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Enzymatic evidence of the depth dependence of stiffening on riboflavin/UVA treated corneas [in German]

Authors:
Thomas Schilde at St. Johannes Hopital Dortmund
Thomas Schilde
M Kohlhaas
M Kohlhaas
M Kohlhaas
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Eberhard Spoerl at Universitätsklinikum Carl Gustav Carus Dresden
Eberhard Spoerl
Lutz E Pillunat at Universitätsklinikum Carl Gustav Carus Dresden
Lutz E Pillunat
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Abstract

It has been shown that the treatment of keratoconus with riboflavin/ultraviolet A (UVA) causes significant stiffening of the cornea due to cross-linking. The aim of this study was to evaluate how deep the mechanical stabilization after collagen cross-linking could be shown biochemically. Ten out of 20 enucleated porcine eyes were treated with riboflavin as a photosensitizer and UVA (370 nm, 3 mW/cm2, 30 min). The other 10 eyes served as controls. With a Microkeratom device, two flaps with a thickness of 200 microm and a diameter of 8 mm were cut off from each eye and put in a collagenase solution (NaCl plus collagenase A, 1:1). The surfaces of the flaps were measured digitally every day to characterize the dissolving behavior. The resistance (regarding corneal collagen against enzymatic digestion) of the treated superficial flaps was considerably higher (p=0.001) compared to those that were cut secondarily and to the control flaps. But even the flaps from deeper layers showed a significant increase in resistance (p=0.02) compared with the untreated flaps. The half-life of the surfaces of the treated superficial flaps was 220 h; of those cut secondarily, it was 80 h. Both untreated flaps had a half-life of 50 h. The biochemical study showed that the treatment of the cornea with riboflavin/UVA leads to significant collagen cross-linking not only in the anterior slice of 200 microm but also in the following 200 microm. This locally limited cross-linking effect may be explained by the absorption behavior for UVA of the riboflavin-treated cornea; 65% of UVA irradiation is absorbed in the first 200 microm and only 25-30% in the next 200 microm. Therefore, deeper-lying structures and especially the endothelium are not affected.
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Depth dependence of stiffening on riboflavin/UVA treated corneas
Schilde, T.; Spoerl, E.; Kohlhaas, M.; Pillunat, L. E.
Department of Ophthalmology, University of Dresden
Fetscherstr. 74, Germany-01307 Dresden
Purpose:
Treatment of keratoconus with riboflavin/UVA is based on a significant stiffening of the cornea. This second study
should show until which depth the mechanical stabilization after collagen-crosslinking would be provable
biochemically.
Method:
10 out of 20 enucleated porcine eyes were treated with riboflavin as photosensitizer and UVA (370nm, 3mW/cm²,
30min), the other 10 eyes served as controls. With a microkeratom two flaps of 200µm thickness were cut from
each eye and afterwards laid in collagenase solution (NaCl + collagenase; 1:1). The surfaces of the flaps were
measured digitally and compared daily to characterize the solvent behaviour.
Results:
The resistance (regarding corneal collagen against enzymatic digestion) of the treated flaps, that were cut firstly
was considerably (p=0,001) higher compared to those which were cut secondly and to the control flaps. But even
the flaps that were cut secondly showed a significant increase of resistance (p=0,02) in comparison with the
untreated flaps. The half-life of the surfaces of the treated flaps which were cut firstly was 220 hours, of those cut
secondly 80 hours. The both untreated flaps had a half-life of 50 hours.
Conclusions:
The biochemical study showed, that treatment of the cornea with riboflavin/UVA leads to a significant collagen-
crosslinking not only in the anterior slice of 200µm but also in the following 200µm. This locally limited
crosslinking-effect may be explained by the absorption behaviour for UVA of the riboflavin-treated cornea. 65% of
UVA-irradiation is absorbed in the first 200µm and only 25-30% in the next 200µm. Therefore, deeper laying
structures and especially the endothelium are not influenced.
Department of Ophthalmology
University of Dresden
Fetscherstr. 74
01309 Dresden
Germany
Contact:
Thomas Schilde
Thomas.Schilde@web.de
Commercial Relationship:
T. Schilde, None;
E. Spoerl, None;
M. Kohlhaas, None;
L.E. Pillunat, None
Depth Dependence of Stiffening
on Riboflavin/UVA Treated Corneas
T. Schilde; E. Spoerl; M. Kohlhaas; L.E. Pillunat
Department of Ophthalmology, University of Dresden, Dresden, Germany
Purpose: It was shown that treatment of keratoconus with riboflavin/UVA is based on a significant stiffening of the cornea (ARVO 2004
abstr.nr.2888). Aim of this study was to evaluate how deep the mechanical stabilization after collagen-crosslinking could be shown
biochemically.
Methods: 10 out of 20 enucleated porcine eyes were treated with riboflavin as photosensitizer and UVA (370nm; 3mW/cm²; 30 min), the
other 10 eyes served as controls. With a microkeratom two flaps of 200µm thickness were cut from each eye and afterwards were put in
collagenase solution (NaCl + collagenase A). The surfaces of the flaps were measured digitally and compared daily to characterize the
solvent behavior.
Results: The resistance (regarding corneal collagen against enzymatic digestion) of the treated superficial flaps was considerably higher
(P=0.001) compared to those which were cut secondly and to the control flaps. But even the flaps from deeper layers showed a significant
increase of resistance (P=0.02) in comparison with the untreated flaps. The half-life of the surfaces of the treated superficial flaps was 220
hours, of those cut secondly 80 hours. The both untreated flaps had a half-life of 50 hours.
Fig.1: experimental equipment Fig.2: riboflavin/UVA application Fig.3: cutting flaps with a Fig.4: dissected flaps (200µm) Fig.5: half-life of the
microkeratom surfaces
Fig.6: solvent behaviour: Fig.7: solvent behaviour: Fig.8: digitally measurement Fig.9: digitally measurement Fig.10: results
treated superficial flaps treated superficial flaps calibrate the computer- of the solved surfaces
after 3 hours after 171 hours program
Conclusion: This biochemical study showed that treatment of cornea with riboflavin/UVA leads to a significant collagen-crosslinking not
only in the anterior slice of 200µm but also in the following 200µm. This locally limited crosslinking-effect may be explained by the
absorption behaviour for UVA of the riboflavin-treated cornea. 65% of UVA- irradiation is absorbed in the first 200µm and only 25 – 30% in
the next 200µm. Therefore, deeper laying structures and especially the endothelium seem to be not influenced.
0
10
20
30
40
50
60
70
80
1 21 28 46 68 73 127 146 194 240 315
time in h
surface in mm²
koflap1
koflap2
uvflap1
uvflap2
P=0.3P=0.01
deeper
layers
P=0.025080flap from
flap
P=0.00150220superficial
untreatedtreated
Purpose: Treatment of keratoconus with riboflavin/UVA is based on a significant stiffening of the cornea. Aim of the study was to examine to which depth of the
cornea the stiffening effect is present biomechanically.
Methods: 20 enucleated porcine eyes were treated with riboflavin as photosensitizer and UVA (370 nm, 3 mW/cm², 30 min) and 20 eyes served as controls. Two
consecutive flaps of 200 μm thickness were cut with a microkeratom from each. Each flap was divided into stripes of 5 mm width and 7 mm length. Stress-strain curves
were measured with a material tester to characterize the stiffening effect. For statictics ANOVA was used. Additionally, the same procedure was realized at a pair of
human corneas.
Fig.1: UVA-radiation Fig. 2: rotormicrokeratom Fig. 3: cornea stripe Fig. 4: stress-strain measuring Fig. 5: biomechanical surveyors table
Results: Stress at 5 % strain was 261±133x10³ N/m² for the superficial treated flaps in comparison to 104±52x10³ N/m² corresponding control flaps. This difference
was statistically significant (P=0.0001). The deep treated flaps showed a stress of 105±56x10³ N/m² compared to the corresponding control flaps with 103±62x10³
N/m². This difference was not statistically significant (P=0.94). The stiffness of the superficial flap was statistically significant increased in comparison to the deeper
flaps (P = 0.001). The stiffness between the superficial and deep control flaps was not statistically significant different (P=0.9).
The same stiffening effect was also observed in the human eye. Contrary to the porcine cornea the superficial flap of the human cornea is stiffer than the second flap.
Fig. 6: Fig. 7
Conclusions: Treatment of the cornea with riboflavin/UVA leads to a significant stiffening only in the anterior 200 μm of the cornea. This locally limited stiffening
effect might be explained by the absorption behaviour of the riboflavin-treated cornea for UVA. 65 - 70 % of UVA-irradiation is absorbed in the first 200 μm and only 20
% in the next 200 μm.
Evaluation of the stiffening effect of riboflavin/UVA treatment in
different depth of the cornea
G.Unger, T. Schilde, E. Spoerl, M. Kohlhaas, L.E. Pillunat
Department of Ophthalmology, University of Dresden, Dresden, Germany
Department of Ophthalmology
University of Dresden
Fetscherstr. 74
01309 Dresden
Germany
Contact:
Gabriele Unger
gabriele.unger@uniklinikum-
dresden.de
Commercial Relationship:
G. Unger, none
T. Schilde, none
E. Spoerl, none
M. Kohlhaas, none
L.E. Pillunat, none
0
200
400
600
800
1000
1200
0 2 4 6 8 10
Strain in %
first treated flaps
first untreated flaps
second untreated flaps
second treated flaps
porcine cornea
0
200
400
600
800
1000
1200
0 2 4 6 8 10
Strain in %
Stress in 10³ N/m²
first untreated flap
first treated flap
second untreated flap
second treated flap
human cornea (65 years)
... This melting processes can be halted by two mechanisms: by decreasing the production of degrading enzymes or by increasing the strength of the cornea to the enzymatic digestion. The later effect is achieved by combined Riboflavin/UVA CXL of the corneal stroma [10][11][12][13]. Thus, the corneal stroma becomes more resistant to digestive enzymes from microbial and inflammatory origin [14]. ...
... Thus, the corneal stroma becomes more resistant to digestive enzymes from microbial and inflammatory origin [14]. The structural and biomechanical effects of Riboflavin/UVA CXL in the corneal stroma have been evaluated both clinically and experimentally [10][11][12][13]15,16]. For in vitro evaluations light, electron and confocal microscopy, x-ray scattering and second harmonic generation imaging, 2-dimensional fast Fouriertransform analysis stress-strain biomaterial tester and enzymatic digestion have been used [17]. ...
Enzymatic Digestion of Fresh-Frozen Human Cornea After Riboflavin/Ultraviolet-A Collagen Crosslinking
Article
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  • Jun 2022
Objective: Crosslinking (CXL) of the cornea by using riboflavin (RBF)/ultraviolet-A (UVA) has been developed for stiffening the collagenous matrix of the cornea. Collagenases and other metalloproteinases are known to play an important role in the pathogenesis of corneal ulceration. Our aim in this study was to show and compare the morphological and histopathological changes of the CXL human corneas against enzymatic digestion by collagenase, trypsin and pepsin solutions.Materials and Methods: Eighteen corneas from nine fresh frozen cadavers were included in this study. The crosslinked group (n=12) was treated with the combined riboflavin (RBF) and ultraviolet-A (UVA) and the control group (n=6) was only treated with RBF. After treatment corneas were exposed to collagenase, trypsin and pepsin enzymes. For each enzyme group one cornea was evaluated morphologically and the other one was evaluated microscopically. Results: The collagenase treated control group, digestion of the cornea was noted in the first days and completed on the 5th day. The control cornea treated with pepsin was fully digested after 14 days. In the trypsin treated CXL cornea complete digestion was noted after 18 days. In both groups, enzyme activity was observed to be parallel to each other morphologically, but CXL cornea was evaluated to be more resistant to enzymatic digestion. Conclusion: In conclusion, CXL increases resistance of the cornea against enzymatic digestion. In addition to the biomechanical support, this study was also showing the histopathological changes of CXL procedure and the results of enzymatic digestion, supporting new treatment options in the corneal diseases.
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... Ultraviolet A light was utilized to ina-ctivate microorganisms in drinking water and to disinfect blood products [6]. Riboflavin prompts a change in properties of the collagen and affects the corneal stroma, which stabilizes it and increases its stiffness and resistance to enzymatic bacterial degradation avoiding the progression of corneal melting [7]. In practice, the standard riboflavin/ultraviolet A crosslink protocol seems to produce only short-term improvement in pain, irritation, and discomfort and sometimes reducing the corneal thickness [8]. ...
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... 12 It induces a change in the property of the collagen and has a stiffening effect on the corneal stroma which stabilizes it and increases its resistance to enzymatic degradation avoiding the progression of corneal melting. 16,17 This is also utilized to prevent melting of the corneal graft used as a carrier in Boston keratoprosthesis. 18 The photoactivation of riboflavin damages the RNA and DNA of microorganisms by oxidation processes causing lesions in their chromosomal strands. ...
Benefit of collagen cross linking of the donor corneal button and the graft host junction during therapeutic penetrating keratoplasty - A pilot study
Article
Full-text available
  • Mar 2021
Collagen crosslinking (CXL), used as a treatment modality in infectious keratitis, can be further studied for the prevention of recurrent infection in cases undergoing therapeutic penetrating keratoplasty (TPK). To study the role of collagen crosslinking of the donor corneal tissue and the graft host junction during TPK in preventing recurrence of infection. This was a prospective, clinical, interventional pilot case study done from May 2017 to July 2018. Fifteen cases and 15 controls who had infectious keratitis and needed TPK were included. Pediatric patients, one eyed patients, perforated ulcers and those with any other significant co-morbidity were excluded. In patients who were enrolled as cases, the donor corneal graft and the graft host junction were subjected to CXL using modified Dresden protocol just after the TPK. TPK was done as per standard protocol. The patients were followed up for at least four months duration and the recurrences in both the groups were recorded. The Odds Ratio of the cases not developing a recurrent infection vis-a-vis the controls has been calculated along with the Confidence Interval using the SPSS Statistics software. Recurrence of infection was found in one case and in four controls within one month of TPK. CXL done on donor corneal tissue and the graft host junction intra operatively, has a role in reducing recurrence of infection after TPK.
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... 8,9 A decade ago, Wollensak and associated have introduced a novel minimally invasive technique using riboflavin\UVA corneal collagen cross-linking (CXL), which has been shown to enhance corneal biomechanical stiffening, stabilize and even arrest progression of keratoconus in adult eyes, with no permanent deleterious effects, thus rendering corneal transplantation unnecessary in some patients. [10][11][12][13][14][15][16][17][18] It seems that keratoconus has a tendency to progress faster in children than in adults, and therefore the consequence of unsatisfactory visual acuity or contact lens intolerance may be encountered sooner. Vinciguerra and associates have recently reported their results of a 2 years follow-up after corneal CXL in patients younger than 18 years with documented progressive keratoconus. ...
Corneal Cross-linking in Patients Younger than 18 Years: Long-term Follow-up in Three Israeli Medical Centers
Article
  • Aug 2014
Purpose To report refractive, topographic and safety outcomes of corneal cross-linking (CXL) in patients younger than 18 years of age with progressive keratoconus. Materials and methods In this retrospective study, we enrolled 31 eyes of 21 children aged 11 to 17 years that underwent corneal riboflavin-ultraviolet A induced CXL due to progressive keratoconus at three different ophthalmology departments in Israel. They were followed for 3 to 48 months (average 23 ± 13.6 months). Evaluated parameters were uncorrected visual acuity (UCVA), best spectacle corrected visual acuity (BSCVA), manifest refraction, pachymetry, slit- lamp examination and corneal topography at baseline and at 1,3,6,12,24 and 48 months. Results We found a nonsignificant improvement in UCVA and BSCVA with a small reduction of manifest cylinder and no significant change in spherical equivalent or K-values. Following CXL, stability of UCVA and BSCVA at the last follow-up examination was found in 71 and 77% of treated eyes, respectively. No permanent adverse events have been recorded throughout the study period. Conclusion In our series, CXL was a safe procedure in the pediatric population. Stabilization of progressive keratoconus was achieved in visual acuity, refractive and topography parameters with no improvement in corneal indices in contrary to adult CXL treatment. How to cite this article Bakshi E, Barequet IS, Aizenman I, Levinger S, Avni I, Zadok D. Corneal Corss-linking in Patients Younger than 18 Years: Long-term Follow-up in Three Israeli Medical Centers. Int J Kerat Ect Cor Dis 2014;3(2):84-87.
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... 4 It induces a change in the property of collagen and has a stiffening effect on corneal stroma which stabilizes it and increases its resistance to enzymatic degradation avoiding progression of corneal melting. 15,16 This is also utilized to prevent melting of corneal graft used as a carrier in Boston Keratoprosthesis. 9 The photoactivation of riboflavin damages the RNA and DNA of micro-organisms by oxidation processes causing lesions in their chromosomal strands. ...
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Kollagen-Crosslinking der Hornhaut mit UV-A und Riboflavin zur Behandlung des Keratokonus – 10-Jahres-Ergebnisse
Thesis
  • Jan 2022
Ziel der Studie: Evaluation der Effektivität und Sicherheit des Kollagen-Crosslinkings der Hornhaut mit Riboflavin und UV-A (CXL) bei progressivem Keratokonus über einen Nachbeobachtungszeitraum von bis zu 10 Jahren. Design: Retrospektive klinische Längsschnittstudie Methoden: 131 Augen von 131 Patienten (männlich:weiblich = 95:36) erhielten an der Universitäts-Augenklinik Würzburg zwischen 2006 und 2016 ein Kollagen-Crosslinking (CXL) nach dem Dresdner Standardprotokoll, bestehend aus einer Abrasio, Applikation von iso-osmolaren Riboflavin/Dextran-Augentropfen für 30 min und anschließender UV-A-Bestrahlung mit 3 mW/cm2 für 30 min. Die retrospektive Nachbeobachtung betrug 1 (n=103 Augen) bis maximal 10 Jahre (n=44 Augen). Einschlusskriterien waren eine Zunahme des maximalen Hornhautkrümmungsradius (Kmax, gemessen mittels Pentacam HR) >1 dpt und eine Hornhautdicke >400 µm nach Abrasio. Für parametrische bzw. nichtparametrische Daten wurde der T-Test bzw. der Wilcoxon-Vorzeichen-Rangsummentest durchgeführt. Ergebnisse: 1 bis 3 Jahre präoperativ nahm der Median von Kmax und K2 signifikant um 1,5 dpt (p=0,001) und 1,1 dpt (p<0,001) bis zur Behandlung zu. Die apikale Hornhautdicke nahm nach 1-3 Jahren präoperativ um 12 µm ab (p=0,003). Postoperativ stieg der Median von K2 zunächst bis nach 1 Jahr um 0,1 dpt, nahm dann aber über den weiteren Nachbeobachtungszeitraum kontinuierlich ab, nach 10 Jahren lag er um 0,85 dpt (p=0,021) unter dem Ausgangswert. Die mittlere apikale Hornhautdicke nahm nach 3, 7 bzw. 10 Jahren um 11 µm (p<0,001), 9 µm (p=0,014) und 3 µm (p=0,358) ab. Der mediane Kmax zeigte Schwankungen ohne signifikante Veränderung. Der mittlere bestkorrigierte Visus (logMAR) nahm nach 5 Jahren signifikant um 0,13 und nach 10 Jahren um 0,08 ab (p=0,013 und p=0,010). Der Anteil der Non-Responder, definiert durch einen postoperativen Anstieg von Kmax>2 dpt, nahm von 16% nach 5 auf 33% nach 10 Jahren zu. Risikofaktoren waren ein junges Alter, hoher Astigmatismus, eine dünne Hornhaut und atopische Dermatitis. 4 Augen erhielten im Verlauf komplikationslos eine Revernetzung, woraufhin sich keine weitere Krankheitsprogression zeigte. Fazit: Die CXL-Behandlung kann die Progression des Keratokonus verlangsamen oder stoppen. Allerdings war ab 5 Jahren nach dem Eingriff eine Abnahme des Anteils der Responder zu beobachten. Regelmäßige Nachkontrollen sollten daher besonders auch im Langzeitverlauf durchgeführt werden, um eine erneute Progression frühzeitig erkennen und behandeln zu können.
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Corneal collagen cross-linking for bacterial infectious keratitis
Article
  • Jun 2020
Background: Infectious keratitis is an infection of the cornea that can be caused by bacteria, viruses, fungi, protozoa, or parasites. It may be associated with ocular surgery, trauma, contact lens wear, or conditions that cause deficiency or loss of corneal sensation, or suppression of the immune system, such as diabetes, chronic use of topical steroids, or immunomodulatory therapies. Photoactivated chromophore for collagen cross-linking (PACK-CXL) of the cornea is a therapy that has been successful in treating eye conditions such as keratoconus and corneal ectasia. More recently, PACK-CXL has been explored as a treatment option for infectious keratitis. Objectives: To determine the comparative effectiveness and safety of PACK-CXL with standard therapy versus standard therapy alone for the treatment of bacterial keratitis. Search methods: We searched the Cochrane Central Register of Controlled Trials (CENTRAL) (which contains the Cochrane Eyes and Vision Trials Register) (2019, Issue 7); Ovid MEDLINE; Embase.com; PubMed; Latin American and Caribbean Health Science Information database (LILACS); ClinicalTrials.gov; and the World Health Organization (WHO) International Clinical Trials Registry Platform (ICTRP). We did not use any date or language restrictions in the electronic search for trials. We last searched the electronic databases on 8 July 2019. Selection criteria: We included randomized controlled trials (RCTs), quasi-RCTs, and controlled clinical trials (CCTs) of PACK-CXL for bacterial keratitis. We included quasi-RCTs and CCTs as we anticipated that there would not be many RCTs eligible for inclusion. Data collection and analysis: Two review authors working independently selected studies for inclusion in the review, assessed trials for risk of bias, and extracted data. The primary outcome was proportion of participants with complete healing at four to eight weeks. Secondary outcomes included visual acuity, morphology, adverse events, and treatment failure at four to eight weeks. Main results: We included three trials (two RCTs and one quasi-RCT) in this review for a total of 59 participants (59 eyes) with bacterial keratitis. Trials were all single-center and were conducted in Egypt, Iran, and Thailand between 2010 and 2014. It is very uncertain whether PACK-CXL with standard antibiotic therapy is more effective than standard antibiotic therapy alone for re-epithelialization and complete healing (risk ratio (RR) 1.53, 95% confidence interval (CI) 0.88 to 2.66; participants = 15). We judged the certainty of the evidence to be very low due to the small sample size and high risk of selection and performance bias. The high risk of selection bias reflects the overall review. Masking of participants was not possible for the surgical arm. No participant had a best-corrected visual acuity of 20/100 or better at eight weeks (very low certainty evidence). There is also no evidence that use of PACK-CXL with standard therapy results in fewer instances of treatment failure than standard therapy alone (RR 0.50, 95% CI 0.05 to 4.98; participants = 32). We judged the certainty of evidence to be low due to the small sample size and high risk of selection bias. There were no adverse events reported at 14 days (low certainty evidence). Data on other outcomes, such as visual acuity and morphological characteristics, could not be compared because of variable time points and specific metrics. Authors' conclusions: The current evidence on the effectiveness of PACK-CXL for bacterial keratitis is of low certainty and clinically heterogenous in regard to outcomes. There are five ongoing RCTs enrolling 1136 participants that may provide better answers in the next update of this review. Any future research should include subgroup analyses based on etiology. A core outcomes set would benefit healthcare decision-makers in comparing and understanding study data.
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Safety and efficacy of anterior chamber paracentesis with simultaneous intracameral administration of 0.5% pilocarpine in dogs and cats with glaucoma of various etiologies
Article
Full-text available
  • Nov 2019
Purpose. In animals with glaucoma to determine the technique for conducting ACP with simultaneous administration of a 0.5 % pilocarpine solution, to identify the dynamics of IOP reduction, as well as effectiveness of this procedure both after and in the long term. Based on analysis of data obtained, highlight the complications of this procedure. Materials and methods. Study included 37 animals (39 eyes): 24 dogs and 13 cats with glaucoma, which, according to anamnesis, developed no more than 10 days without any preliminary ocular hypotensive therapy. All animals underwent ACP without general anesthesia or sedation. Immediately before procedure, animals were instilled with a local anesthetic in conjunctival sac (0.5% proxymethacaine). ACP was performed under 6-fold magnification using a 27G headlamp with 9 hours for right eye and 3 hours for left eye. A puncture was carried out at periphery of cornea 1 mm from limbus. Volume of aspirated AH was no more than 2 drops. After that, needle was removed from the AC. Next step was intracameral injection of 0.5% solution of pilocarpine through hole of paracentesis. Volume of injected solution did not exceed 0.3 ml. After paracentesis and administration of pilocarpine solution, subconjunctival administration of dexamethasone solution in combination with gentamicin and novocaine was performed. Immediately after ACP, antihypertensive therapy was prescribed from a combination of eye drops: synthetic analogues of prostaglandins F2α (travoprost 0.004%) in combination with non-selective beta-blockers (timolol maleate 0.5%), as well as carbonic anhydrase inhibitors (brinzolamide 1%) every 12 hours. Results. Average IOP was in animals before ACP 60.26 ± 16.32 mmHg; immediately after procedure ― 10.15 ± 3.23 mm Hg; 1 hour after ACP ― 13.28 ± 3.09 mm Hg; after 7 days ― 17.61 ± 6.03. Immediately after intracameral administration of a 0.5% pilocarpine solution, we observed a rapid and pronounced narrowing of the pupil within 1…2 minutes. Myosis was noted for at least 1 hour after procedure. There were no adverse reactions from administration of pilocarpine solution in AC. Among complications after immediately 1 hour after the ACP, we encountered aqueous flare in 12 eyes (31% of cases) and moderate hyphema in 5 eyes (13% of cases). 7 days after procedure, no complications were observed. Conclusion. This study shows that ACP with simultaneous intracameral administration of 0.5% pilocarpine solution is effective in dogs and cats without any significant side effects. These results allow us to offer ACP as a procedure for an emergency reduction in IOP, especially in acute attacks of glaucoma in dogs and cats.
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Spoerl E, Huhle M, Seiler T. Induction of cross-links in corneal tissue
Article
Full-text available
  • Jan 1998
The aim of this study was to investigate the possibility of induction of cross-links in corneal tissue in order to increase the stiffness as a basis for a future conservative treatment of keratectasia. Collagenous biomaterials can be stabilized by chemical and physical agents. The epithelium of enucleated porcine eyes was removed. Eight test groups, 10 eyes each, were treated with UV-light (lambda=254 nm), 0.5% riboflavin, 0.5% riboflavin and UV-light (365 nm) blue light (436 nm) and sunlight, and the chemical agents-glutaraldehyde (1% and 0.1%, 10 min) and Karnovsky's solution (0.1%, 10 min). Strips of 5 mm in width and 9 mm in length were cut from each cornea and the stress-strain behaviour of the strips was measured to assess the cross-linking process. For comparison, ten untreated corneas were measured by the same method. Compared to untreated corneas treatment with riboflavin and UV-irradiation as well as weak glutaraldehyde or Karnovsky's solutions resulted in an increased stiffness of the cornea. The biomechanical behaviour of the cornea can be altered by glutaraldehyde, Karnovsky's solution, and with riboflavin and UV-irradiation which offers the potential of a conservative treatment of keratoconus. To optimize this effect further investigation is necessary regarding the dose-response and in-vivo application.
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Increased rigidity of cornea caused by intrastromal cross-linking
Article
Full-text available
  • Dec 1997
To increase the stability of the cornea by artificial cross-linking (radiation or chemical agents) and to investigate a future therapy for keratoconus. The epithelium of enucleated porcine eyes was removed. Ten eyes in each of eight test groups were treated with UV light (lambda = 254 nm), 0.5% riboflavin and UV light (365 nm), blue light (436 nm) and sunlight, and the chemical agents glutaraldehyde (1% and 0.1%, 10 min) and Karnovsky's solution (0.1%, 10 min). Strips of 5 mm in width and 9 mm in length were cut from each cornea and the stress-strain behaviour of the strips was measured. For comparison, eight groups of ten untreated corneas each were measured by the same method. Compared to untreated corneas riboflavin and UV irradiation as well as glutaraldehyde and Karnovsky's solution treatment resulted in significantly increased stiffness of the cornea (p < 0.05). The biomechanical behaviour of the cornea can be altered by low-concentration glutaraldehyde, Karnovsky's solution, and by riboflavin and UV irradiation, which offers potential conservative treatment of keratoconus. To optimize this effect further investigation is necessary regarding the dose-effect relation and the in-vivo conditions.
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Studies on the stabilization of the cornea in rabbits
Article
Full-text available
  • Apr 2000
The mechanical stabilization of the cornea in keratoconus may delay progression of this disease. The cross-linking techniques optimized in corneas of enucleated porcine eyes were investigated under in vivo conditions in rabbits to estimate the biocompatibility and duration of the stiffening effect. Twenty-eight rabbits were treated monocularly, the fellow eye serving as control. The epithelium was mechanically removed and 19 eyes were treated with riboflavin plus ultraviolet irradiation (365 nm, 2 mW/cm2) for 45 min and 9 eyes with 0.075% glutaraldehyde for 20 min. After treatment, the eyelids were sutured for 3 days. The healing process was controlled by slit-lamp examination and photographically documented. After 1 month, 20 animals and after 3 months 8 animals were sacrificed, the eyes enucleated, and the stress-strain relation of the corneas measured and compared to the fellow eye. The epithelium was closed after 4-5 days. The transparency of the corneas remained clear during follow-up, and there were no signs of inflammatory reaction. Stress for a strain of 6% was higher in the treated corneas by a factor of 1.3 +/- 0.66 (P = 0.319) in the glutaraldehyde group and by a factor of 1.6 +/- 0.75 (P = 0.0408) in the riboflavin group at 1 month, and by 1.3 +/- 0.48 (P = 0.07) at 3 months after treatment. The cross-linking technique using riboflavin plus UV irradiation is suitable for at least temporarily stiffening the cornea in vivo and seems to be a promising method for conservative treatment of keratectasia.
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Treatment of keratoconus by collagen cross linking
Article
Full-text available
  • Feb 2003
We were able to show a significant increase in corneal stiffness of rabbit and porcine eyes after combined riboflavin/UVA-induced collagen cross-linking. In this study,we tried to treat keratoconus patients with this method to stop the progression of corneal ectasia. We treated 16 eyes of 15 patients with progressive keratoconus and mostly moderate keratectasia (48-56 dpt). After removal of the epithelium (7 mm X), riboflavin solution was applied on the cornea, which was irradiated with UVA (370 nm,3 mW/cm(2)) at a distance of 1 cm for 30 min.Post-operative follow-up controls were conducted every 3 months in the first year and then every 6 months, always including visual acuity testing, corneal topography and measurements of endothelial cell density. The follow-up time was between 1 and 3 years. Progression of keratectasia was stopped in all patients. Best corrected visual acuity and the maximal keratometry values improved slightly in about 50% of the cases. In all patients corneal transparency, the degree of keratectasia registered by corneal topography and the density of endothelial cells remained unchanged within the follow-up time. No negative side-effects were observed. Our results show that collagen cross linking might be a useful conservative treatment modality to stop the progression of keratoconus. By this means the need for keratoplasty might be significantly reduced. Given the simplicity of the technique and minimal costs of the treatment it might also be well suited for developing countries.Further studies are envisaged to exclude long-term side effects and to evaluate the long term durability of the mechanical stiffness effect.
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Increased resistance of crosslinked cornea agaist enzymatic difestion
Article
Full-text available
  • Aug 2004
Collagen-crosslinking using combined riboflavin/ UVA treatment has been developed by us as a new treatment for keratoconus by stiffening the collagenous matrix. Recently, we have started to use the same method for the treatment of corneal ulcers. The aim of the present study was to evaluate the influence of the crosslinking treatment on the resistance of the cornea against enzymatic degradation. 60 enucleated porcine eyes were treated with the photosensitizer riboflavin and UVA-irradiation (370 nm; irradiance of 1, 2 or 3 mW/cm2) for 30 minutes and compared with 20 untreated control eyes. After crosslinking treatment, the corneal buttons were trephined and exposed to pepsin, trypsin and collagenase solutions. The extent of the corneal digestion was monitored daily. Selected cases were examined by light microscopy. The corneal buttons crosslinked with riboflavin/ UVA at 3 mW/cm2 were dissolved only by day 13 following pepsin digestion and by day 14 following collagenase treatment versus 6 days in the untreated control corneas. Digestion by trypsin was observed on day 5 in buttons crosslinked at 3 mW/cm2 compared to day 2 in the control corneas. Microscopically, a prolonged preservation especially of the anterior portion of the crosslinked corneas could be demonstrated. Photochemical crosslinking of the cornea using riboflavin and UVA results in a markedly increased resistance versus collagen digesting enzymes. The findings support the use of the new method in the treatment of corneal ulcers.
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Endothelial cell damage after riboflavin-ultraviolet-A treatment in the rabbit
Article
To evaluate the possible cytotoxic effect of combined riboflavin-ultraviolet-A (UVA) treatment on the corneal endothelium. Department of Ophthalmology, Technical University of Dresden, Dresden, Germany. The right eyes of 34 New Zealand White rabbits were treated with riboflavin and various endothelial UVA doses ranging from 0.16 to 0.9 J/cm2 (0.09 to 0.5 mW/cm2, 370 nm) and postoperative enucleation times of 4 hours and 24 hours. The endothelial cells were evaluated in histological sections. The terminal deoxynulceotidyl transferase deoxy-UTP-nick-end labeling (TUNEL) technique and transmission electron microscopy were used to detect apoptosis. There was no endothelial damage in the 6 rabbit eyes enucleated at 4 hours. In those enucleated at 24 hours, there was significant necrosis and apoptosis of endothelial cells in the corneas treated with an endothelial dose of > or =0.65 J/cm2 (0.36 mW/cm2), which is about twice the endothelial UVA dose used in the treatment of keratoconus patients. In rabbit corneas with a corneal thickness less than 400 microm, the endothelial UVA dose reached a cytotoxic level of > or =0.65 J/cm2 (0.36 mW/cm2) using the standard surface UVA dose of 5.4 J/cm2 (3 mW/cm2). Pachymetry should be routinely performed before riboflavin-UVA treatment; in thinner corneas, irradiation should not be done because of the cytotoxic risk to the endothelium.
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Light-induced endocapsular polymerization of injectable lens refilling materials
Article
The use of advanced phacoemulsification techniques enables the surgeon to remove opacified lens material while preserving a nearly complete capsular bag. For this reason, several groups are presently working on lens refilling techniques. We have developed a method for the injection of a liquid monomer into the lens capsule. The material can be polymerized by exposure to cold light (wavelength, 400-500 nm) within the capsular bag. Using cell-culture experiments, we selected an optically clear material that has cytotoxic properties when placed in direct contact with cells but does not release cytotoxic substances into the surrounding medium. We have shown that reflux of the liquid monomer into the anterior chamber can be avoided by using a material that can be polymerized within the capsule. Initial experiments in rabbits indicate that the technique can be used without inducing a significant postoperative inflammatory response or leading to morphological changes in the cornea or the retina.
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Biochemical properties of keratoconus and normal conneas
Article
  • Nov 1980
The uniaxial tensile strength was determined for six keratoconus and seven normal corneas. Strip specimens were stretched at a constant speed in a materials testing machine while recording load and deformation. Load-deformation curves were transformed into load-strain and stress-strain curves. The following parameters were derived from the load-strain and stress-strain curves: the maximum load and stress values and the corresponding strain value, the values for maximum stiffness, and the relative energy absorption of the specimens during testing. With the exception of the strain value at maximum load or stress, these parameters were smaller in the keratoconus group than in the normal group. The load and stress values at corresponding strain values were less in keratoconus corneas than in normal corneas both for the initial and for the linear parts of the curves.Biochemically, no difference was found in the concentration of uronic acid in the two groups. Similarly no difference was found in the concentration of hydroxyproline in the two groups. The solubility of pepsin treated collagen in the keratoconus corneas was greater than that in the normal corneas, however, the solubility was less than that in tendons.
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Photopolymerization of dental composite matrices
Article
The kinetic behaviour of dental composite is traditionally studied, considering only the isothermal behaviour, whereas a fast and highly non-isothermal bulk polymerization is expected as a consequence of the significant heat developed due to the exothermic nature of the polymerization reaction. In this paper the photopolymerization kinetics of a commercial dental composite activated by visible light are analysed by differential scanning calorimetry. This technique is applied to determine the degree of reaction and the glass transition temperature of thin layers of the composite matrix, at different isothermal cure temperatures. A phenomenological kinetic model is then integrated with an energy balance in order to analyse the cure behaviour of thicker composite layers. The full model results indicate that non-isothermal cure conditions may be achieved, obtaining higher values for the glass transition temperature and the degree of reaction.
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Relative strength of photodynamic biologic tissue glue in penetrating keratoplasty in cadaver eyes
Article
  • Jan 1999
To compare the relative strength of photodynamic biologic tissue glue (PBTG) with that of 10-0 nylon sutures in closing penetrating keratoplasty (PKP) wounds in cadaver eyes. Corneal Research Laboratory, University of Chicago, Chicago, Illinois, USA. A 7 mm central corneal trephination was performed on 8 recently enucleated human eyes. Each corneal button was removed and then resutured in its original position, using 4 interrupted 10-0 nylon cardinal sutures and PBTG (n = 7) or 16 interrupted 10-0 nylon sutures (n = 1). Wound leak and incisional bursting pressures were determined. The average pressure at which wound leak and iris prolapse occurred was 124 mm Hg (range 70 to 180) and 185 mm Hg (range 90 to 300), respectively. The pressure required to create wound leak increased as the time between glue application and pressurization of the globe lengthened (mean 22 minutes, range 5 to 60 minutes), indicating that the bonding and stability of the adhesive increased with time. In contrast, the control eye developed wound leak at 70 mm Hg and iris prolapse at 300 mm Hg. Photodynamic biologic tissue glue was comparable to sutures in providing adequate corneal wound strength in PKP in a cadaver eye model. Wound closure with PBTG may reduce the number of sutures required in corneal transplantation and decrease the incidence of suture-related complications and allograft rejection.
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