Dihydrofluorescein diacetate is superior for detecting intracellular oxidants: comparison with 2',7'-dichlorodihydrofluorescein diacetate, 5(and 6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate, and dihydrorhodamine 123.

To detect intracellular oxidant formation during reoxygenation of anoxic endothelium, the oxidant-sensing fluorescent probes, 2',7'-dichlorodihydrofluorescein diacetate, dihydrorhodamine 123, or 5(and 6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate were added to human umbilical vein endothelial cells during reoxygenation. None of these fluorescent probes were able to differentiate the controls from the reoxygenated cells in the confocal microscope. However, dihydrofluorescein diacetate demonstrated fluorescence of linear structures, consistent with mitochondria, in reoxygenated endothelium. This work tests the hypothesis that dihydrofluorescein diacetate is a better fluorescent probe for detecting intracellular oxidants because it is more reactive toward specific oxidizing species. To investigate this, dihydrofluorescein diacetate was exposed to various oxidizing species (hydrogen peroxide, superoxide [KO2], peroxynitrite, nitric oxide, horseradish peroxidase, ferric iron, xanthine oxidase, cytochrome c, and lipoxygenase) and compared with the three other popular probes. Though oxidized dihydrofluorescein has higher molar fluorescence, comparison of the reactions of dihydrofluorescein with these other three probes in a cell-free system indicates that dihydrofluorescein is sometimes less fluorescent than the other probes. In addition, we find that the reactivity of all of the probes is very complex. Based on the results reported here, it is no longer appropriate to think of these probes as detecting a specific oxidizing species in cells, such as H2O2, but rather as detectors of a broad range of oxidizing reactions that may be increased during intracellular oxidant stress. Cell-loading studies indicate that dihydrofluorescein achieves higher intracellular concentrations than the second brightest intracellular probe, 2',7'-dichlorodihydrofluorescein. This fact and its higher molar fluorescence may account for the superior brightness of dihydrofluorescein diacetate. Dihydrofluorescein diacetate may be a superior fluorescent probe for many cell-based studies.

The photic sneeze reflex and ocular anesthesia.

BACKGROUND AND PURPOSE: Intravenous sedation to minimize discomfort from local anesthetic injection has many potential complications including severe involuntary sneezing (i.s.). This prospective study evaluates the occurrence of i.s. and a history of photic sneezing (p.s.). METHODS AND MATERIALS: All patients receiving local anesthesia (retrobulbar or periocular injections) after intravenous thiopentone for eye surgery during eight months were asked about p.s. and observed for i.s. RESULTS: The 557 patients (40% males) had a mean age of 69.9 years and 14% recalled p.s. (29.5% males). I.s. developed in 5.2% of the 557. Only 7.6% of those with p.s. developed i.s. After periocular injections 23.8% developed as compared to 4.5% after retrobulbar injections. (P < 0.001). There was no relationship between p.s. and i.s. (p = 0.43). CONCLUSION: I.s. is not linked to p.s., with males and females at equal risk for either. I.s. is more common after periocular injections.

Hook and loop fastener on loose prisms assists in measuring ocular deviation.

It is difficult to hold a pair of loose prisms in one hand. Doing so can lead to inaccuracy, which worsens with large-angle exotropia or vertical deviations. The authors applied a self-adhesive hooked Velcro (Velcro Sticky Back Tape, Velcro USA Inc., Manchester, NH) strip to the base and the top of loose prisms. They applied the complementary looped Velcro to a wooden bar. As a result, a pair of prisms could be suspended horizontally and/or vertically while being held in one hand. Forty consecutive patients undergoing strabismus surgery without adjustable sutures were retrospectively studied to evaluate the accuracy of this method. Clinical use confirmed its ease and convenience. Of 19 patients with exotropia and 15 patients with esotropia (mean ages 41.9 and 15.7 years, respectively; mean deviations 44.7 and 49.8 D, respectively), 4 patients with abducens paralysis, and 2 patients with trochlear palsies, 1 surgery achieved less than 10 D of residual deviation in all but 2 (5.9%). This simple, inexpensive system can assist with the clinical evaluation of ocular deviation.

Salvaged viscoelastic reduces irrigation frequency during cataract surgery.

A more viscous solution may decrease the need for frequent irrigations to maintain a clear cornea during cataract surgery. Fifty-four consecutive cataract patients were prospectively randomized to receive a drop of viscoelastic or saline on the cornea at the start of surgery. The surgical scrub nurse was instructed to irrigate only when the cornea appeared hazy or at the surgeon's request. The number of irrigations, ultrasound time and energy, and surgery times were compared using the Student's t test. Only one ampoule of viscoelastic was used for each case. When the patients who received viscoelastic were compared with the patients who received saline, the mean numbers of irrigations were 2 and 18, whereas the mean numbers of irrigations per minute were 0.17 and 1.21, highly significant (P < .0001) differences. The duration of surgery, ultrasound time, and ultrasound energy were less with viscoelastic, but were not statistically significant. Viscoelastic on the cornea reduces the frequency of irrigation without increasing cost.

Ibuprofen protects human endothelial cell prostaglandin H synthase from hydrogen peroxide.

Human endothelial cells exposed to H2O2 demonstrate decreased prostacyclin (PGI2) synthesis due to decreased prostaglandin H synthase (PGH synthase) activity. We tested the hypothesis that PGH synthase activity could be protected from H2O2 by a reversible nonsteroidal anti-inflammatory drug. Experiments demonstrate that ibuprofen if present during H2O2 exposure, protects endothelial cell PGH synthase against the decrease in prostaglandin formation caused by H2O2. Additional studies demonstrated that decreasing arachidonic acid release from cell phospholipids during H2O2 exposure did not protect PGI2 synthesis following H2O2 exposure. In other experiments, ibuprofen did not chelate Fe2+ in a conformation that inhibited the reactivity of Fe2+. In addition, ibuprofen did not scavenge HO. However, we demonstrate that ibuprofen significantly protects purified PGH synthase cyclooxygenase activity from the effects of H2O2. The results confirm the hypothesis. These findings suggest that ibuprofen displaces oxidant species from the cyclooxygenase site of PGH synthase, thereby preventing oxidation of the functional groups important for PGH synthase activity.

Extracellular iron (II) can protect cells from hydrogen peroxide.

We hypothesized that exposure of cells to H2O2 plus Fe2+ would increase formation of cell-derived lipid peroxides that would inactivate prostaglandin H synthase, resulting in decreased prostaglandin synthesis. Therefore, we treated human endothelial cells with 0-100 microM H2O2 followed immediately by addition of 0-200 microM Fe2+. After oxidant exposure, cells were stimulated with 20 microM arachidonic acid to induce prostaglandin I2 (PGI2) synthesis. Adding 100 microM H2O2 prior to arachidonic acid decreased PGI2 synthesis more than 80%. However, to our surprise, the addition of Fe2+, in increasing amounts, progressively protected PGI2 synthesis against the harmful effects of H2O2. A ratio of one part H2O2 to two parts Fe2+ offered almost complete protection, whereas Fe3+ did not protect PGI2 synthesis from H2O2. We found that 100 microM H2O2 was not cytolytic; however, 250 microM H2O2 was cytolytic; Fe2+ protected against this cytotoxicity. In addition, extracellular Fe2+ prevented the rise in intracellular calcium caused by H2O2 and extracellular Fe2+ preserved intracellular glutathione in H2O2-exposed cells. Electron paramagnetic resonance spin trapping demonstrated that extracellular Fe2+ generated the hydroxyl free radical, HO. outside the cell. We speculate that extracellular Fe2+ protects the intracellular space from H2O2 by initiating the Fenton reaction outside the cell. This reductive cleavage of H2O2 generates HO. in the extracellular space, where much of the HO. will react with noncellular components, thereby protecting the cell interior.

Prostaglandin E2 synthesis after oxidant stress is dependent on cell glutathione content.

The role of glutathione in protecting prostaglandin (PG) generation after exposure of fibroblasts to oxidant stress was investigated. Exposure of 3T3 fibroblasts to H2O2, followed by washing and then 20 microM arachidonic acid, caused a dose-dependent decrease in PG synthesis as assessed by radioimmunoassay. PGE2 production decreased from 3.7 +/- 1.1 to 0.15 +/- 0.04 pmol/microgram protein, and prostacyclin (PGI2) formation decreased from 0.56 +/- 0.03 to 0.06 +/- 0.03 pmol/microgram protein after exposure to 200 microM H2O2. Decreasing intracellular glutathione with 50 micrograms/ml 1,3-bis(chloroethyl)-1-nitrosourea (BCNU) enhanced the H2O2-induced decrease in PGE2 synthesis. Another glutathione-depleting agent, 1-chloro-2,4-dinitrobenzene (CDNB), also potentiated the H2O2-induced decrease in PGE2 formation. However, although PGI2 production was decreased by H2O2, neither BCNU nor CDNB potentiated this decrease. Without oxidant stress, extreme glutathione depletion decreased PGE2 synthesis and caused PGI2 synthesis to exceed PGE2. In summary, oxidant stress decreases both PGE2 and PGI2 formation. However, the primary effect of decreasing cell glutathione during oxidant stress is a reduction in PGE2 formation, not PGI2. This implies that the predominant effect of glutathione depletion during oxidant stress is on the PGE2 isomerase(s) and not PGH synthase or PGI2 synthase.

Effect of glutathione on endothelial prostacyclin synthesis after anoxia.

We previously observed decreased prostacyclin (PGI2) formation after reoxygenation of anoxic endothelium. In the present study, the effects of glutathione on endothelial prostaglandin (PG) H synthase activity after reoxygenation were explored. Intracellular glutathione content decreased 70% after 24 h of anoxia; reoxygenation did not produce any additional decrease in glutathione content. Intracellular glutathione was maintained in the reduced state by the endothelium even during the oxidant stress caused by reoxygenation or the addition of peroxide. Glutathione depletion produced by DL-buthionine-(S,R)-sulfoximine (BSO), 1,3-bis(chloroethyl)1-nitrosourea (BCNU), or incubation in a sulfhydryl-free medium resulted in increased sensitivity of PGH synthase to the effects of added H2O2. However, glutathione depletion resulting from BSO or culture in sulfhydryl-free medium during anoxia did not increase the sensitivity of PGH synthase to reoxygenation. In addition, anoxia did not make the endothelium more sensitive to H2O2. Glutathione peroxidase and glutathione reductase activities were preserved after anoxia-reoxygenation. When glutathione reductase was inhibited with BCNU during reoxygenation, PGI2 release was decreased further. These findings demonstrate that, although anoxia decreases endothelial glutathione content, the endothelium is able to utilize its remaining glutathione to protect against additional oxidant stress because glutathione peroxidase and glutathione reductase retain their activity.

Morphologic effects of hypervolemic administration of DBBF hemoglobin in the rat.

Conscious rats were given either 14 g/dl bis(3,5-dibromosalicyl) fumarate cross-linked hemoglobin (DBBF-Hb) in lactated Ringer's (LR) as an intravenous bolus (40, 50, or 60% of blood volume), 12.5 g/dl human serum albumin (HSA) in LR as a control for oncotic effects, or LR as a control for injection volume. The high dose HSA and DBBF-Hb rats experienced pulmonary edema after injection; one rat in each of these groups died soon after dosing. Rats were killed after 48 hours for histopathology. Only the 60% HSA and the 50% and 60% DBBF-Hb rats had treatment-related lesions. In the liver, randomly distributed mononuclear cell aggregates occasionally surrounded a necrotic hepatocyte. Liver lesions in 60% DBBF-Hb rats were the largest and most numerous, but in all groups were qualitatively similar. Hearts from HSA and DBBF-Hb rats had similar mild inflammatory lesions. We conclude that bolus administration of DBBF-Hb causes morphologic lesions in rats only at volumes sufficient to cause pulmonary edema. Hepatic and cardiac changes with high volumes of DBBF-Hb resembled those in rats given a corresponding bolus of HSA, suggesting that vascular overload with a hyperoncotic solution, rather than cytotoxicity of DBBF-Hb, caused the injury.