An-Najah Staff

Background: Reactive oxygen species (ROS) are formed as a natural byproduct of the normal metabolism of oxygen and have important roles in cell signaling and homeostasis. However, during times of oxidative stress, ROS levels can rise dramatically. This may result in significant damage to cell structures. In this work we are interested to show the effect of different ROS on the morphology of fresh human RBCs.
Methods: The RBCs were incubated with different reaction mixtures at room temperature and exposed to cool fluorescent light (800 lux). Then, cells were isolated and scanned by a scanning electron microscope.
Results: When incubated with photoilluminated riboflavin, RBCs lost their biconcave shape and adopted a spherocytes shape. The formation of spherocytes is usually associated with spectrin deficiency. In the presence of Cu(II) and riboflavin, RBCs appeared with spikes of different sizes on their surface showing the formation of “acanthocytes”, which is usually prevalent in abetalipoprotienemia. Moreover, addition of NaN3 to riboflavin-Cu(II) system resulted in completely damaged RBCs.  Away from the above combinations, when RBCs are incubated with riboflavin-aminophylline combination, they appeared with spikes of equal lengths and sizes on their surface “echinocytes”, which usually appear in different diseases like pyruvate kinase deficiency and uremia.
Conclusion: Red blood cells undergo different morphological changes when incubated in each of the above combinations, most probably due to the formation of different ROS and these ROS could be involved in different pathological consequences.

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Purpose. To assess and compare changes in the biomechanical properties of the cornea following different corneal collagen cross-linking protocols using scanning acoustic microscopy (SAM).
Methods. Ten donor human corneal pairs were divided into two groups consisting of five corneal pairs in each group. In group A, five corneas were treated with low-fluence (370 nm, 3 mW/cm²) cross-linking (CXL) for 30 minutes. In group B, five corneas were treated with high-fluence (370 nm, 9 mW/cm²) CXL for 10 minutes. The contralateral control corneas in both groups had similar treatment but without ultraviolet A. The biomechanical properties of all corneas were tested using SAM.
Results. In group A, the mean speed of sound in the treated corneas was 1677.38 ± 10.70 ms⁻¹ anteriorly and 1603.90 ± 9.82 ms⁻¹ posteriorly, while it was 1595.23 ± 9.66 ms⁻¹ anteriorly and 1577.13 ± 8.16 ms⁻¹ posteriorly in the control corneas. In group B, the mean speed of sound of the treated corneas was 1665.06 ± 9.54 ms⁻¹ anteriorly and 1589.89 ± 9.73 ms⁻¹ posteriorly, while it was 1583.55 ± 8.22 ms⁻¹ anteriorly and 1565.46 ± 8.13 ms⁻¹ posteriorly in the untreated control corneas. The increase in stiffness between the cross-linked and control corneas in both groups was by a factor of 1.051×.
Conclusions. SAM successfully detected changes in the corneal stiffness after application of collagen cross-linking. A higher speed-of-sound value was found in the treated corneas when compared with the controls. No significant difference was found in corneal stiffness between the corneas cross-linked with low- and high-intensity protocols.

Purpose. To explore the biomechanical changes induced by repeated cross-linking using scanning acoustic microscopy (SAM).
Methods. Thirty human corneas were divided into three groups. In group A, five corneas were cross-linked once. In group B, five corneas were cross-linked twice, 24 hours apart. In group C, five corneas were cross-linked three times, 24 hours apart. The contralateral controls in all groups had similar treatment but without UV-A. The speed of sound, which is directly proportional to the square root of the tissue's elastic modulus, was assessed using SAM.
Results. In group A, the speed of sound of the treated corneas was 1677.38 ± 10.70 ms⁻¹ anteriorly and 1603.90 ± 9.82 ms⁻¹ posteriorly, while it was 1595.23 ± 9.66 ms⁻¹ anteriorly and 1577.13 ± 8.16 ms⁻¹ posteriorly in the controls. In group B, the speed of sound of the treated corneas was 1746.33 ± 23.37 ms⁻¹ anteriorly and 1631.60 ± 18.92 ms⁻¹ posteriorly, while it was 1637.57 ± 22.15 ms⁻¹ anteriorly and 1612.30 ± 22.23 ms⁻¹ posteriorly in the controls. In group C, the speed of sound of the treated corneas was 1717.97 ± 18.92 ms⁻¹ anteriorly and 1616.62 ± 17.58 ms⁻¹ posteriorly, while it was 1628.69 ± 9.37 ms⁻¹ anteriorly and 1597.68 ± 11.97 ms⁻¹ posteriorly in the controls. The speed of sound in the anterior (200 × 200 μm) region between the cross-linked and control corneas in groups A, B, and C was increased by a factor of 1.051 (P = 0.005), 1.066 (P = 0.010), and 1.055 (P = 0.005) respectively. However, there was no significant difference among the cross-linked corneas in all groups (P = 0.067).
Conclusions. A significant increase in speed of sound was found in all treated groups compared with the control group; however, the difference among the treated groups is not significant, suggesting no further cross-links are induced when collagen cross-linking treatment is repeated.   

The photodynamic action of riboflavin is generally considered to involve the generation of reactive oxygen species, whose production is enhanced when Cu(II) is present in the reaction. In the present study we report that photoactivated riboflavin causes K⁺ loss from fresh human red blood cells (RBC) in a time dependent manner. Addition of Cu(II) further enhances the K⁺ loss and also leads to significant hemolysis. Riboflavin in a 2:1 stoichiometry with Cu(II) leads to maximum K⁺ loss and up to 45% hemolysis. Bathocuproine, a specific Cu(I)-sequestering agent, when present in the reaction, inhibits the hemolysis completely. Free radical scavengers like superoxide dismutase, potassium iodide and mannitol inhibited the hemolysis up to 55% or more. However, thiourea was the most effective scavenger showing 90% inhibition. These results suggest that K⁺ leakage and hemolysis of human RBC are basically free radical mediated reactions.

The effect of aminophylline on human red blood cells (RBC) has been studied. Under in vitro condition, aminophylline alone does not hemolyse RBC. However, in the presence of riboflavin and visible light, aminophylline causes hemolysis of RBC. This hemolysis depends on the concentration of both riboflavin and aminophylline. Using different free radical scavengers we show that RBC hemolysis is caused by reactive oxygen species. Studies using bovine serum albumin show that riboflavin-aminophylline combination can also cause protein degradation in vitro.

Riboflavin (RF) upon irradiation with fluorescent light generates reactive oxygen species like superoxide anion, singlet and triplet oxygen, flavin radicals and substantial amounts of hydrogen peroxide (H2O2). H2O2 can freely penetrate cell membrane and react with a transition metal ion like Cu(ll), generating hydroxyl radical via the modified metal-catalyzed Haber-Weiss reaction. Earlier, it was reported that trypsin-chymotrypsin mixture served as an indirect antioxidant and decreased free radical generation. Thus, in the present study, we used photoilluminated RF as a source of ROS to investigate the effect of free radicals on the activity of trypsin. We also compared the damaging effect of photoilluminated RF and RF-Cu(ll) system using trypsin as a target molecule. RF caused fragmentation of trypsin and the effect was further enhanced, when Cu(II) was added to the reaction. Results obtained with various ROS scavengers suggested that superoxide radical, singlet and triplet oxygen were predominantly responsible for trypsin damage caused by photoilluminated RF. On the other hand, when Cu(ll) was added to the reaction, hydroxyl radical was mainly responsible for trypsin damage. A mechanism of generation of various ROS in the reaction is also proposed. Trypsin did not show any antioxidant effect with RF alone or with RF-Cu(II) combination.