Heat shock protein concentration and clarity of porcine lenses incubated at elevated temperatures.

PURPOSE: To quantify the concentration of heat shock proteins in lenses in lens organ culture at elevated temperatures, and to examine the relation between elevated temperature and lens clarity. METHODS: Pig lenses obtained from a local abattoir were dissected aseptically and incubated in medium M199 without serum for 4 days to stabilize, and lenses with protein leakage of less than 10 mg/l were obtained for heat shock exposure. Heat shock was performed by incubation for 1 h in M199 without serum at various temperatures ranging from 37 °C to 55 °C. After incubation for 24 h, cataract blurring of the images was assessed using Scantox™ and Scion Image analysis of the lens photographs. Lens homogenates were subsequently analyzed for Hsp70 and Hsp27 with western blotting. RESULTS: The degree of cataract blurring of the images increased with increasing temperature, but the two functional measures provided different results. Focal length inconsistency, as assessed with the back vertex distance standard error of the mean (BVD SEM; the variability in focal lengths measured at 20 equally spaced locations across the lens, Scantox™), increased nearly linearly with the heat treatment temperature. In contrast, decreased clarity, evident by a fuzzy image with lower contrast, was not markedly altered as the temperature rose until a threshold of approximately 47.5 °C. The inducible isoform of the Hsp70 family (Hsp70) of heat shock proteins was increased at all temperatures above the control except those above 50 °C. Changes in Hsp27 were less clear as the protein content increased only at the incubation temperatures of 39 °C and 48.5 °C. CONCLUSIONS: The porcine lens demonstrates subtle changes in the variability of the focal length, and the variability increases as the incubation temperature rises. In contrast, lens clarity is relatively stable at temperatures up to 47.5 °C, above which dramatic changes, indicative of the formation of cataracts, occur. The lens content of Hsp70 was elevated in lenses exposed to heat shock only up to 50 °C. These data suggest that in a stressful environment, Hsp70 may be associated with protection against loss of clarity. In addition, the functional measures BVD SEM and clarity assess different qualities of the lens, with the former likely more sensitive to subtle changes in the protein structure.

Exercise is the primary factor associated with Hsp70 induction in muscle of treadmill running rats.

AIM: The cytoprotective, inducible stress protein, Hsp70, increases in muscles of rodents subjected to strenuous treadmill running. Most treadmill running protocols employ negative reinforcement to encourage animals to exercise. As these stimuli may themselves activate stress responses, the present investigation was conducted to determine their contribution to the exercise-induced expression of Hsp70. METHODS: Twenty-one male Sprague-Dawley rats were randomly divided into three equal groups including an exercise group (EX), which ran on a treadmill at 30 m min(-1) for 60 min; a stimulation group (STIM), which was not allowed to run, but was stimulated with compressed air and mild electric shock concurrently with their exercising cohort; and a control group (CON), which was housed in the treadmill room during the exercise period. Animals were killed 24 h post-experiment and hearts (H), soleii (SOL) and white gastrocnemii (WG) were harvested and analysed for Hsp70 content (mean% +/- SEM of standard). RESULTS: Significant increases in Hsp70 (as a % of standard) were noted in H and WG (H = 77.4 +/- 8.5; WG = 93.9 +/- 18.4) of EX but not in STIM (H = 32.5 +/- 4.6; WG = 32.0 +/- 3.4) or CON (H = 20.5 +/- 3.7; WG = 32.4 +/- 7.4). In SOL, Hsp70 expression in EX (126.7 +/- 6.2) was different from STIM (98.3 +/- 10.9) only. This occurred, despite the fact that all groups were exposed to a stressful environment and exhibited elevated (P < 0.001) temperatures (EX -41.2 +/- 0.1 degrees C > STIM -40.5 +/- 0.2 degrees C > CON -39.0 +/- 0.1 degrees C) indicative of a general stress response. CONCLUSIONS: These data suggest that exercise per se, rather than environmental conditions or noxious stimuli, are responsible for the induction of Hsp70 in rat muscle during treadmill running.

Modelling cortical cataractogenesis 21: in diabetic rat lenses taurine supplementation partially reduces damage resulting from osmotic compensation leading to osmolyte loss and antioxidant depletion.

The concentration of taurine and the amino acids, glutathione, cysteine, ascorbate and ATP were determined in the lenses of rats made diabetic with streptozotocin. In the clear lenses, prior to vacuole formation after 1 or 2 weeks of diabetes, the increase in concentration of sorbitol and the total decrease of all these osmolytes were not significantly different. The major components of the osmolytes lost were taurine and amino acids, which together accounted for over 75% of the total osmolyte loss. Since glutathione, ascorbate, taurine and cysteine have been reported to have antioxidant activity, it appears that their loss may potentiate damage occurring as a result of free radicals generated by nonenzymic glycation by the Maillard reaction. Amino acids also lost as a result of the osmotic compensation, are estimated to be responsible for almost half of the antioxidant activity lost. To test this hypothesis, normal and streptozotocin diabetic female Wistar rats were given taurine at 0.05% or 0.10% (w/w) in the diet. This treatment resulted in small only marginally significant increases in serum taurine levels. At the end of 6 weeks the rats were examined for weight gain or loss and at the time of killing, blood was collected for measurement of serum glucose. gamma-Crystallin levels were determined in vitreous and aqueous humours using a radioimmunoassay. A lens from each rat was homogenized in 8 m guanidinium chloride for adenosine triphosphate (ATP) analysis. In normal rats, a small amount of gamma-crystallin was found in the vitreous humour, and an even smaller amount in the aqueous humour. Diabetes caused a 4- to 5-fold increase in the vitreous humour and a 4-fold increase in gamma-crystallin in the aqueous humour. Diabetes also led to a significant worsening in general body condition, loss of body weight, formation of cataracts, and decrease in lens ATP levels. Addition of taurine to the diet of diabetic animals resulted in a significant decrease of gamma-crystallin leakage into the vitreous but not the aqueous humour. Taurine had no effect on the lens ATP levels. Neither streptozotocin diabetes nor taurine in the diet appeared to affect the weight of the lenses.

Modelling cortical cataractogenesis XXIV: uptake by the lens of glutathione injected into the rat.

PURPOSE: Work of several groups including ours has shown that injection of glutathione may help to prevent the formation of cataract in the rat lens both in vitro and in vivo. These experiments were initiated to investigate the mechanism by which injected glutathione reaches the lens in vivo. The route is uncertain, but might involve either aqueous or vitreous humors, in contact with the lens anterior and posterior, respectively. Kannan's work has indicated that glutathione can be taken up ex vivo from the aqueous, by perfused isolated lens, but has not investigated; (1) whole animal glutathione injections, (2) the relative proportion of reduced and oxidized glutathione, and (3) the possibility that uptake can occur from the vitreous (in contrast to the aqueous humor) route. METHODS: 3H- or 35S-glutathione was injected into rats intraperitoneally and the radioactivity in serum and lens homogenates followed. RESULTS: The 3H-radioactivity reached a peak in the serum approximately 20-30 min after injection. Counts were also found in the lens, aqueous and vitreous humors. HPLC using a C18 Bondapak column (37 x 300 mm) indicated that the majority of the 3H-radioactivity in the lens was found in a component of a lower molecular weight than glutathione, but 8.1% of the counts occurred in the peak corresponding to reduced glutathione. Analysis of the unidentified radioactive component revealed a mobility the same as that of a dipeptide. Further analysis suggested this contained the amino acids cysteine and glycine bound in peptide linkage. These results suggest that glutathione may be degraded by the gamma-glutamyl cycle, and the action of transpeptidase produced cysteinylglycine. To confirm these results, similar experiments were undertaken using 35S-glutathione injection, to test whether a differently labelled form would be able to enter the lens. Homogenates prepared from the lens 20 min after 35S-glutathione injection were fractionated by HPLC. The glutathione peak contained 4.5% of the radioactivity in the lens extract. This amount was similar in quantity to the value for 3H-glutathione uptake by the lens. The average of the two values indicated that 6.3% of the total lens label was glutathione. The source of the labelled glutathione taken up by the lens was investigated by determining its concentration in the aqueous and vitreous humors and serum. The dipeptide appeared to be the major radiolabelled form occurring in the serum. This may explain its high level in the lens, as a result of uptake from other sources. Analysis using HPLC revealed that reduced glutathione (GSH) was the predominant chemical species of glutathione in the aqueous humor. In the vitreous humor, oxidized glutathione (GSSG) was the major species. The ratio of GSSG:GSH in the vitreous varied between 2:1 and 4:1. CONCLUSIONS: Over a 4 h period the lens could obtain 12.3% of its total GSH from the injected GSH, using the specific activity of the labelled glutathione to calculate the actual uptake of glutathione by the lens, suggesting a half-time of 16.25 h for replenishing GSH from external sources. The probable route of glutathione entry was by blood plasma and aqueous since the specific activity of the vitreous humor was too low for the vitreous to be a possible source of the lens GSH.

Cysteine and ascorbate loss in the diabetic rat lens prior to hydration changes.

PURPOSE: Glutathione (GSH) loss precedes vacuole formation in the diabetic rat lens, but the cause of this loss is not known. Cysteine availability is a rate limiting factor to glutathione biosynthesis in rat and human lenses but its concentration is not known; therefore free cysteine was measured prior to lens hydration in the diabetic rat lens. GSH can regenerate ascorbate from dehydroascorbate within the lens and potentially modulate the ascorbate pool; therefore ascorbate loss is also a possibility that has not been examined previously. METHODS: Diabetes was induced in Wistar rats to provide a slowly progressing model of cortical cataract. Age-matched control rats were injected with buffer vehicle only. Lens condition was monitored by binocular slit-lamp microscope after pupil dilation. Lens cysteine and glutathione were measured in the same lens, while ascorbate and total ascorbate (ascorbate + dehydroascorbate) of the contralateral lens were quantified by high performance liquid chromatography electrochemical detection. The 1- and 2-week periods of diabetes were chosen as they both preceded lens hydration changes and Na+/K+ changes, to avoid leakage due to ruptured cell membranes. RESULTS: Lens weights were not significantly different compared to controls at either the 1- or 2-week periods, and lenses were completely free of initial vacuole formation. Lens GSH concentration was diminished by 72% compared with controls after 1 week of diabetes and 74% after 2 weeks of diabetes. Lens free cysteine was decreased by 62% and 78% compared with controls after 1 and 2 weeks of diabetes, respectively. Total lens ascorbate concentration was decreased by 34% after 1 week of diabetes and 48% after 2 weeks of diabetes. Dehydroascorbate levels represented less than 10% of the total lens ascorbate pool in all experimental groups. GSH and ascorbate concentration were correlated after 1 week of diabetes (p < 0.005) and after 2 weeks of diabetes (p < 0.001). GSH and cysteine concentration were also correlated after 1 week of diabetes (p < 0.001) and after 2 weeks of diabetes (p < 0.001). CONCLUSIONS: Decreased free cysteine, in the diabetic rat lens, precedes hydration changes and vacuole formation, contributing to decreased glutathione content. While cysteine was not abundant in the lens, its concentration is greater than previously supposed. The lens ascorbate pool was also diminished prior to lens hydration.

Endogenous superoxide-like species and antioxidant activity in ocular tissues detected by luminol luminescence.

A new luminescent method was used to detect the reactive oxygen species in aqueous and vitreous humors and in homogenates of the lens and retina of laboratory rats. Superoxide-like activity per microgram protein increased in all tissues with weight of the rat, a good indicator of animal age. Superoxide dismutase, centrophenoxine, soluble vitamin E (D-alpha-Locopherol (polyethlyene glycol 1000) succinate, and N'-diphenyl-p-phenylenediamine (DPPD) reduced the luminescence. Catalase had no effect. These results are consistent with the detected species being superoxide-like.

A new technique for enhancing luminol luminescent detection of free radicals and reactive oxygen species.

A novel technique is reported which makes use of (1) an improved method for solubilizing luminol at neutral pH along with (2) the addition of dimethyl sulfoxide to stabilize superoxide anion in the solutions in which luminescence is detected. These improvements resulted in (1) very low blank values of luminescence and (2) an approximately 6-fold increase in sensitivity of detection of peroxide and superoxide, as well as stabilizing the superoxide radical anion. The technique can also be used to evaluate the availability of antioxidants in biological homogenates and fluids.

Modelling cortical cataractogenesis. 13. Early effects on lens ATP/ADP and glutathione in the streptozotocin rat model of the diabetic cataract.

A possible contribution to cell toxicity in the diabetic lens due to early ATP loss is not well characterized prior to the appearance of vacuoles in the lens. Changes in lens ATP levels at longer periods of hyperglycaemia (6-8 weeks) have been reported. We used [31P]NMR analysis of lens extracts at three time periods, comparing diabetic to concurrent control groups at 1, 2 and 4 weeks of hyperglycaemia. With this design, significant alterations (> 10%) in the ATP/ADP ratio can be monitored. NMR analysis revealed a decreased ATP/ADP ratio at all time periods, averaging a 38% decrease. Luminescent determination of ATP levels indicates that this decrease is mainly caused by a decrease of 25% in ATP concentration. The early loss of GSH was large and not accompanied by an appearance of GSSG, as monitored by HPLC electrochemical detection. A 1-week experiment with animals receiving daily insulin treatment was carried out to control for effects of STZ on the lens. This treatment resulted in normal lens GSH levels and a near normal [31P]NMR profile.

Modelling cortical cataractogenesis. XII: Supplemental vitamin A treatment reduces gamma-crystallin leakage from lenses in diabetic rats.

Normal and streptozotocin diabetic female Wistar rats were given normal diets with the following additions: 0, or 12,500 iu/kg food vitamin A (retinyl palmitate). At the end of 6 weeks, the rats were examined for weight gain or loss, general body condition, and cataracts. At sacrifice, blood was collected for measurement of serum glucose. gamma-Crystallin levels were determined in aqueous and vitreous humours using a radioimmunoassay. One lens (the right) was homogenized in 8 M guanidinium chloride for ATP analysis. In normal rats, gamma-crystallin was detected in both aqueous and vitreous humours, with a greater concentration found in the vitreous. Diabetes caused a 4-5 fold increase in gamma-crystallin in both aqueous and vitreous humours. Diabetes also led to a significant loss of body weight, and decrease in lens ATP levels. Addition of vitamin A to the diet resulted in reduction in gamma-crystallin leakage into the aqueous and vitreous humours. Vitamin A at 12,500 iu/kg food resulted in an increase in lens ATP for the diabetic rats. Neither streptozotocin diabetes nor vitamin A in the diet appeared to affect the weight of the lenses after 6 weeks. It is suggested that childhood vitamin A deficiency leading to latent fiber cell damage may be an important factor contributing to the high incidence of cataracts in the third world.

Modelling cortical cataractogenesis. XI. Vitamin C reduces gamma-crystallin leakage from lenses in diabetic rats.

Normal and streptozotocin diabetic female Wistar rats were given vitamin C (VC) at 0.3% or 1.0% (w/w) in the diet: 1% dietary VC resulted, in 12-24 hr, in significant increases in serum ascorbate levels and lens ascorbate concentrations in normal rats. The increase was biphasic, with VC concentrations falling to a lower level which was still significantly elevated compared to controls in the period of 1.7-4 days for serum and 1.7-5 days for lenticular VC. At the end of 10 weeks the rats were examined for weight gain or loss, general body condition and cataracts. At the time of killing, blood was collected for measurement of serum glucose. Alpha-crystallin levels were determined in vitreous and aqueous humours using a radioimmunoassay. One lens from each rat was fixed for either scanning electron microscopy or light microscopy; the other lens was homogenized in 8 M guanidinium chloride for adenosine triphosphate analysis. In normal rats, a small amount of gamma-crystallin was found in the vitreous humour, and an even smaller amount in the aqueous humour. Diabetes caused a five-fold increase in the vitreous humour and a 2.5-fold increase in gamma-crystallin in the aqueous humour. Diabetes also led to a significant worsening in general body condition, loss of body weight, formation of cataracts, and decrease in lens adenosine triphosphate levels. Addition of VC to the diet of diabetic animals resulted in reduction in cataracts and a decrease of gamma-crystallin leakage into the aqueous and vitreous humours. VC had no effect on lens adenosine triphosphate levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Modelling cortical cataractogenesis. X. Evaluation of lens optical function by computer based image analysis using in vitro rat lens elevated glucose model.

As a model system rat lenses (Wistar) were isolated and cultured intact in medium M199 or M199 with 55 mM glucose. Glucose-stressed lenses developed opacities within 20 hours while control lenses remained clear. The comparative functionality of lenses was examined by two different video/computer-based imaging systems: one based on image analyses of laser light after transmission through the lens (Scanning Lens Monitor), and the other based on image analyses of the projection of white light transmitted through the lens (Kevex Image Analysis System). The combination of both methods detect changes in focal length and light scattering. On the basis of these results, these techniques can be used in animal studies for grading lenses by functional properties to complement morphologically based grading (slit-lamp) of cataracts provided by a veterinary pathologist.

Esthesiometric evaluations of corneal anesthesia and prolonged analgesia in rabbits.

Esthesiometry was used to evaluate prolonged corneal analgesia, induced by repeated intramuscular injections in New Zealand White rabbits of 2.3 to 4.9 kg body weight of two analgesics: meperidine (10 mg/kg) or buprenorphine (200 micrograms/kg) following short term anesthesia induced by ketamine (35 mg/kg) and xylazine (5 mg/kg). The surface pressure was calculated, using data supplied by the manufacturer, for the fiber length just sufficient to elicit the corneal reflex. Thus the corneal reflex was used as a proxy for pain. For the 12 injections of meperidine, at 2 hr intervals, the surface pressure peaked one hour after each injection of meperidine, and subsequently declined at the next hour when the injection was repeated resulting in a saw-tooth pattern of responsiveness. For the 5 injections of buprenorphine, at 5 hr intervals, the surface pressure to elicit the corneal reflex rose after a second injection to overlap the pressures generated by meperidine. The consistent analgesia continued until after 24 hours. Accordingly long-term analgesia may be conveniently measured using a quantitative technique, esthesiometry. Comparison of the two regimens suggests buprenorphine is preferred over meperidine in experiments that require sustained analgesic coverage for periods of 24 hours.

Modeling cortical cataractogenesis: IX. Activity of vitamin E and esters in preventing cataracts and gamma-crystallin leakage from lenses in diabetic rats.

Normal and streptozotocin diabetic female Wistar rats were given vitamin E in the diet as the tocopherol, acetate, or succinate form (2,850 IU/kg food). At the end of 6 weeks, the rats were examined for weight gain or loss, general body condition, and cataracts. At sacrifice, blood was collected for measurement of serum glucose, and gamma-crystallin levels were measured in aqueous and vitreous humors using a radioimmunoassay. One lens was homogenized in 8 M guanidinium chloride for ATP analysis. In normal rats, gamma-crystallin was detected in both aqueous and vitreous humors, with the higher concentration in the vitreous humor. Diabetes caused a sixfold increase in gamma-crystallin in both the aqueous and vitreous humors. Diabetes also led to a significant worsening in general body condition, loss of body weight, formation of cataracts, and decrease in lens ATP levels. Addition of vitamin E and vitamin E succinate, but not vitamin E acetate, to the diet resulted in reduction of gamma-crystallin leakage into the vitreous humors and an increase in body weight. There was no improvement noted for the lens ATP levels, the general body condition, or visual cataract score. Neither streptozotocin-induced diabetes nor vitamin E in the diet appeared to affect the weight of the lenses.

Modelling cortical cataractogenesis VIII: effects of butylated hydroxytoluene (BHT) in reducing protein leakage from lenses in diabetic rats.

Normal and streptozotocin diabetic female Wistar rats were given butylated hydroxytoluene at 0-, 0.067- or 0.50% w/w in the diet. At the end of 10 weeks, the animals were examined for weight gain or loss, general body condition, and cataracts. After death, blood was collected for measurement of serum glucose. gamma-Crystallin was determined in aqueous and vitreous humours using a radioimmunoassay. One lens from each rat was homogenized in 8 M guanidinium chloride for adenosine triphosphate analysis. In normal rats, there is a small amount of gamma-crystallin found in the vitreous humour, and an even smaller amount in the aqueous humour. Diabetes caused a 2.5-fold increase of gamma-crystallin in the aqueous humour and a five-fold increase in the vitreous humour. Diabetes also led to a significant worsening in general body condition, loss of body weight, decrease in lens adenosine triphosphate levels, and formation of cataracts. Addition of butylated hydroxytoluene (BHT) to the diet resulted in improved general body condition, reduction in cataracts, decrease of gamma-crystallin leakage into the vitreous humour, and weight gain. There was no effect of dietary butylated hydroxytoluene on levels of lens adenosine triphosphate. Neither the diabetic state nor treatment with butylated hydroxytoluene affected the weight of the lenses.