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PubMed2001

Dry eye after laser in situ keratomileusis.

PURPOSE: To determine whether patients undergoing laser in situ keratomileusis have postoperative dry eye. METHODS: In this retrospective, interventional case series, 124 eyes of 64 consecutive patients who underwent laser in situ keratomileusis were examined for a dry eye symptom, Schirmer test with anesthesia, tear clearance rate, tear break-up time, vital staining for ocular surface, corneal sensitivity, and blink rate. All values were compared before and after surgery (1 month, 3 months, 6 months, and 1 year) and values of P <.05 were considered statistically significant. RESULTS: Subjective score for dryness was increased after laser in situ keratomileusis. Tear function index (Schirmer value/tear clearance rate) and break-up time were decreased until 1 month (tear function index) and 3 months (break-up time) after laser in situ keratomileusis but recovered to preoperative levels thereafter. Fluorescein and rose bengal scores were unchanged at all follow-up points except for improvement of the rose bengal score at 6 months. Corneal sensitivity was decreased at 1 month and 3 months, and returned to the preoperative level at 6 months after laser in situ keratomileusis. Blink rate was decreased at 3 months, 6 months, and 1 year after laser in situ keratomileusis. CONCLUSIONS: Patients undergoing laser in situ keratomileusis develop dry eye with compromised tear function for at least 1 month after surgery. Use of artificial tears in the early postoperative period may help to prevent unwanted symptoms and ocular surface damage.

Adult↗
PubMed1998

Evaluation of North American antibody detection tests for diagnosis of brucellosis in goats.

The sensitivities and specificities of 17 antibody detection tests for brucellosis in goats were estimated. Tests evaluated included the U.S. Department of Agriculture (USDA) card test with 8% cell concentration (8%Card), USDA rapid automated presumptive test (RAP), Mexican rose bengal plate tests with 8 and 3% cell concentrations (8%RB and 3%RB), French rose bengal plate test with 4.5% cell concentration (4.5%RB), USDA standard plate test (SPT), USDA buffered acidified plate agglutination test (BAPA), USDA and Mexican rivanol tests (URIV and MRIV), USDA standard tube tests with Brucella abortus and Brucella melitensis antigens (SATA and SATM), serum enzyme-linked immunosorbent assay (ELISA), USDA cold-fixation complement fixation tests with B. abortus and B. melitensis antigens (CFA and CFM), USDA and Mexican milk ring tests (UBRT and MBRT), and a milk ELISA. Test sensitivity was evaluated by using two groups of 10 goats experimentally infected with B. melitensis or B. abortus and monitored for 24 weeks. Specificity was evaluated by using 200 brucellosis-free nonvaccinated goats from 10 California herds. The 3%RB was considered a good screening test because of high sensitivity at week 24 postinfection (90%), ease of performance, and low cost. The cold-fixation CFA and CFM had 100% specificity in the field study and were considered appropriate confirmatory tests. The milk ELISA was significantly more sensitive (P < 0.05) than the UBRT and significantly more specific (P < 0.05) than the MBRT. The milk ELISA also had the advantage of objectivity and ease of interpretation.

Agglutination Tests↗
PubMed1995

Comparative evaluation of microagglutination test and serum agglutination test as supplementary diagnostic methods for brucellosis.

The diagnosis of brucellosis in cattle and small ruminants requires the use of more than one serological test. The complement fixation test (CFT), the rose bengal test (RBT), and the serum agglutination test (SAT) are among the most useful tests for routine diagnosis. The microagglutination test (MAT) was developed as a simpler and more efficient test than the SAT. The relative efficacy of this test compared with that of the SAT was evaluated by using brucella-free sheep and goats prior to and after vaccination treatment. The specificities of the MAT and the SAT were 100%. Of the ewes and goats with a vaccination history, one ewe, expectedly a negative responder, had reactions in the MAT, the complement fixation test, and the rose bengal test but not in the SAT, suggesting a lower sensitivity of the SAT in this case. The calculated sensitivities of the MAT and the SAT were 93.9%. The agreement between MAT and SAT results from nonresponders was examined by using sera from unvaccinated lambs and kids (95.2% agreement), unvaccinated ewes and goats (84.4%), and ewes and goats with a vaccination history (43.9%). For the latter group higher levels of agglutination units were observed by the MAT than by the SAT in 51.5% of the samples. In testing sera from positive reactors after vaccination neither method was superior (MAT values were greater than SAT values for 23.5% of the samples, and MAT values were less than SAT values for 21.9% of the samples). Comparison of the methods on the individual sample level revealed a significant correlation between the MAT and the SAT (r = 0.96 +/- 0.005; P < 0.001). Since the MAT is simpler to perform than the SAT and can potentially be automated, the inclusion of the MAT as a supplementary test in brucellosis control programs is recommended.

Agglutination Tests↗
PubMedDate not supplied

Effects of laser photocoagulation on corneal neovascularization in rabbits.

BACKGROUND: Corneal neovascularization is a common clinical entity. Although visual acuity is usually impaired and corneal function compromised, there has been only limited success in the clinical management of this condition. We evaluated the efficacy of laser photocoagulation of neovascularization in the rabbit cornea. METHODS: New vessel formation was provoked by the placement of sutures in the corneas. Rose bengal was injected intravenously and new vessels in the upper part of the corneas were treated with an argon laser. The lower halves were used as controls. Eighteen rabbits were divided into 2 groups. In group A neovascularization was treated 28 days after suture removal, when corneal inflammation had regressed. In group B treatment was performed 3 days after suture removal, when the cornea still exhibited marked inflammation. Postoperatively, the corneas were studied by slit-lamp microscopy, fluorescein angiography, and light, as well as electron microscopy. RESULTS: In group A, treatment led to the immediate occlusion of the vessels and to their gradual disappearance during the course of 3 months. In group B, no occlusion was seen during the 3-month follow-up period. The main histologic findings in the occluded vessels were endothelial cell disruption and degeneration, and the formation of clots. CONCLUSION: Our results suggest that argon laser photocoagulation using rose bengal is an effective method of occluding corneal new vessels, providing there is no corneal inflammation at the time of treatment.

Animals↗
PubMed1998

Ocular surface and environmental changes.

PURPOSE: To investigate if ocular surface and precorneal tear film are influenced by the environment. METHOD: We studied the environmental influences on the ocular surface using the tests Break-up time, Schirmer-1 test and Rose Bengal staining. We correlated the values of the above tests among three groups of normal people from different places in Greece with different climates and levels of atmospheric pollution. Group A consisted of 57 persons coming from an area with a dry and warm climate and heavy atmospheric pollution. Group B consisted of 55 normal persons coming from an area with a dry and warm climate and a low level of atmospheric pollution. Group C consisted of 55 persons coming from an area with a humid and cool climate and a low level of atmospheric pollution. RESULTS: Schirmer-1 test and Break-up time are influenced by the climatic conditions but they are not influenced by the atmospheric pollution, while Rose Bengal staining is not influenced either by the climate or by the atmospheric pollution. CONCLUSION: The precorneal tear film is much more influenced by the climatic conditions than by the atmospheric pollution.

Adolescent↗
PubMed2003

Evaluation of the clinical course of dry eye syndrome.

OBJECTIVE: To assess subjective symptoms, tear function factors, and ocular surface morphology in the clinical course of patients with dry eye syndrome under treatment within an observation period of up to 8 years. METHODS: In 97 patients (78 women and 19 men) with ocular discomfort, a clinical diagnosis of dry eye syndrome was made based on typical symptoms and a reduced tear film breakup time of less than 10 seconds. Subsequent evaluations revealed a diagnosis of aqueous tear deficiency in 9 patients, meibomian gland dysfunction in 32 patients, and aqueous tear deficiency combined with meibomian gland dysfunction in 30 patients, aqueous tear deficiency associated with Sjögren syndrome in 12 patients, and aqueous tear deficiency and meibomian gland dysfunction associated with Sjögren syndrome in 14 patients. Follow-up assessments were performed 12 to 94 months (mean follow-up, 40 months) after the initial diagnosis. MAIN OUTCOME MEASURES: In different subgroups of dry eye tear film breakup time, Schirmer test without local anesthesia (Schirmer I), fluorescein and rose bengal staining, impression cytology, as well as subjective dry eye symptoms and frequency of tear substitute application were compared at baseline and after a follow-up of 1 to 8 years (mean, 3.3 years). RESULTS: At baseline, tear film function and ocular surface test results found more pathologic abnormalities and more severe subjective symptoms in patients with aqueous tear deficiency associated with Sjögren syndrome and aqueous tear deficiency and meibomian gland dysfunction associated with Sjögren syndrome compared with the other groups who had dry eye syndrome. No differences in frequency of tear substitute application were observed. At follow-up, tear breakup time, Schirmer I test results, and corneal fluorescein staining improved compared with baseline values, whereas rose bengal staining and impression cytology of the conjunctival surface remained almost unchanged. Subjective symptoms and frequency of artificial tear application were reduced. CONCLUSIONS: Within the observation period of up to 8 years, the dry eye syndrome improved or stabilized under appropriate treatment. Although no patient was completely cured, subjective reports as well as frequency of artificial tear application were reduced.

Adult↗
PubMed1996

Aqueous tear production in patients with neurotrophic keratitis.

The purpose of this study was to determine whether corneal epithelial defects and epitheliopathy in patients with unilateral dysfunction of the ophthalmic division of the trigeminal nerve (neurotrophic keratitis) is associated with reduced aqueous tear production. Sensation of the skin, cornea, and nasal mucosa, aqueous tear production by Schirmer 1 testing, nasal-lacrimal reflex, and exposure zone rose bengal staining were evaluated in the affected and fellow eyes of subjects with neurotrophic keratitis (n = 5), eyes of subjects who had recent herpes zoster ophthalmicus (HZO) and who did not develop neurotrophic keratitis (n = 4), and normal controls (n = 10). Sensation in the brow and upper lid skin and nasal mucosa was absent on the affected side of patients with neurotrophic keratitis, but was intact in all other groups. Corneal sensation and Schirmer 1 test values were significantly reduced (p < or = 0.05) in eyes with neurotrophic keratitis compared with the other groups. The nasal-lacrimal reflex was absent on the involved side of subjects with neurotrophic keratitis but was intact in subjects with HZO without keratopathy, and in normal controls (p < 0.008). Rose bengal keratitis staining scores were significantly increased in eyes with neurotrophic keratitis compared with the other groups (p < 0.05). We conclude that neurotrophic keratitis is associated with reduced cutaneous, nasal mucosal, and corneal sensation on the affected side, as well as marked reduction in aqueous tear production with loss of the nasal-lacrimal reflex. It is possible that the corneal epithelial pathology in neurotrophic keratitis is due in part to aqueous tear deficiency.

Herpes Zoster Ophthalmicus↗
PubMed1998

Effects of lacrimal drainage occlusion with nondissolvable intracanalicular plugs on hydrogel contact lens wear.

PURPOSE: To investigate effects of lacrimal drainage occlusion with intracanalicular silicone plugs in hydrogel lens wearers with the symptom of dryness. Effects of rewetting drops in combination with the plugs were also evaluated. METHODS: A 6-week, prospective, double masked, placebo-controlled study design was followed. Thirty-five patients (26 females and 9 males) were entered into the study based on specific symptoms and signs of dry eye with hydrogel lens wear. Baseline symptoms, noninvasive prelens tear film break-up time, lens water content, fluorescein, and rose bengal staining were recorded. Herrick Lacrimal Plugs were inserted into both canaliculi of one previously randomized eye by an independent investigator. The fellow eye was manipulated in a sham procedure. Symptoms and signs were followed over 4 weeks. Nonpreserved rewetting drops were used in both eyes during the 5th week. Symptoms and signs were again followed. RESULTS: Symptoms improved more significantly in the plugged eye than the control eye after insertion of the plugs, and again after use of the drops. This improvement diminished in some of the patients. Prelens tear film break-up time was not significantly changed. Water content of all lenses combined increased only in the plugged eye with the use of drops. Fluorescein staining decreased significantly in the plugged eye. Rose bengal staining showed a slight decrease in the plugged eye. CONCLUSIONS: Intracanalicular silicone plugs offer improvement of symptoms and signs of dryness in this population. There seems to be some decrease in improvement of symptoms with the plugs over time. Concurrent use of rewetting drops increases the benefits.

Adult↗
PubMed2005

Topical combination of NGF and DHA increases rabbit corneal nerve regeneration after photorefractive keratectomy.

PURPOSE: To investigate the effect of nerve growth factor (NGF) in combination with docosahexaenoic acid (DHA) on corneal nerve regeneration in a rabbit model after PRK and correlate the findings with functional tear test. METHODS: Unilateral PRK was performed on 21 New Zealand albino rabbits. Three groups, each consisting of six rabbits, were randomized to receive twice-weekly treatments with DHA, NGF, or NGF plus DHA delivered by collagen shield. A fourth group, the control, received treatment with albumin. Rabbits were observed for 8 weeks, and tear secretion tests were conducted every 15 days. The eyes were prepared for immunostaining. Monoclonal antibodies for class II beta-tubulin, calcitonin gene-related peptide (CGRP), substance P (SP), and Ki-67 were used. Cell nuclei were stained with 4',6'-diamino-2-phenylindole (DAPI). Positive staining areas in relation to total area were calculated with image-analysis software. RESULTS: There were no significant differences in the tear-secretion test results among the four groups. However, none of the eyes treated with NGF plus DHA showed rose bengal staining 30 days after PRK, compared with 50% in the control group and 33% in the DHA-treated group. A higher percentage of Ki-67-positive cells, a marker of cell proliferation, was observed in the DHA plus NGF-and NGF-treated groups compared with DHA alone or the control. Eight weeks after PRK, tubulin-positive and CGRP-positive epithelial and subbasal nerve bundle areas were significantly higher in the DHA plus NGF group than in the control and NGF or DHA alone. No differences were noted in the SP-positive nerve bundles between the different treatments and the control treatment. CONCLUSIONS: NGF plus DHA treatment after PRK in rabbits is associated with increased corneal nerve surface area, increased epithelial proliferation, and decreased rose bengal staining compared with NGF, DHA, or vehicle control alone. The combination of NGF plus DHA yields faster nerve recovery after PRK and may have therapeutic usefulness in the treatment of post-PRK dry eye and other neurotrophic keratopathies.

Administration, Topical↗
PubMed1987

Argon laser-induced arterial photothrombosis. Characterization and possible application to therapy of arteriovenous malformations.

The common carotid, femoral, and middle cerebral arteries in the rat have been occluded thrombotically by means of a rose bengal dye-sensitized photochemical reaction initiated in vascular endothelium by the 514.5-nm beam of an argon laser, focused for maximum excitation efficiency of the photosensitizer according to a derived criterion. The total energy required for vessel occlusion was approximately 1 joule (J) for the middle cerebral artery and 140 to 180 J for the femoral and carotid arteries. At energy fluences (energy deposited per unit area) of 3.5 kJ/sq cm for the middle cerebral artery and 35 kJ/sq cm for the larger arteries, occlusion was observed within 3 minutes. The middle cerebral artery thrombus consisted entirely of aggregated platelets; in the larger arteries the thrombi were composed of platelet aggregates and groups of red blood cells interspersed within a matrix of coagulum. Vessels irradiated similarly in the absence of rose bengal dye displayed no morphological or functional damage. Because the photochemical reaction is mediated by electronic-state transitions, the process of photothrombosis (as opposed to photocoagulation) can be initiated in vessels with high flow rates without the requirement of increased temperature. The photothrombotic technique may be useful in the treatment of arteriovenous malformations owing to its significant enhancement of the efficiency and permanency of vessel occlusion.

Animals↗
PubMed2004

Efficacy and retention rate of two types of silicone punctal plugs in patients with and without Sjögren syndrome.

OBJECTIVE: To compare the efficacy, retention rates, and complications of two types of silicone lacrimal punctal plugs in patients with or without Sjögren syndrome. METHODS: We studied 36 patients with keratoconjunctivitis sicca (KCS) including 17 cases with Sjögren syndrome (SS) and 19 without SS. The fluorescein and rose bengal staining scores and the Schirmer values with and without nasal stimulation were evaluated before and after insertion of the Eagle Plugs and the punctal plugs (FCI Punctal Plugs). The retention rates and complications of these plugs were also investigated. RESULTS: The staining scores were significantly improved after the insertion of the plugs, but the Schirmer values did not increase significantly in either SS or non-SS patients. A spontaneous loss of the plugs was observed in 29% of all plugs within 1 month after insertion. The Eagle Plugs were lost more frequently, and plugs in the upper punctum were lost more often for the Eagle Plugs. There was one case of granulomatous proliferation and two cases of punctal infection with the FCI Punctal Plugs. CONCLUSIONS: Both types of punctal plugs led to an improvement of the fluorescein and rose bengal staining scores in eyes with KCS. The difference in the retention rate and complications between the two types of plugs was probably caused by the differences in the material and the design of the plugs. Close monitoring is necessary to check for loss of plugs and to prevent complications.

Adult↗
PubMedDate not supplied

A murine photochemical stroke model with histologic correlates of apoptotic and nonapoptotic mechanisms.

INTRODUCTION: The neuronal cell death that occurs after ischemia-induced cerebral infarction (stroke) contains elements of apoptosis and necrosis, an intermediary form of the two, and a distinct excitotoxic process. We previously developed a photochemical model of stroke in the rat. We have now adapted this model for use in the mouse. The present manuscript describes the mouse model. METHODS: Minimal beam intensity (0.1 W/cm(2)) cold white light (8 min exposure) was used to evoke discrete infarcts in the parietal lobes of 11 mice sensitized by the administration of fresh Rose Bengal (10 mg/kg by rapid iv infusion). RESULTS: At 2 h, five out of five mice and at 6 h, six out of six mice demonstrated light microscopic histologic features like those in the rat model. These included a superior ischemic zone with shrunken and pyknotic nuclei, a middle transition zone of edematous vacuolated neuropil but normal neurons with open chromatin and retained Nissl granules, and an inferior zone with normal neurons. There was widespread nuclear terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling (TUNEL) in the superior infarct zone in 11/11 mice. However, in the edematous vacuolated transition zone, 11/11 mice had TUNEL positive and negative nuclei randomly mixed. Light microscopic analysis of that same transition zone showed no pyknosis or chromatin bodies in the TUNEL positive or negative cells. DISCUSSION: In mice, photoactivation of Rose Bengal evoked similar infarct and transition zone patterns found previously in rats, with TUNEL evidence of apoptotic and nonapoptotic events. Thus, it will be possible to use this model for further quantitative study of apoptotic and excitotoxic events in wild and transgenic mice.

Animals↗
PubMed2001

Calcium as a modulator of photosensitized killing of H9c2 cardiac cells.

Illumination of H9c2 rat heart cells in the presence of Rose Bengal resulted in dose-dependent cell killing (assessed by trypan blue staining) and modification of ionic currents flowing through the heart cell membrane. Inhibitors of voltage-gated ionic currents were shown to have little effect on cell killing. Ionic current measurements were used to assess the increase in leak conductance of these cells, which has been suggested to be a causal factor in killing of other cell types (1). Inhibitors of voltage-gated ionic currents, including the sodium channel blocker tetrodotoxin (100 microM) and the calcium channel blocker lanthanum (10 microM) were shown to have little effect on cell killing. The potassium channel inhibitor tetraethylammonium (20 mM) inhibited cell killing, but the effect is viewed as being caused by an inhibition of leak current. The time course of block of voltage-activated ionic currents during illumination, in the presence of Rose Bengal, was rapid compared with that for induction of leak current and for cell killing. These observations are consistent with a role for leak current in photosensitized killing of cardiac cells. They are interpreted with respect to calcium influx through the leak current pathway as a trigger for the cellular response.

Animals↗
PubMed2003

The effects of chronic smoking on the ocular surface and tear characteristics: a clinical, histological and biochemical study.

PURPOSE: To investigate the effects of chronic smoking on the ocular surface and tear characteristics. METHODS: The Schirmer I-test, tear film break-up time, rose Bengal staining scores, impression cytology, tear lysozyme concentration, eye irritation symptoms, and eye irritation indices were determined for 44 eyes in 44 healthy, chronic smokers who had smoked six or more cigarettes per day for the previous year. Thirty-seven eyes in 37 healthy, non-smokers were assessed in the same masked manner for comparison. Cytological specimens were obtained from the temporal interpalpebral bulbar conjunctiva by 'impression' technique. Goblet cells were counted in the specimens obtained and squamous metaplasia was graded following epithelial cell morphology assessment. RESULTS: In chronic smokers, we found decreased tear film break-up time (p=0.022) and tear lysozyme concentration (p=0.013), and increased Schirmer I-test values (p=0.047), squamous metaplasia scores (p=0.016), eye irritation scores (p<0.001) and eye irritation indices (p=0.013), as compared with the control group. There were no statistically significant differences in goblet cell counts (p=0.710) or rose Bengal staining scores (p=0.827). CONCLUSION: These findings suggest that chronic smoking has a negative effect on the ocular surface and affects some tear characteristics. The chronic ocular irritative effects of cigarette smoking may lead to defects in ocular surface defence.

Adult↗
PubMed1994

Photosensitization-induced retinopathy in the newborn beagle.

PURPOSE: Photosensitization is a mechanism by which oxygen and light may interact to generate free radicals capable of tissue injury. It has been proposed as a possible etiologic mechanism in the development of retinopathy of prematurity. The authors report the effects of light exposure and a photosensitizer, rose bengal (RB), on the developing retina of the beagle puppy. METHODS: Seven purebred beagle puppies (2 or 7 days old) were given RB (7.5 mg/ml) intravenously (0.9 ml bolus followed by a 74 microliters/min constant infusion), and one eye was exposed to filtered light delivered by a modified slit lamp at 30 mW/cm2 for 5, 15, 25, 35, and 45 minutes. The fellow eye was not irradiated and served as an RB-only control. Three beagles were exposed to light (one eye only) for 15 to 120 minutes in the absence of RB to provide light-only and no-treatment control eyes. Animals were followed clinically by indirect ophthalmoscopy and fluorescein angiography, and eyes were processed for light microscopy. RESULTS: Clinical and/or histologic abnormalities were found in all seven eyes exposed to light in the presence of rose bengal, as follows: vitreous hemorrhage in four eyes, incomplete peripheral retinal vascularization in four eyes, fibrovascular/fibrocellular proliferation with traction on the retina in two eyes (including preretinal neovascularization in one eye), total or partial retinal detachment in two eyes, dilated peripheral vessels compatible with shunt vessels in one eye, retinal dysplasia with loss of normal architecture and formation of rosettes in five eyes. All 13 control eyes showed normal, complete retinal vascularization. CONCLUSIONS: The failure to progress to the end-stage tractional retinal detachment seen in human infants is a shortcoming of existing animal models. Photosensitization injury to the developing retina in this animal model can produce a spectrum of retinal pathology that includes extraretinal vascularization and subsequent retinal detachment.

Animals↗
PubMed1991

Effects of oxidant stress on steady-state background currents in isolated ventricular myocytes.

Free radicals and oxidant stress have previously been shown to induce depolarization, transient action potential prolongation, and automaticity. We have investigated the ionic basis of these electrophysiological changes in isolated rabbit ventricular cells. Oxidant stress was generated by the photoactivation of rose bengal, and, in current-clamp experiments, the effects of oxidant stress on the action potential were confirmed. In voltage-clamp studies, oxidant stress decreased both inward and outward current through the inward-rectifier potassium channel, and the slope conductance (measured at the voltage-axis intercept near the resting membrane potential) was decreased from 40 +/- 8 to 25 +/- 6 nS (n = 6). Transient inward currents were induced on repolarization after a depolarizing clamp step, suggesting that the cells were calcium overloaded. In addition, oxidant stress activated a steady-state membrane conductance that showed a slight outward-going rectification and a reversal potential of approximately 0 mV. Evidence is presented to indicate that this reflects an increase in the conductance of the calcium-activated nonselective cation channel. The slope conductance of this calcium-activated channel (measured at the voltage-axis intercept) increased with prolonged exposure to oxidant stress (from 0.5 to 12 nS after 7 min), indicating that the intracellular free calcium increased gradually during the maintained application of rose bengal. These results suggest that oxidant stress depolarizes the cell membrane by reducing the inward-rectifier potassium current and by activating a calcium-activated membrane conductance. Both factors may contribute to the oxidant stress-induced changes in action potential duration and automaticity.

Action Potentials↗
PubMed1993

Measurement of Na(+)-K+ pump current in isolated rabbit ventricular myocytes using the whole-cell voltage-clamp technique. Inhibition of the pump by oxidant stress.

Free radical-induced oxidant stress has been implicated in ischemia and reperfusion-induced injury in the heart. A number of studies have reported that oxidant stress reduces the activity of isolated Na+,K(+)-ATPase enzyme. We have studied the effects of oxidant stress on the Na(+)-K+ pump current recorded in isolated rabbit ventricular myocytes using the whole-cell voltage-clamp technique. Singlet oxygen and superoxide were generated by the photoactivation of rose bengal (50 nM). The compositions of Tyrode's and pipette solutions were designed to block channel currents and electrogenic Na(+)-Ca2+ exchange. Cells were dialyzed with a pipette solution containing 30 mM sodium via wide-tipped (1-2-M omega) electrodes, and outward Na(+)-K+ pump current was recorded during a voltage-ramp protocol. The validity of using such a ramp protocol was confirmed by comparison with steady-state Na(+)-K+ pump current measurements made at the end of 200-msec square-clamp steps. Active currents were abolished by potassium-free Tyrode's solution or ouabain (100 microM), and Na(+)-K+ pump current was defined as the Ko-sensitive fraction of recorded currents. The activation of Na(+)-K+ pump current by intracellular sodium and extracellular potassium revealed a concentration of potassium necessary for half-maximal activation of 18.7 mM for Nai and 1.88 mM for Ko. Oxidant stress inhibited Na(+)-K+ pump current at all voltages, such that after a 10-minute exposure to photoactivated rose bengal, Na(+)-K+ pump current measured at 0 mV was reduced by approximately 50%. The voltage dependence of Na(+)-K+ pump current was, however, not profoundly affected by oxidant stress. Passive membrane currents recorded in the absence of all major electrogenic ion channels, exchangers, or pumps were unaffected by oxidant stress. This observation suggests that, over the time course during which Na(+)-K+ pump inhibition and calcium overload occur, oxidant stress does not cause nonspecific membrane damage and changes in the passive resistance of the lipid bilayer. The inhibition of Na(+)-K+ pump activity by oxidant stress may contribute to ischemia/reperfusion injury and reperfusion-induced cellular calcium overload.

Animals↗
PubMed1995

Hemoglobin-induced lipid peroxidation in the retina: a possible mechanism for macular degeneration.

To investigate a possible link between subretinal hemorrhage and macular degeneration, oxyhemoglobin (HbO2) or methemoglobin (metHb) was incubated with retinal homogenate and unsaturated phospholipid peroxidation was monitored by (a) assay of thiobarbituric acid-reactive substances (TBARS), (b) luminescence originating from an energy transfer of lipid-degraded products to rose bengal, and (c) the decrease in composition of highly unsaturated fatty acids of phospholipids. TBARS formation and rose bengal luminescence in the case of metHb-induced lipid peroxidation were about 1.5 times greater than those in HbO2-induced lipid peroxidation. alpha-Tocopherol, a lipid-soluble antioxidant, and docosahexaenoic acid, a major unsaturated fatty acid, were slightly more rapidly decomposed after a 60-min incubation with metHb than with HbO2 at the same concentration. Atomic absorption analysis revealed that an equal concentration of iron was released from both HbO2 and metHb during incubation with retinal homogenates. The released iron may promote microsomal phospholipid peroxidation in the presence of endogenous ascorbate or NADPH-dependent cytochrome P-450 reductase because ascorbate oxidase and p-chloromercuribenzoic acid (an inhibitor of sulfhydryl enzymes) inhibited metHb- or HbO2-induced lipid peroxidation. MetHb-induced lipid peroxidation in retina was inhibited by KCN or NaN3, which binds to FeIII of metHb. KCN or NaN3 had no effect on HbO2-induced lipid peroxidation, because conversion of HbO2 to metHb, which can proceed in HbO2 incubated with phospholipid liposome, did not occur in retinal homogenates. It is concluded that metHb induces peroxidation of retinal unsaturated phospholipids (1) directly and (2) by releasing iron.

Animals↗