disulfide-cross-linked proteins in the urea-insoluble fraction of lens cortex and nucleus. When lenses were treated with 100 atmospheres of 100% O2 for 24 hr. intermolecular disulfide-linked proteins formed in both the cortical and nuclear regions. Under these conditions the level of reduced glutathione and the activity of glyceraldehyde-3 phosphate dehydrogenase (G-3PD) were depleted by greater than 95% in both regions. The lenses were hazy in appearance but not opaque. Two-dimensional diagonal electrophoresis followed by immunoblotting indicated that the majority of the cross-linked proteins were beta- and gamma-crystallins. Also involved in the cross-linking was the enzyme G-3PD but not the main intrinsic membrane protein. MIP26 kDa. Treatment of the nuclear urea-insoluble fraction of O2-treated lenses with sodium borohydride showed a nearly fourfold increase in the level of protein disulfide compared to that present in the same fraction of either fresh lenses or N2-treated controls. It was determined that an increase of approximately one disulfide group per 10(5) Da molecular weight corresponded to cross-linking of nearly 20% of the urea-insoluble protein present in the O2-treated lenses. Experiments carried out at 8 atmospheres O2 were used to determine the region of the lens in which urea-insoluble disulfide first formed after exposure to O2. After 8 hr of treatment of lenses with 8 atmospheres O2 an increase in protein disulfide was observed in the urea-insoluble proteins of the lens nucleus but not of the cortex. Under these conditions, the level of glutathione had decreased by 62% in the nucleus compared to only 13% in the cortex. Increasing the culture time to 16 hr under 8 atmospheres O2 produced a further increase in protein disulfide in the nuclear region. The formation of a small amount of cross-linked protein in the cortex and a significantly greater decrease of G-3PD activity in the lens nucleus compared to the cortex. The overall results of the study demonstrate that exposure of lenses to hyperbaric O2 leads to disulfide-cross-linking of crystallins in the urea-insoluble fraction and that the initial formation of protein disulfide as well as the initial loss of glutathione occurs first in the lens nucleus.(ABSTRACT TRUNCATED AT 400 WORDS)

From E.M. microprobe analysis to NMRD studies of the lens.

We describe how electron microscope X-ray emission spectroscopy (XES) was the basis for recent studies of lens cytoplasm using proton nuclear magnetic resonance, NMR, relaxation methods. Although electron microscopy and NMR are very different techniques, the phosphorus distributions that were observed in whole lens using XES led to studies of proton relaxation rates in lens cytoplasm. Proton NMR is sensitive to water-protein interactions that exist in transparent lens cytoplasm. The cytoplasm is transparent because the interactions produce Fourier components in the density fluctuations of lens cytoplasm that remain small relative to the wavelength of light (1). New information on the interactions that are involved in the development and maintenance of transparent cytoplasm may be obtained using methods of proton NMRD (2).