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Keywords:

  • UVR;
  • cornea;
  • epithelium;
  • rabbit

Abstract.

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Purpose: It is known that the corneal epithelium strongly absorbs ultraviolet radiation (UVR). The aim of the present study was to examine the protective role of corneal epithelium against UVR damage by comparing the biological effect of UVR exposure on whole corneas with that on de-epithelialized corneas.

Methods: Six New Zealand albino rabbit corneas were exposed to UVR centred around 280 nm at a dose that causes biomicroscopically significant keratitis (012 J/cm2). Three corneas underwent manual de-epithelialization prior to UVR exposure. A control group of three rabbits underwent only manual de-epithelialization. The animals were killed 76 hours after treatment. The corneas were stained with haematoxylin and evaluated by light microscopy.

Results: Corneas that underwent only the exposure to UVR showed a loss of epithelial cells in the treated area. No damage to keratocytes or the stroma was detected. Corneas that underwent manual de-epithelialization showed a loss of epithelial cells, and also keratocytes in the anterior quarter of the corneal stroma. However, corneas that were exposed to UVR after manual de-epithelialization showed very deep stromal damage. The keratocytes disappeared through the entire thickness of the stroma in the UVR-exposed area.

Conclusion: Exposure to UVR at 280 nm alone does not result in any deep damage to the corneal stroma and keratocytes. Manual de-epithelialization causes the disappearance of anterior keratocytes. However, the stromal damage caused by UVR in the de-epithelialized corneas was very deep. The corneal epithelium serves to protect the deeper corneal structures against UVR damage, probably by absorbing a substantial amount of the UVR energy applied to the eye.


Introduction

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Acute exposure of the mammalian eye to an adequate dose of ultraviolet radiation (UVR) results in the development of photokeratitis after a few hours (Widmark 1889; Martin 1912; Friedenwald et al. 1948; Pitts et al. 1969, 1977; Pitts 1970). Chronic exposure to UVR may be associated with an increased risk of several corneal disorders, including pterygium, climatic droplet keratopathy and climatic proteoglycan stromal keratopathy (Klintworth 1977; Moran & Hollows 1984). The degree of damage produced by acute exposure to UVR depends on several factors, such as the spectrum of UVR applied and exposure duration (Pitts et al. 1969; Pitts 1970). Wavelengths below 290 nm are almost completely absorbed by the epithelium and do not penetrate the deeper, underlying structures of the eye (Kinsey 1948; Sherashov 1970). In contrast, the middle UVR waveband (300–320 nm) is known to be absorbed by the corneal stroma and lens (Kinsey 1948; Sherashov 1970). We have shown in previous studies that exposure of the rabbit cornea to UVR in the 280 nm wavelength range to above threshold for keratitis results in apoptosis and loss of corneal epithelial cells (Podskochy & Fagerholm 1998; Podskochy et al. 2000). Exposure to UVR with longer wavelengths (310 nm) results in much deeper corneal damage, with apoptosis and disappearance of keratocytes throughout the entire thickness of the corneal stroma, as well as epithelial damage (Podskochy & Fagerholm 1998; Podskochy et al. 2000). The peak of apoptosis occurs approximately 24 hours after the UVR exposure (Podskochy et al. 2000).

Kolozsvari et al. (2002) recently reported that the corneal epithelium has a significantly higher absorption coefficient for UVR with wavelengths shorter than 300 nm than the stroma. The corneal epithelium may thus protect the underlying structures by absorbing considerable amounts of UVR. This suggestion is particularly important because photorefractive keratectomy (PRK), where the epithelium, Bowman's layer and some of the anterior stroma are removed by excimer laser, has become a routine ophthalmic procedure. After PRK the remainder of the corneal stroma is left unprotected against UVR, at least temporarily, until the epithelium is healed.

The purpose of the present study was to evaluate the protective role of the corneal epithelium against UVR by comparing the biological effect of UVR exposure on whole corneas with that on de-epithelialized corneas.

Material and Methods

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Ultraviolet radiation source

The UVR source was a 150-W xenon high pressure lamp, powered by universal power supply, Oriel 68805 (Oriel, Stratford, Connecticut, USA). The lamp was mounted in an Oriel 7340 lamp housing. The lamp power supply system was stabilized with a light intensity controller (Oriel 68850). The radiation from the source was focused at the monochromator entrance slit by the housing optics. The exit optical beam was focused by a quartz lens producing a beam size of approximately 4 × 10 mm2 at the centre of the rabbit cornea. The spectral irradiance was centred around 280 nm with a half maximum width of 10 nm with a calibrated Oriel 77250 monochromator. The irradiance in the focus area used during the rabbit exposures was measured with a calibrated LaserMate/Q detector (Coherent, Auburn, CA, USA).

Animals

Nine female adult New Zealand albino rabbits were used, in accordance with the ARVO Resolution on the Use of Animals in Research and with permission from the local ethics committee for the use of animals in research. Each rabbit received intramuscular Ketalar (ketamine hydrochloride 30 mg/kg) and Rompun (xylazine 5 mg/kg) anaesthesia prior to the experiment. One eye of each rabbit was treated and the second eye was used as a control. The rabbits were divided into three groups. The first group (three rabbits) was exposed to 280 nm UVR at a dose producing biomicroscopically significant keratitis (0.12 J/cm2, or 10 times the corneal threshold dose for keratitis). The photokeratitis threshold doses for different wavelengths were reported by Pitts (1970). In the second group (three rabbits), the corneal epithelium was scraped manually with a corneal knife before exposure to UVR at 280 nm as described above. The third group (three rabbits) underwent only manual de-epithelialization by scraping.

The rabbits were killed with an overdose of pentobarbital intravenously 76 hours after treatment. The corneas were excised from the treated eyes and untreated fellow eyes. The corneas were fixed in 10% formaldehyde, embedded in paraffin and sectioned at 5 µm. The sections were stained with Mayers haematoxylin and evaluated in the light microscope.

Results

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

The untreated control corneas appeared normal without any damage to any of the corneal layers (Fig. 1A). Corneas that underwent only the exposure to UVR showed a loss of epithelial cells in the treated area (Fig. 1B). No damage to keratocytes, stroma or endothelium was detected. Corneas that underwent manual de-epithelialization showed a loss of epithelial cells and keratocytes in the anterior quarter to a half of the corneal stroma (Fig. 1C). The remaining stroma was populated by keratocytes. However, corneas that were exposed to UVR after manual de-epithelialization showed very deep stromal damage. The keratocytes had disappeared throughout the entire thickness of the stroma in the UVR-exposed area (Fig. 1D). These corneas had also experienced a loss of endothelial cells and the corneas themselves appeared swollen.

image

Figure 1. (A) Normal cornea. (B) Cornea exposed to 280-nm UVR, showing loss of epithelial cells. (C) Cornea after manual de-epithelialization, showing disappearance of keratocytes in the anterior quarter of the stroma. (D) Cornea which underwent manual de-epithelialization before exposure to 280-nm UVR, showing extensive damage to the stroma where the keratocytes had disappeared throughout its entire thickness.

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Discussion

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

We have shown in previous studies that UVR at 280 nm causes apoptosis and loss of epithelial cells without causing any deep damage to underlying corneal layers (Podskochy & Fagerholm 1998; Podskochy et al. 2000). Apoptosis after UVR exposure is activated through the Fas/Fas ligand system (Podskochy & Fagerholm 2002). Wilson et al. (1996) showed that the disappearance of keratocytes in the anterior part of the stroma after epithelial scraping is explained by apoptosis mediated by cytokines released from damaged epithelial cells. In the present study, corneas were exposed to UVR after the epithelium had been scraped off. This resulted in very deep damage to the corneas. Keratocytes disappeared throughout the entire thickness of the cornea, and even a loss of endothelial cells was detected. These results confirm that the corneal epithelium protects deeper, underlying ocular structures against excessive UVR in the 280 nm range.

Kolozsvari et al. (2002) recently showed that the anterior portion of the cornea, including the epithelium and Bowman's layer, absorbs the biggest proportion of UVR at wavelengths shorter than 300 nm. The significant UVR absorption in the epithelium appears to be due to high amounts of tryptophan residues and high ascorbate content (Mitchell & Cenedella 1995; Ringvold 1997; Ringvold 1998; Brubaker et al. 2000). Recent data suggest that the high amount of ascorbate alone in the epithelium might absorb 77% of incident radiation at wavelengths likely to be dangerous (Brubaker et al. 2000).

In summary, these results show that removal of the corneal epithelium makes the underlying ocular structures more susceptible to damage by UVR in the 280 nm range. Further investigations are needed to establish whether the corneal epithelium has equally protective properties against UVR at other wavelengths.

Acknowledgement

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

This study was supported by funds from the Swedish Medical Research Council and Kronprinsessan Margaretas Arbetsnämnd.

References

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References