A Histological Examination of Corneal Epithelium After Iontophoresis with Different Riboflavin Solutions

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A Histological Examination of Corneal Epithelium After Iontophoresis with Different Riboflavin Solutions

Na Li, Zhengjun Fan, Xiujun Peng

Department of Ophthalmology, The Sixth Medical Center of PLA General Hospital, Beijing 100048, China

Introduction

Keratoconus is a progressive eye disorder that affects the cornea. Currently, corneal collagen crosslinking (CXL) with riboflavin is a promising therapy. Riboflavin, as a photosensitizer, can be activated by ultraviolet radiation in the cornea to prevent damage to intraocular tissues. However, riboflavin, being a water-soluble macromolecule, has difficulty permeating the corneal epithelial barrier. Conventional de-epithelial eye-dripping methods have drawbacks like pain, long recovery time, and infection risks. Transepithelial corneal CXL is a research focus, and improving riboflavin’s transepithelial penetration is crucial. Although iontophoresis has been reported, its mechanism wasn’t clear. In this study, we compared the permeability effects of riboflavin in different solvents via iontophoresis and analyzed corneal epithelial cell layer changes.

Materials and Methods

Animals and Groups

We selected 32 New Zealand white rabbits (2.0 – 2.5 kg), healthy with no eye diseases. Only the right eye of each was used. Rabbits were randomized into eight groups (four rabbits each). Three test subgroups received iontophoresis of 0.1% riboflavin in balanced saline (with NaCl, KCl, CaCl₂, and MgCl₂), normal saline, or distilled water for 5 minutes. Three eye-dripping-alone groups got eye-dripping with the same riboflavin solutions for 5 minutes. The de-epithelial eye-dripping group had eye-dripping with 0.1% riboflavin dextran solution for 30 minutes after de-epithelization. The normal cornea control group had no treatment.

Iontophoresis Procedure

In the iontophoresis group, rabbits were anesthetized with intramuscular injection of sumianxin and ketamine. Placed laterally with the right eye up. Skin in the right foreleg was prepared, disinfected, and connected to the skin electrode. An eye speculum opened the eyelid, an eyecup was placed and stabilized on the cornea with vacuum suction. The corneal electrode of the iontophoresis apparatus was connected, and riboflavin solution was injected. The iontophoresis current was 1 mA for 5 minutes. After the apparatus stopped, devices were removed.

Eye-Dripping-Alone Procedure

Similar to the iontophoresis group in anesthesia and positioning. An eye speculum opened the eyelid, an eyecup was placed. Riboflavin solution was injected and after 5 minutes, the eyecup was removed.

De-Epithelial Eye-Dripping Procedure

Same anesthesia and positioning as iontophoresis. An eye speculum opened the eyelid, 8.5 mm corneal epithelium was removed from the center, and then the eye was dripped with 0.1% dextran solution every 3 minutes for 30 minutes.

Observations and Examinations

After operations, corneal stroma and aqueous humor yellowing, and corneal epithelium status were observed under a slip-lamp. For comparing stroma yellowing, an 8.5 mm corneal stroma button was excised, washed, and compared. For pathological examination, an 8.5 mm corneal button was excised, washed, fixed with formalin (for light microscopy) or formaldehyde (for electron microscopy). At the end, animals were euthanized.

Results

Slip-Lamp Observations

In the iontophoresis group, after 5 minutes:

  • Balanced saline and normal saline subgroups: Corneal epithelium smooth, stroma faint yellow, no edema.
  • Distilled water subgroup: Mild fog-like corneal epithelium edema, stroma significantly yellow. In the eye-dripping-alone group: Corneal epithelium smooth, stroma slightly yellow, no edema in each subgroup. In the de-epithelial eye-dripping group: Corneal stroma significantly yellow, no edema.

Corneal Stroma Button Yellowing Comparison

  • Distilled water iontophoresis and de-epithelial eye-dripping groups: Corneal stroma significantly yellow.
  • Balanced saline and normal saline iontophoresis groups: Corneal stroma faint yellow.
  • Eye-dripping-alone group subgroups: Corneal stroma slightly yellow.

Microscopic Examinations

  • Light microscope: No corneal epithelium in the de-epithelial eye-dripping group, normal structure in others.
  • Electron microscope:
    • Balanced saline and normal saline iontophoresis groups: Intact corneal epithelial cell layer, slightly dilated intercellular space, reduced tight junctions and desmosomes.
    • Distilled water iontophoresis group: Cellular edema in corneal epithelial cell layer, intercellular space significantly reduced (nearly non-existent), unclear tight junctions and desmosomes.
    • Eye-dripping-alone group: Intact corneal epithelial cell layer, intercellular space not significantly changed, slightly decreased desmosomes.

Discussion

The degree of yellowing of the corneal stroma after iontophoresis of 0.1% riboflavin distilled water solution for 5 minutes was similar to the conventional de-epithelial eye-dripping method. But iontophoresis with balanced saline and normal saline and eye-dripping alone had unfavorable permeability effects.

Why Distilled Water Iontophoresis was Superior

  • Least parasitic ions: Minimized resistance for riboflavin ion migration in the electric field.
  • Low osmotic pressure: Slight corneal epithelium edema affected the barrier function.

Corneal Epithelial Barrier Function

The corneal epithelial barrier has intercellular and intracellular pathways. Hydrophilic drugs (like riboflavin) enter via the intercellular pathway. In the eye-dripping-alone group, only slight desmosome decrease and no significant intercellular space change indicated little permeability increase. In balanced saline and normal saline iontophoresis groups, decreased tight junctions and desmosomes, and slightly dilated intercellular space meant increased permeability. In the distilled water iontophoresis group, cell edema increased cell membrane permeability (enhancing the intracellular pathway) to promote riboflavin penetration despite reduced intercellular space.

Conclusion

Although we got definite results, the molecular mechanism of corneal epithelial membrane permeability changes and riboflavin’s transport across the cell membrane need further study.

References

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How to cite this article: Li N, Fan Z, Peng X. A histological examination of corneal epithelium after iontophoresis with different riboflavin solutions. Chin Med J 2022;135:753–755. doi: 10.1097/CM9.0000000000001579

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