A Histological Examination of Corneal Epithelium After Iontophoresis with Different Riboflavin Solutions
Corneal collagen crosslinking (CXL) with riboflavin has emerged as a groundbreaking therapy for controlling the progression of keratoconus, a condition characterized by the thinning and bulging of the cornea. Riboflavin, a photosensitizer, plays a crucial role in this process by absorbing ultraviolet (UV) radiation and preventing it from damaging intraocular structures. For CXL to be effective, it is essential to maintain a sufficient concentration of riboflavin in the corneal stroma during the treatment. However, riboflavin, being a water-soluble macromolecule, faces significant challenges in penetrating the corneal epithelial barrier. Traditionally, this barrier has been overcome by de-epithelialization, a process that involves removing the corneal epithelium. While effective, this method is associated with several complications, including pain, prolonged recovery time, and an increased risk of infection. As a result, transepithelial corneal CXL, which avoids the removal of the epithelium, has become a focus of research. Iontophoresis, a technique that uses an electric current to enhance the penetration of drugs, has been explored as a potential solution to improve the transepithelial delivery of riboflavin. This study aimed to compare the permeability effects of riboflavin in different solvents when delivered via iontophoresis and to analyze the histological changes in the corneal epithelial cell layer to identify the most effective solvent and understand the underlying mechanisms.
The study involved 32 healthy New Zealand white rabbits, weighing between 2.0 and 2.5 kg, with no pre-existing eye conditions. Only the right eye of each rabbit was used for the experiment. The rabbits were randomly divided into eight groups, each consisting of four animals. Three subgroups of the test group received iontophoresis with 0.1% riboflavin in balanced saline solution (containing NaCl, KCl, CaCl2, and MgCl2), 0.1% riboflavin in normal saline solution, and 0.1% riboflavin in distilled water for 5 minutes, respectively. Three additional groups received eye-dripping alone with the same riboflavin solutions for 5 minutes. A de-epithelial eye-dripping group was treated with 0.1% riboflavin dextran solution for 30 minutes after the removal of the corneal epithelium. A control group with normal corneas received no treatment. At the end of the experiment, the central corneal epithelium was removed from two eyes in each group to observe the yellowing of the corneal stroma. Additionally, corneas were collected from two eyes in each group for histological examination using optical and electron microscopy.
For the iontophoresis procedure, the rabbits were anesthetized using a mixture of sumianxin and ketamine administered via intramuscular injection. The animals were placed in a lateral position with the right eye facing upward. The skin on the right foreleg was prepared, disinfected, and connected to a skin electrode. An eye speculum was used to open the eyelid, and an eyecup was placed and stabilized using vacuum suction on the center of the cornea. The corneal electrode of the iontophoresis apparatus was then connected, and the riboflavin solution was injected until it was slightly higher than the electrode nest. The iontophoresis apparatus was activated with a current of 1 mA for 5 minutes. After the procedure, the devices were removed, and the eyes were examined.
In the eye-dripping-alone groups, the rabbits were anesthetized and positioned similarly to the iontophoresis group. The eyelid was opened using an eye speculum, and the eyecup was placed and stabilized using vacuum suction. The riboflavin solution was injected and left in place for 5 minutes before the eyecup was removed. In the de-epithelial eye-dripping group, the corneal epithelium was removed from the central 8.5 mm area, and the eye was dripped with 0.1% dextran solution every 3 minutes for 30 minutes.
Following the procedures, the corneal stroma and aqueous humor were observed for yellowing using a slit-lamp. An 8.5 mm corneal stroma button was excised from the central part of the cornea, washed with balanced saline, and compared for the degree of yellowing. For pathological examination, the corneal button was fixed with formalin and examined under a light microscope or cut into smaller blocks, fixed with formaldehyde, and examined under an electron microscope. At the end of the experiment, the animals were euthanized.
Under slit-lamp observation, the corneal epithelium in the iontophoresis group was smooth, and the stroma appeared faint yellow with no edema in the balanced saline and normal saline subgroups. However, mild fog-like edema was observed in the corneal epithelium, and the stroma was significantly yellow in the distilled water subgroup. In the eye-dripping-alone group, the corneal epithelium was smooth, and the stroma was slightly yellow with no edema in all subgroups. The de-epithelial eye-dripping group showed significantly yellow corneal stroma with no edema.
When comparing the yellowing of corneal stroma buttons, the distilled water iontophoresis and de-epithelial eye-dripping groups exhibited significantly yellow stroma, while the balanced saline and normal saline iontophoresis groups showed faint yellow stroma. The eye-dripping-alone groups displayed slightly yellow stroma.
Light microscopy revealed no corneal epithelium in the de-epithelial eye-dripping group, while the other groups showed normal epithelial structure. Electron microscopy showed intact corneal epithelial cell layers in the balanced saline and normal saline iontophoresis groups, with slightly dilated intercellular spaces and reduced tight junctions and desmosomes. In the distilled water iontophoresis group, the corneal epithelial cell layer exhibited cellular edema, significantly reduced intercellular spaces, and unclear tight junctions and desmosomes. The eye-dripping-alone group showed intact corneal epithelial cell layers with no significant changes in intercellular spaces and slightly decreased desmosomes.
The study concluded that the iontophoresis effect of distilled water was superior to that of balanced saline and normal saline, likely due to the minimal presence of parasitic ions in distilled water, which reduced resistance to the migration of riboflavin ions in the electric field. Additionally, the low osmotic pressure of distilled water caused slight edema in the corneal epithelium, which may have affected the barrier function of the corneal epithelium. The corneal epithelial barrier function is divided into intercellular and intracellular pathways. Since riboflavin is a water-soluble macromolecule, it primarily penetrates the corneal stroma via the intercellular pathway. The electron microscopy results supported this, showing that the eye-dripping-alone group had insignificant changes in intercellular spaces, while the iontophoresis groups exhibited increased permeability due to reduced tight junctions and desmosomes. In the distilled water iontophoresis group, the cell edema likely increased the permeability of the cell membrane, enhancing the intracellular pathway and promoting riboflavin penetration.
Despite these findings, the molecular mechanisms underlying the changes in corneal epithelial membrane permeability and the transport of riboflavin across the cell membrane require further investigation. The study provides valuable insights into the potential of iontophoresis as a method for enhancing the transepithelial delivery of riboflavin in corneal collagen crosslinking, offering a promising alternative to de-epithelialization with fewer associated complications.
doi.org/10.1097/CM9.0000000000001579
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