Pinelli Euro Opthalmic_Layout 1 21/01/2010 10:37 Page 67
Anterior Segment Cornea
Tensioactive-mediated Transepithelial Corneal Cross-linking –
First Laboratory Report
Roberto Pinelli, Hytham IB El-Shawaf and Mohammad M Al Marzouky
Istituto Laser Microchirurgia Oculare, Brescia
Abstract
Purpose: The aim of this study was to investigate the possibility of delivering riboflavin into the corneal stroma for cross-linking with the
aid of a tensioactive substance (benzalkonium chloride), without removing the epithelium, in order to make corneal cross-linking (CXL) a
less invasive technique. Methods: Rabbit corneas were cross-linked in vivo without removal of the epithelium, using riboflavin solution
0.1% to which benzalkonium chloride solution was added. The corneas were then examined by light and electron microscopy for evidence
of collagen cross-linking. Control non-treated eyes were compared with treated ones. Results: The treated corneas were stiff and yellow
in colour, and on light microscope examination they showed straight compact collagen fibres in comparison with control corneas in
approximately 50% of the corneal thickness. Electron microscopy revealed regular arrangement of fibres in the outer 200µm with reduced
inter-fibre distances in the treated corneas, but with no noticeable differences between control and treated corneas in the inner 100µm.
Conclusion: The addition of benzalkonium chloride to the riboflavin solution facilitated its entry to the corneal stroma through the
epithelium, and CXL occurred without epithelium removal.
Keywords
Benzalkonium chloride, collagen fibres, corneal cross-linking, epithelium, riboflavin, ultraviolet A
Disclosure: The authors have no conflicts of interest to declare.
Received: 21 July 2009 Accepted: 20 November 2009
Correspondence: Roberto Pinelli, Istituto Laser Microchirurgia Oculare (ILMO), Via Cefalonia, 70, 25124 Brescia, Italy. E: pinelli@ilmo.it
It has been over five years since riboflavin–ultraviolet A (UVA) Materials and Methods
corneal collagen cross-linking treatment was introduced.
1
Long- The cross-linking was carried out at the Zooprophylaxis Institute,
term stability of up to six years has been reported for this treatment Brescia, Italy, on four male albino rabbits (Oryctolagus cuniculus),
in stopping the progression of keratoconus (KC).
1,2
It is estimated approximately 60–90 days old, weighing 2–2.5kg. The rabbits were
that >1,000 corneal cross-linking (CXL) procedures are performed anaesthetised with intraperitoneal injection of xylazine and
monthly worldwide.
3
Other available treatment modalities for KC ketamine 15mg/kg.
include glasses, contact lenses, intracorneal rings, epikeratoplasty
and thermal keratoplasty. However, the effect of these treatments is Clinical treatment consisted of the standard reported CXL treatment,
1
only temporary as they do not stop the progression of KC, and the with the following variation: the anaesthetic drops used were
final choice left is penetrating keratoplasty with many potential benzalkonium chloride (BAK)-free, there was no epithelium removal
intra- and post-operative complications.
2
and the riboflavin solution used contained 0.02% BAK (patented in
Italy by Dr Roberto Pinelli) as a penetration enhancer. In each rabbit
Post-laser-assisted in situ keratomileusis (LASIK) ectasia, reported one eye was treated and the other eye served as a control.
since 1998
4
and characterised by progressive corneal steepening
(usually inferiorly), increase in myopia and astigmatism with A lethal dose of Tanax (Intervet, Munich, Germany), a solution used for
consequent loss of uncorrected as well as best corrected visual painless death in pets and laboratory animals, was used to kill
acuity,
5
is a new area for CXL with promising early results.
6,7
Other the rabbits. The procedure was performed in compliance with the
recent applications of CXL are in the treatment of bullous Association for Research in Vision and Ophthalmology (ARVO)
keratopathy
3
and infective keratitis.
8
As the efficacy of CXL is statement for the use of animals in ophthalmic and vision research,
becoming more established and new applications for its usage are as well as Italian animal welfare law.
appearing, the number of patients seeking this treatment continues
to grow. We see the standard protocol for CXL as moderately invasive The eyes were then enucleated, rapidly washed in 0.1M phosphate
as it requires mechanical removal of the epithelium; this has buffer solution and immersed in 4% paraformaldehyde and 2.5%
motivated us to develop a variant of the standard technique in which glutaraldehyde in 0.1M cacodylate buffered solution (pH 7.4) at 4°C
the epithelium is not removed, in order to offer patients a less (Sigma-Aldrich). After 30 minutes of fixation, the corneas were
invasive, safer and faster CXL while retaining the efficacy of the removed from each eye, sectioned into central and peripheral
standard technique. fragments and transferred into electron microscopy vials (EMS)
© TOUCH BRIEFINGS 2009 67
Page 1  |  Page 2  |  Page 3  |  Page 4  |  Page 5  |  Page 6  |  Page 7  |  Page 8  |  Page 9  |  Page 10  |  Page 11  |  Page 12  |  Page 13  |  Page 14  |  Page 15  |  Page 16  |  Page 17  |  Page 18  |  Page 19  |  Page 20  |  Page 21  |  Page 22  |  Page 23  |  Page 24  |  Page 25  |  Page 26  |  Page 27  |  Page 28  |  Page 29  |  Page 30  |  Page 31  |  Page 32  |  Page 33  |  Page 34  |  Page 35  |  Page 36  |  Page 37  |  Page 38  |  Page 39  |  Page 40  |  Page 41  |  Page 42  |  Page 43  |  Page 44  |  Page 45  |  Page 46  |  Page 47  |  Page 48  |  Page 49  |  Page 50  |  Page 51  |  Page 52  |  Page 53  |  Page 54  |  Page 55  |  Page 56  |  Page 57  |  Page 58  |  Page 59  |  Page 60  |  Page 61  |  Page 62  |  Page 63  |  Page 64  |  Page 65  |  Page 66  |  Page 67  |  Page 68  |  Page 69  |  Page 70  |  Page 71  |  Page 72  |  Page 73  |  Page 74  |  Page 75  |  Page 76  |  Page 77  |  Page 78  |  Page 79  |  Page 80  |  Page 81  |  Page 82  |  Page 83  |  Page 84  |  Page 85  |  Page 86  |  Page 87  |  Page 88  |  Page 89  |  Page 90  |  Page 91  |  Page 92  |  Page 93  |  Page 94  |  Page 95  |  Page 96  |  Page 97  |  Page 98  |  Page 99  |  Page 100  |  Page 101  |  Page 102  |  Page 103  |  Page 104  |  Page 105  |  Page 106  |  Page 107  |  Page 108  |  Page 109  |  Page 110  |  Page 111  |  Page 112  |  Page 113  |  Page 114  |  Page 115  |  Page 116