Heidi Rhian Laviers
email: laviers@hotmail.com

Fatma Omar

Issa Muhsin Burhan

Hakika Jecha

Clare Gilbert

Evaluation of distribution of presbyopic correction through primary health care centres in Zanzibar, East Africa

Keywords: Presbyopia, Primary Eye Care, Community Distribution

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Presbyopia, the age related decline in visual acuity for near objects, is an extremely common condition which is readily correctable by spectacles. It has been estimated that 1.04 billion people in 2005 were presbyopic, 517 million of whom were without adequate spectacle correction. [START_REF] Holden | Global Vision Impairment due to Uncorrected Presbyopia[END_REF] As the world's population increases, ages and becomes more literate the demand for near spectacle correction will increase. Age is overwhelmingly the most important risk factor, but climate, geographic location, gender and ethnicity also contribute to the observed variation in the age of onset and degree of presbyopia. [START_REF] Holden | Global Vision Impairment due to Uncorrected Presbyopia[END_REF][START_REF] Miranda | The geographic factor in the onset of presbyopia[END_REF][START_REF] Nirmalan | A population-based assessment of presbyopia in the state of Andhra Pradesh, South India: The Andhra Pradesh eye disease study[END_REF][START_REF] Burke | Population-based study of Presbyopia in Rural Tanzania[END_REF] In terms of priority setting for provision of services for presbyopia, the World Health Organization (WHO) has recommended that if less than one-third of those affected have near correction, the population would be ranked as a high priority for service delivery. [START_REF] Deafness | Elimination of avoidable visual disability due to refractive errors[END_REF][START_REF] Laviers | Presbyopic spectacle coverage, willingness to pay for near correction and the impact of correcting uncorrected presbyopia in adults in Zanzibar, East Africa[END_REF] In developing countries, particularly in rural areas, spectacles are available for purchase through optical suppliers and hospitals, but usually located in urban areas where prices are high. Strategies to improve presbyopic spectacle coverage in rural areas of developing countries, include community distribution schemes, using trained "Village Entrepreneurs", a model developed by Vision Spring [START_REF]The economist[END_REF][START_REF] Levin | Scojo Foundation provides affordable eye glasses to the poorest countries[END_REF] or "Vision Guardians" used by Vision Centres in India [START_REF] Rao | An infrastructure model for the implementation of VISION2020: The right to sight[END_REF] .

In 2007 a study of presbyopia was undertaken within a Rapid Assessment of Avoidable Blindness survey in Zanzibar [START_REF] Nirmalan | A population-based assessment of presbyopia in the state of Andhra Pradesh, South India: The Andhra Pradesh eye disease study[END_REF] . Those with good distance VA and uncorrected presbyopia were given spectacles and the majority were traced and re-interviewed 6 months later [START_REF] Laviers | Presbyopic spectacle coverage, willingness to pay for near correction and the impact of correcting uncorrected presbyopia in adults in Zanzibar, East Africa[END_REF] . The prevalence of presbyopia among the 381 participants aged 40 years and above was 89.2%, but at baseline less than 1 in 5 had spectacles. Barriers to accessing services included spectacles not being a priority and lack of money. At follow up the mean satisfaction was high at 89.5%. The mean amount participants were willing to pay for spectacles increased from 2.17 USD at baseline to 3.14 USD at follow up. The impact of correcting presbyopia on visual function (VF) produced an effect size of 2.90 and effect sizes ranging from 1.15 to 3.90 for mean quality of life (QOL) scores.

Zanzibar, which has a population of approximately 1 million, consists of two main islands, Unguja and Pemba, and is situated off the East coast of Tanzania. [START_REF] Burke | Population-based study of Presbyopia in Rural Tanzania[END_REF] Zanzibar has a good primary health system, with Primary Health Care Units (PHCUs) and Centres (PHCCs) spread across the country, the latter being larger with more highly qualified staff. Medical officers (MO) with one year training in PHC, run and generally stay at the facilities and are ideally placed to supply the community with near spectacles and basic eyecare.

The pilot project to incorporate distribution of presbyopic correction was subsequently introduced into 6 randomly selected clinics. The aim of this study was to evaluate the project so as to make recommendations for scaling up across Zanzibar and elsewhere in East Africa.

METHODS:

Establishing the pilot project Four PHCUs and two PHCCs were randomly selected from 16 facilities with limited PEC training and were trained in dispensing presbyopic correction. This further training (1 day at the tertiary hospital) consisted of power-point presentations, demonstrations, group discussions, case studies and a manual was distributed. Topics included the findings of the presbyopic study, WHO blindness definitions and major causes, basic anatomy of the eye, history taking, and examination skills. Equipment was demonstrated and they were taught record keeping and referral criteria. They underwent refresher training in measuring distance VA using the E chart at 6m. They were instructed to dispense the least binocular add required for participants to read/see N8 font clearly at 40cms using a logMAR E chart under standard lighting conditions (at least 480 lux by light meter), provided the person had a presenting distance VA of at least 6/18 in both eyes.

The MOs were given log books to record patient details: eye/visual complaints, gender, age, general health, reason for attendance, presenting distance VA monocularly and binocularly, near vision with and without distance spectacles, power of near spectacles dispensed, frame preference and referral details. They charged a preset value of $3.50 per pair of spectacles based on the baseline research.

Two days following training each MO was visited for half a day to reinforce training and resolve problems. Each MO was given 200 pairs of plastic or metal ready-made spectacles (powers range +0.50D to +4.00D) and a stock take was carried out. The spectacles were supplied by the International Centre for Eye Education at a cost of 2.7USD. The team met all village leaders to discuss their role in creating community awareness.

Methods used in the evaluation:

The project was evaluated 6 months later by a team of two optometrists, the Zanzibar PEC coordinator and National Eye Care coordinator. The evaluation consisted of an assessment of the planning, process and product of the project by reviewing documents and one to one interviews with the MOs, village leaders and service users. The evaluation team held a final focus group meeting.

Ethical approval was obtained from the London School of Hygiene and Tropical Medicine ethics committee and Zanzibar Ministry of Health, and adhered to the tenets of the Declaration of Helsinki. All participants gave written informed consent and those requiring treatment were treated or referred accordingly.

Data management:

Statistical analyses were carried out using STATA 9 software. Data regarding levels of satisfaction with certain aspects of training, field work or service received were analysed by summing scores of 1 to 5 for each question and producing a total score, linearly transformed to give a score out of 100. 6

RESULTS.

Evaluation of plan

The training curriculum was developed by the PEC coordinator and the two optometrists. This project entailed integration into an existing infrastructure and although the logistics had been planned in detail little had been documented.

Recommendations: A structured approach to planning will be required from the outset if the project is to be scaled up, including clearly documented aims and objectives, a plan of action and defined monitoring indicators.

Evaluation of process

Selection criteria: The facilities selected for the study had high attendance and acceptance by the community with the exception of two villages.

Recommendations: Selection for future expansion should consider the following: infrastructure, logistics, population density, capabilities and location.

Training: MOs were very satisfied with the eyecare component of the course and materials provided (Table 1).They were less complementary about training in stock control, accounting and record keeping and felt that more time should have been allocated to reinforce these skills. Village leader's views: Awareness was successfully created through community meetings and announcements at the mosque. Village leaders were content with the information they received and their extra responsibilities.

Supply and revolving fund: The current system for banking in Zanzibar involves one account for the eye department, requiring the eyecare coordinator to permit the release of funds.

Recommendations: A revolving fund would be simpler to organise and monitor as a separate account allowing for the existing PEC infrastructure and funding in place. This would involve three monthly collections of monies by the PEC coordinator from the clinics and depositing in the PEC account. Supply of spectacles requires a bank transfer organised by the eyecare coordinator with the main stock held at the tertiary centre. The cost of spectacles should remain at the current price of 3.50USD.

Referral system: The agreed referral criteria included a distance VA of <6/18 in either eye or a near addition of +4.00D. Participants referred during the pilot study could not be followed up as the tertiary hospital does not differentiate between self-referrals and those who had been referred.

Recommendations: Monitoring mechanisms need to be introduced to track the referral and management of patients through the system.

Evaluation of product

A total of 574 patients presented with eye/vision complaints to the six facilities serving a total population of 15,700; giving an average annual attendance rate of 73/1,000 population/year (Table 3). In two villages the attendance rate was considerably lower, due to lack of service awareness and low population density. More men presented than women although not statistically significant, and the majority of patients were aged 40-50 years (Table 4). in the better eye). In 24 of these 43 individuals, VA improved to 6/18 in one or both eyes with a pinhole (Figure 1). Conjunctivitis was the commonest reason for attending in all age groups (49.7%)(Table 5).

Almost a third (30.1%) complained exclusively of reduced near vision; all 173 patients were prescribed near spectacles and 3 individuals were referred.

Replicability

In keeping with VISION 2020 priorities, the aim is to reduce the unmet need for presbyopic correction to less than one third of the population and make this a long term sustainable outcome. This will have an impact on PEC and refractive error provided the referral mechanisms and infrastructure are in place. The plan to scale up the project to the national level will require operational research and modification as the project evolves (Figure 2). This project has demonstrated how programmes can build on existing health systems and provides an example of integration of service delivery through increased capacity building of existing staff. Organising a revolving financial fund, stock take and delivery at the tertiary hospital.

Figure 2 :

 2 Figure 2: Planning process at the national level.

Table 1 : Medical Officer's level of satisfaction with training, where higher percentages indicate a higher level of satisfaction.

 1

	Training	%	95% CI
	History taking	96.7	95.2-98.1
	Ophthalmic examination	86.7	84.0-89.4
	Distance visual acuity	86.7	84.0-89.4
	Near visual acuity	90.0	87.6-92.4
	Prescribing near spectacles	86.7	84.0-89.4
	Referral mechanisms	93.3	91.3-95.3
	Stock take	70.0	66.3-73.7
	Accounting	66.7	62.9-70.4
	Record keeping	76.7	73.3-80.05
	Materials	90.0	87.6-92.4
	Duration of training	40.0	36.1-43.9

Assessment of training was carried out by observation of examination skills, completion of record cards, history taking and book keeping; any difficulties were raised at the individual facility visit the next day. Recommendations: Future training should take place over two days allowing for more practical training to reinforce topics taught in the lecture sessions. The training should be formalised, with clear goals, objectives and learning outcomes, with formal assessments, particularly of practical skills and book keeping. As the MOs are not trained in ophthalmoscopy, limitations of their responsibilities should be clarified in any future programme.

Table 2 : Level of satisfaction with aspects of field work where higher percentages indicate a higher level of satisfaction.

 2

	Field work	%	95% CI
	Community awareness	83.3	80.4-86.3
	Acceptance by villagers	90.0	87.6-92.4
	Examination procedure	93.3	91.3-95.3
	Equipment	93.3	91.3-95.3
	Referral links	60.0	66.3-73.7
	Links with PEC coordinator 100.0	-
	Stock take	70.0	76.8-83.2
	Spectacles supply	83.3	91.3-95.3
	Spectacle prices	100.0	-
	Record keeping	73.3	80.4-86.3
	Recommendations: More regular follow up was required by the PEC coordinator to provide
	post training support.

Table 3 : Number of patients attending each unit in 6 month study period. Village Patients with eye/ vision complaints Population in village served by PHCU or PHCC Annual rate of attendance / 1,000 population

 3

		N	%	N	%
	Michamwi	73	12.7	1,292	8.2	113
	Uroa	26	4.5	2,387	15.2	22
	P/ Michangani	112	19.5	2,227	14.2	101
	Machanduchi	92	16.0	5,220	33.3	35
	Myuni	57	10.4	876	5.6	130
	Kivunge	213	37.1	3,697	23.5	115
	Total	574	100	15,699	100	73

Table 4 : Distribution of patients with eye/vision complaints by age group and gender.

 4

		Males	Females	Total
	Age							P-
	(years)	No.	%	No.	%	No.	%	value
	0-9	42 15.7	48	15.7	90	16.7 0.434
	10-19	37 13.8	36	11.8	73	12.9
	20-29	24	9	37	12.1	61	12.2
	30-39	34 12.7	43	14.1	77	12.5
	40-49	71	26	76	24.9	147 24.9
	50-59	32 11.9	30	9.8	62	9.9
	60-69	21	7.8	24	7.9	46	7.7
	70-79	7	2.2	11	3.6	17	3
	80-100	1	0.37	0	0	1	0.2
	Total	269 100	305	100	574	100
	Only 7.5% of those attending the clinics had reduced distance VA at presentation (i.e. <6/18

Figure 1: Distribution of participants according to reasons for attendance and management.

			Patients
			attended 574
	PEC		Near problems	Distance problems
	372 (64.8 %)		173 (30.1%)	29 (5%)
	Treated	Referred	Dispensed 173	Treatment	Refraction
	298	74	(100.0%)	1 (3.4%)	referral 28
	(80.1%)	(19.9%)			(96.6%)

Acknowledgements

With thanks to Sightsavers International for their financial contribution, the International Centre for Eye Health in project development, Mnazi Mmoja Hospital for its support and the Zanzibar Ministry of Health for their approval.

Licence for publication:

The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd and its Licensees to permit this article (if accepted) to be published in BJO editions and any other BMJPGL products to exploit all subsidiary rights, as set out in our licence (http://group.bmj.com/products/journals/instructions-forauthors/licence-forms/).

Spectacle powers of +1.50Ds and +2.00Ds and metal frames (61.9%) were most frequently dispensed (Table 6). Recommendations: Medication to treat common conditions should be available for each clinic and spectacles stocks need to be replenished regularly.

Participant views on the scheme

Of the 173 participants given near spectacles, 41 individuals were traced; all still had their spectacles and always had them with them. Satisfaction rates were high (Table 7) and all recommended adoption in other villages.

CONCLUSIONS

The implications of this evaluation in terms of scaling up the provision of near spectacle correction across Zanzibar will be considered in relation to relevance, effectiveness, equality, sustainability and replicability.

Relevance

The project is highly relevant and timely given that correction of presbyopia is now a priority with WHO and the global magnitude and need is increasing.

Effectiveness

Due to good planning, training in the Eye Department was effective. A large number of patients attended the service and had been provided with spectacles, or referred. However, a system for monitoring referral mechanisms will be required.

Equality

Gender: Both male and female MOs were randomly selected for training. On reviewing the patient figures no significant difference was found.

Location: All facilities in the pilot scheme were, by chance, located in rural areas. In future, selection will be based on location, population density and infrastructure.

Sustainability

Human resources: MOs are well place in the community in Zanzibar and many already have PEC awareness. Those interviewed during the evaluation were highly motivated and content to provide an additional service.

Management and equipment: Future management of the project will be the responsibility of the PEC coordinator. All the equipment required is inexpensive and readily available.

Financial: The project used a revolving fund which promotes financial sustainability as long as the small profit is reinvested. Scaling up could either take place slowly by this means or more rapidly with injection of additional capital.

Competing Interest: None to declare.


Gregor Wollensak
email: gwollens@hotmail.com

Eberhard Spörl

Cosimo Mazzotta

Thomas Kalinski

Saadettin Sel

Dr Gregor Pd

Wollensak

Interlamellar cohesion after corneal crosslinking using riboflavin and UVA

Methods: 72 post mortem porcine eyes were divided into six different treatment groups:The untreated control group, the standard crosslinking group, the hypoosmolar crosslinking group, the stromal swelling, the formaldehyde group, and the α-amylase group. An anterior 9

x 4 mm strip of 400 µm thickness was prepared using a lamellar rotating microkeratome. For interlamellar cohesive force measurements a splitting plane was created at 50% depth.

Force-distance profiles were recorded using a microcomputer-controlled biomaterial testing machine.

Results:The mean interlamellar cohesive force was 0.24 N/mm in the untreated control group, 0.26 N/mm in the standard crosslinking group, 0.25 N/mm in the hypoosmolar crosslinking group, 0.23 N/mm in hydrated corneas, 0.27 N/mm in the formaldehyde group without statistically significant difference. Only the values of the α-amylase group were statistically significantly lowered by 31.5% to 0.16 N/mm. Conclusions: Surprisingly, corneal crosslinking does not increase the interlamellar cohesive force. In the α-amylase group the cohesive force was mainly decreased due to the digestion of proteoglycans. Crosslinking seems to stabilize only inter-and intrafibrillar but not interlamellar cohesion.

Corneal crosslinking treatment of progressive keratoconus using the photosensitizer riboflavin and ultraviolet A (UVA) was introduced by Wollensak [START_REF] Wollensak | Riboflavin/ultraviolet-A-induced collagen crosslinking for the treatment of keratoconus[END_REF]

et al. from Germany in

2003 and has become increasingly popular in recent years. [START_REF] Wollensak | Corneal collagen crosslinking: new horizons[END_REF][START_REF] Raiskup-Wolf | Collagen crosslinking with riboflavin and ultraviolet-A light in keratoconus: Long-term results[END_REF] Long-term results have confirmed the earlier positive results. [START_REF] Raiskup-Wolf | Collagen crosslinking with riboflavin and ultraviolet-A light in keratoconus: Long-term results[END_REF] The success of the new crosslinking method (CXL) in the treatment of progressive keratoconus (kc) is based primarily on its biomechanical stiffening effect stabilizing the corneal collagen fibril network and halting the progression of ectasia ("freezing"). [START_REF] Wollensak | Corneal collagen crosslinking: new horizons[END_REF][START_REF] Wollensak | Stress-strain measurements of human and porcine corneas after riboflavin-ultraviolet-A-induced cross-linking[END_REF][START_REF] Mccall | Mechanisms of corneal tissue crosslinking in response to treatment with topical riboflavin and long wavelength ultraviolet radiation (UVA)[END_REF] The crosslinking effect is strongest in the anterior stroma as found in numerous biomechanical, [START_REF] Kohlhaas | Biomechanical evidence of the distribution of cross-links in corneas treated with riboflavin and ultraviolet A light[END_REF] histological 2,7 and hydration studies. [START_REF] Wollensak | Hydration behavior of porcine cornea crosslinked with riboflavin and ultraviolet A[END_REF] Ultrastructurally, the collagen fiber diameter is increased by 12.2% in the anterior stroma after CXL concurrent with intrafibrillar collagen crosslinks [START_REF] Wollensak | Collagen fiber diameter in the rabbit cornea after collagen cross-linking by riboflavin/UVA[END_REF] , and there is almost complete absence of hydration effects in the anterior crosslinked zone compatible with interfibrillar collagen crosslinks. [START_REF] Wollensak | Hydration behavior of porcine cornea crosslinked with riboflavin and ultraviolet A[END_REF] Recent studies have suggested that in keratoconus a reduction in the stromal cohesion may lead to interfibrillar or interlamellar slippage of collagen fibers. [START_REF] Edmund | Corneal tissue mass in normal and keratoconic eyes. In vivo estimation based on area of horizontal optical sections[END_REF][START_REF] Meek | Changes in collagen orientation and distribution in keratoconus corneas[END_REF][START_REF] Mcmonnies | Mechanisms of rubbing-related corneal trauma in keratoconus[END_REF][START_REF] Dawson | Corneal ectasia after excimer laser keratorefractive surgery: Histopathology, ultrastructure, and pathophysiology[END_REF] Therefore, in the present study we tried to investigate the possible changes of interlamellar cohesion after crosslinking to better understand the way CXL may function in the treatment of progressive keratoconus.

MATERIALS AND METHODS

Sample preparation

72 porcine eyes were retrieved from the local abattoir within 24 hours post-mortem. The horizontal meridian was identified by the elliptical shape of the cornea and marked with a marker pen. Only clear corneas were used. The epithelium was carefully removed. Anterior circular flaps of 400 µm thickness and 9 mm diameter were cut using a Draeger lamellar rotating microkeratome (Storz Instrument GmbH, Heidelberg, Germany). Finally, a 4 x 9 mm rectangular central strip was cut along the horizontal meridian of the circular lamellar flap.

Treatment groups

The 72 porcine eyes were divided into six different groups with twelve samples per group:

1.the untreated control group.

2.the standard crosslinking (CXL) group, in which the porcine eyes were crosslinked according to the standard protocol of Wollensak 1 with removal of the epithelium and subsequent application of the standard riboflavin solution (1 mg riboflavin-5-phosphate in 1 ml 20% dextran T-500 dissolved in physiologic saline solution) for 5 minutes before the irradiation, and every 5 minutes during the 30 minutes of irradiation using a commercial UVA diode system (Peschke Meditrade GmbH, Germany) with a surface irradiance of 3 mW/cm² and a focussing distance of 5 cm.

3.the hypoosmolar crosslinking group, which was treated like group 2 except for applying a hypoosmolar 310 mOsmol/L riboflavin solution without dextran, as is used sometimes in thin keratoconus corneas inducing moderate stromal swelling.

4.the group, in which the samples were placed into a moist chamber with physiologic saline for 24 hours inducing stromal swelling alone (without crosslinking).

5.the formaldehyde group with maximum crosslinking, with the samples placed into a 3.5 % neutral buffered formaldehyde solution for 48 hours.

6.the α-amylase group, in which the corneas were kept in an aqueous solution of α-amylase (from Aspergillus oryzae, ≥800 FAU/g , Sigma-Aldrich) for 72 hours at room temperature to digest the proteoglycans.

Pachymetry

After treatment, the thickness of the initially 400 µm thin flaps was determined using ultrasound pachymetry (Pachette, Technomed).

Interlamellar cohesive force measurements

4 x 9 mm anterior corneal strips were incised and carefully cleaved at 50% depth for a 2 mm length on one end of the strip. Once the splitting plane had been created, the anterior and posterior end were fixed in the two clamps of a microcomputer-controlled biomaterial testing machine (MINIMAT; Rheometric Scientific GmbH, Bensheim, Germany; fig 1A andB). After an initial build-up of tension, the stroma split up at a constant rate of 3 mm/min and the cohesive force was measured until the strips were split for a distance of about 7 mm. Forcedistance profiles were recorded and the average force or load determined. As cohesive strength is defined as the force in newtons (N) needed to split a stromal sample of 1 mm width along a plane parallel to the surface, the load values obtained for 4 mm wide strips were divided by 4 (fig 2 A and B, table1). For statistical analysis, the p value of the biomechanical data was calculated using a Student´s t-test for the comparison with the control group .

Histology

Characteristic samples from each treatment group were fixed in 4% formaldehyde for three days and embedded in paraffin. 4 µm thin paraffin cross-sections were cut and stained with hematoxylin-eosin (H & E) and periodic acid-Schiff (PAS).

RESULTS

Gross appearance and pachymetry

The flaps in all treatment groups except for the group 3 and 4 with edema appeared transparent. The swelling effect in group 3 and 4 was rather weak because only the anterior 400 µm of the stroma were used, which is rather resistant to swelling. After swelling, the mean thickness of the hypoosmolar group 3 was increased to 442 ± 23 µm and of the physiologic saline group 4 to 433 ± 27 µm. The pachymetry readings for the control group were 402 ± 18 µm, the CXL group 398 ± 12 µm, the formaldehyde group 395 ± 7 µm and the αamylase group 376 ± 22 µm. The samples from group 2 and 3 had a yellowish tint due to the applied riboflavin solution (fig. 1A). The αamylase treated samples appeared more transparent than the flaps of all the other groups including the controls (fig. 1 B).

Biomechanical cohesive force measurements (table 1)

After an initial build-up of tension, the stroma split up with a rather uniform force.

Characteristic force (load)-extension profiles with localized fluctuations in required tearing force (fig 2 A and B) were registered. [START_REF] Smolek | Interlamellar adhesive strength in human eyebank corneas[END_REF][START_REF] Schmack | Cohesive tensile strength of human LASIK wounds with histologic, ultrastructural, and clinical correlations[END_REF] Only the values of the α-amylase group were statistically significantly different from the control group, with a decrease in cohesive force by -31.5% (p<0.0001; table 1). The slight increase in interlamellar cohesive force after CXL by 5.7% was not statistically significant (p<0.31). The number of samples required to exclude a type II error for the CXL changes would be 60 samples for both the CXL and control group demonstrating a low probability of a false-negative result.

Histology

Torn lamellae could be seen at the interface of the tearing plane in all cases. In the control group (fig. 3A) and even more so in the CXL group, the tissue appeared more compacted (fig 3 B) and the lamellae straigthened, in the edematous group 3 and 4 only a moderate degree of stromal edema was seen, in the α-amylase group the collagen lamellae appeared rather wavy, there was some edema, and the cell nuclei appeared less basophilic (fig 3C).

DISCUSSION

This study has shown that collagen crosslinking using riboflavin and UVA or even the strong crosslinker formaldehyde does not have a significant effect on interlamellar cohesion in the anterior stroma. This finding is very remarkable and surprising. Apparently, collagen crosslinks are mainly intrafibrillar and interfibrillar but not interlamellar. On the other hand, the digestion of proteoglycans by α-amylase led to a significant decrease of interlamellar cohesion by -31.5% demonstrating the important role of the proteoglycan-glycosaminoglycan complexes for interlamellar stabilization.

The lamellar structure of the cornea is rather complex and not yet fully elucidated.

The simplified lamellar concept is that the cornea consists of 300-500 smooth lamellar layers just like a stack of 300 sheets of copy paper that can slide to each other, when subjected to shear. Detailed studies using scanning electron microscopy [START_REF] Radner | Interlacing and cross-angle distribution of collagen lamellae in the human cornea[END_REF][START_REF] Radner | Interlacing of collagen lamellae in the midstroma of the human cornea[END_REF][START_REF] Radner | Altered organization of collagen in the apex of keratoconus corneas[END_REF] , microdissection analysis [START_REF] Radner | Interlacing and cross-angle distribution of collagen lamellae in the human cornea[END_REF][START_REF] Radner | Interlacing of collagen lamellae in the midstroma of the human cornea[END_REF] ,transmission electron microscopy [START_REF] Müller | A new three-dimensional model of the organization of proteoglycans and collagen fibrils in the human corneal stroma[END_REF] , polarised light microscopy or X-ray diffraction [START_REF] Boote | Lamellar orientation in human cornea in relation to mechanical properties[END_REF] could demonstrate that the cornea resembles more a woven textile. [START_REF] Radner | Interlacing and cross-angle distribution of collagen lamellae in the human cornea[END_REF][START_REF] Radner | Interlacing of collagen lamellae in the midstroma of the human cornea[END_REF] The lamellae consist of flattened ribbon-like bundles of collagen fibrils and have about 0.2-2.5 µm thickness and 0.5 -250 µm width. [START_REF] Radner | Interlacing and cross-angle distribution of collagen lamellae in the human cornea[END_REF] The lamellae are aligned parallel to the surface and organized in layers, with a common orientation within the same layer. The diameter of the individual collagen fibers is about 25-35 nm and their interfibrillar distance about 20nm. [START_REF] Müller | A new three-dimensional model of the organization of proteoglycans and collagen fibrils in the human corneal stroma[END_REF] The lamellar fibril bundles are partially interwoven within one layer but also between the layers especially in the anterior stroma. [START_REF] Müller | The specific architecture of the anterior stroma accounts for maintenance of corneal curvature[END_REF] Similar to plywood, [START_REF] Dawson | Corneal ectasia after excimer laser keratorefractive surgery: Histopathology, ultrastructure, and pathophysiology[END_REF][START_REF] Müller | A new three-dimensional model of the organization of proteoglycans and collagen fibrils in the human corneal stroma[END_REF] the lamellae are arranged in layers with crossangles between the lamellar layers. The lamellae of the anterior stroma are more obliquely arranged to each other compared to the more orthogonal crossangles of the posterior stroma with a preferential orientation along the superior-inferior and nasal-temporal meridians. [START_REF] Boote | Lamellar orientation in human cornea in relation to mechanical properties[END_REF] The collagen bands at the limbus are arranged in a more circular pattern fusing with the scleral ring. [START_REF] Boote | Lamellar orientation in human cornea in relation to mechanical properties[END_REF] The interlamellar cohesion is maintained by interlacing lamellae providing structural tissue bridges, and molecular proteoglycan-glycosaminoglcan (GAG´s) complexes , acting like an interlamellar glue, keeping the stromal lamellae from falling apart similar to mortar between bricks. [START_REF] Meek | Changes in collagen orientation and distribution in keratoconus corneas[END_REF][START_REF] Dawson | Corneal ectasia after excimer laser keratorefractive surgery: Histopathology, ultrastructure, and pathophysiology[END_REF][START_REF] Smolek | Interlamellar adhesive strength in human eyebank corneas[END_REF][START_REF] Müller | A new three-dimensional model of the organization of proteoglycans and collagen fibrils in the human corneal stroma[END_REF] Interlamellar cohesion still allows a certain degree of physiological interlamellar sliding movement, especially under shear stress. [START_REF] Elsheikh | Numerical study of the effect of corneal layered structure on ocular biomechanics[END_REF] Interlacing, interlamellar fibril bundles could be nicely shown by Radner et al. using microdissection and scanning electron microscopy. [START_REF] Radner | Interlacing and cross-angle distribution of collagen lamellae in the human cornea[END_REF][START_REF] Radner | Interlacing of collagen lamellae in the midstroma of the human cornea[END_REF] The collagen fibril bundles often branch out in two or three subsidiary branches crossing through fissures between the branches of splitting fiber bundles creating an intensely interwoven meshwork. [START_REF] Meek | Changes in collagen orientation and distribution in keratoconus corneas[END_REF][START_REF] Radner | Interlacing and cross-angle distribution of collagen lamellae in the human cornea[END_REF][START_REF] Radner | Interlacing of collagen lamellae in the midstroma of the human cornea[END_REF] The interlacing collagen lamellae should not be called crosslinks because this term should be reserved for chemical crosslinks. The details of the non-collagenous proteoglycan-glycosaminoglycan (GAG) matrix are not yet fully clear.

Proteoglycans (PG´s) are composed of a protein core with covalently attached sulfated glycosaminoglycan (GAG) chains. In the cornea, the two predominant proteoglycans are keratan sulfate proteoglycans (lumican, keratocan, and mimecan) and dermatan sulfate proteoglycans (decorin). The proteoglycans can be stained with quinolinic phtalocyanin (QP) or cuprolinic blue (CB). [START_REF] Müller | A new three-dimensional model of the organization of proteoglycans and collagen fibrils in the human corneal stroma[END_REF] Müller et al. from Holland could show that in cornea hexagonal arranged collagen fibrils seem to be interconnected with their next-nearest neighbour by six proteoglycan proteins. [START_REF] Müller | A new three-dimensional model of the organization of proteoglycans and collagen fibrils in the human corneal stroma[END_REF] The cross-bridges between collagen fibrils that can be seen in quick freeze-deep etching specimens similar like steps of a ladder probably also represent proteoglycans. [START_REF] Müller | A new three-dimensional model of the organization of proteoglycans and collagen fibrils in the human corneal stroma[END_REF] Based on 3D-electron tomography, Lewis et al. from Cardiff have proposed a more sophisticated and dynamic model in which the PG´s are attached to collagen fibrils but the interfibrillar bridges are randomly tilted and dynamic producing an overall effect of a pseudo-hexagonal arrangement. Also, the GAG chains of the PG´s seem to join together in an antiparallel noncovalent fashion forming partially overlapping, long complexes spanning the distance between more than two collagen fibrils. [START_REF] Lewis | Structural interactions between collagen and proteoglycans are elucidated by three-dimensional electron tomography of bovine cornea[END_REF] The PG´s are considered to be crucial for the resistance to compression and lateral stretch and for transparency. [START_REF] Mcmonnies | Mechanisms of rubbing-related corneal trauma in keratoconus[END_REF][START_REF] Müller | A new three-dimensional model of the organization of proteoglycans and collagen fibrils in the human corneal stroma[END_REF] In this study, the interlamellar cohesive force was measured in the range of 0.139 N/mm centrally to 0.31 N/mm peripherally as reported previously by Smolek et al. and Maurice. [START_REF] Smolek | Interlamellar adhesive strength in human eyebank corneas[END_REF][START_REF] Schmack | Cohesive tensile strength of human LASIK wounds with histologic, ultrastructural, and clinical correlations[END_REF] The tearing force peaks are probably related to abrupt tearing of interlacing collagen lamellae crossing the splitting plane. [START_REF] Smolek | Interlamellar adhesive strength in human eyebank corneas[END_REF] The interlamellar connections appear to behave somewhat like a hook and loop fastener, and the interlamellar tearing effect resembles a zipper effect. Interestingly, after a microkeratome section the peaks canot be found because of the mechanical severing of the interlamellar interlacing fiber bundles. [START_REF] Schmack | Cohesive tensile strength of human LASIK wounds with histologic, ultrastructural, and clinical correlations[END_REF] The absence of an effect of both chemical (formaldehyde) and physical (riboflavin/UVA) crosslinking on interlamellar cohesive force suggests that crosslinking in general mainly affects only the intra-and interfibrillar collagen molecule bonds. In crosslinking by riboflavin and UVA, recent studies by McCall et al. have revealed that the collagen crosslinks induced by CXL are made up of covalent chemical bonds including carbonyl and amine groups [START_REF] Mccall | Mechanisms of corneal tissue crosslinking in response to treatment with topical riboflavin and long wavelength ultraviolet radiation (UVA)[END_REF] whereas most formaldehyde crosslinks are methylene bridges between lysine and a peptide.

The increase in collagen fiber diameter after crosslinking seems to be due to intrafibrillar collagen crosslinks [START_REF] Wollensak | Collagen fiber diameter in the rabbit cornea after collagen cross-linking by riboflavin/UVA[END_REF] and the absence of edema in the crosslinked anterior stroma in hydration studies due to interfibrillar collagen bonds. [START_REF] Wollensak | Hydration behavior of porcine cornea crosslinked with riboflavin and ultraviolet A[END_REF] Possibly, the interlamellar distance and the different orientations of the fibril lamellae prevent the creation of interlamellar collagen bonds. Maybe, also in untreated physiological corneas no significant interlamellar collagen bonds are present; only interlacing lamellae and proteoglycans allowing a certain degree of interlamellar sliding movement.

On the other hand, it cannot be excluded that in vivo additional lamellar interlaces are induced by crosslinking during the postoperative corneal remodeling process which could not be investigated in the present in vitro model and therefore is a methodical limitation of the present study. Concurrently, new needle-shaped hyperreflective bands or bridges have been observed using confocal microscopy in crosslinked human corneas which might be new interlamellar connections. [START_REF] Mazzotta | Corneal healing after riboflavin ultraviolet-A collagen cross-linking determined by confocal laser scanning microscopy in vivo: Early and late modifications[END_REF] In contrast to Smolek´s studies on interlamellar cohesive force [START_REF] Smolek | Interlamellar adhesive strength in human eyebank corneas[END_REF] and similar to the studies on the LASIK flap adhesion [START_REF] Schmack | Cohesive tensile strength of human LASIK wounds with histologic, ultrastructural, and clinical correlations[END_REF] we chose a tearing plane in the anterior stroma at about 200 µm depth because the crosslinking effect is mainly located in the anterior 350 µm of the stroma as has been nicely demonstrated by studies on the hydration pattern, [START_REF] Wollensak | Hydration behavior of porcine cornea crosslinked with riboflavin and ultraviolet A[END_REF] keratocyte apoptosis, 2 collagen fiber diameter [START_REF] Wollensak | Collagen fiber diameter in the rabbit cornea after collagen cross-linking by riboflavin/UVA[END_REF] , and biomechanical stiffening effect. [START_REF] Kohlhaas | Biomechanical evidence of the distribution of cross-links in corneas treated with riboflavin and ultraviolet A light[END_REF] The anterior localization of the tearing plane might also explain the absence of the reduction in cohesive force in the edematous samples because the specific architecture of the anterior stroma prevents major swelling. [START_REF] Wollensak | Hydration behavior of porcine cornea crosslinked with riboflavin and ultraviolet A[END_REF][START_REF] Meek | Changes in collagen orientation and distribution in keratoconus corneas[END_REF] Amylase is known to digest selectively proteoglycans in connective tissue matrices. [START_REF] Quintarelli | The effects of alpha amylase on collagen-proteoglycans and collagen-glycoprotein complexes in connective tissue matrices[END_REF] Accordingly, the amylase treated samples showed a significant reduction in cohesive force by about 31.5% due to the digestion of the proteoglycan bridges whereas the collagen fibril interlaces were still intact as demonstrated by the preserved spike pattern of the tearing curve (fig 2B) and the torn lamellae on histology (fig 3C). The loss of the proteoglycan bridges also explains the wavy appearance of the crimping collagen fibrils after amylase treatment.

It is an interesting question if it is possible to draw conclusions from this study regarding the pathogenesis of keratoconus. Interfibrillar and interlamellar slippage of corneal layers leading to the ultimate destruction of the lamellar configuration of the stroma [START_REF] Radner | Altered organization of collagen in the apex of keratoconus corneas[END_REF] has been proposed as a pathogenetic hypothesis. [START_REF] Mcmonnies | Mechanisms of rubbing-related corneal trauma in keratoconus[END_REF][START_REF] Dawson | Corneal ectasia after excimer laser keratorefractive surgery: Histopathology, ultrastructure, and pathophysiology[END_REF] Similarly, in post-LASIK and post-PRK ectasia a reduction in lamellar number possibly due to interlamellar slippage and lamellar thinning due to interfibrillar slippage has been described ("interfiber fracture"). [START_REF] Dawson | Corneal ectasia after excimer laser keratorefractive surgery: Histopathology, ultrastructure, and pathophysiology[END_REF] The present study has shown that there is probably no direct effect of crosslinking on interlamellar slippage. Given the apparent efficiency of crosslinking in the treatment of progressive keratoconus it might be causiously concluded that the interfibrillar slippage effect is more important in the pathogenesis of keratoconus or post-LASIK ectasia or that the stabilization of the interfibrillar collagen connections also reduces interlamellar slippage to a significant extent. In addition to collagen crosslink changes enzymatic proteoglycan degradations might play a role as shown by the reduction in cohesive force in the amylase treated group, and crosslinking of PG´s might increase their resistance to enzymatic degradation.

Our results can nicely explain the absence of major changes after corneal crosslinking treatment in applanation tonometry readings [START_REF] Wollensak | Riboflavin/ultraviolet-A-induced collagen crosslinking for the treatment of keratoconus[END_REF] and ocular response analyzer (ORA) [START_REF] Goldich | Can we measure corneal biomechanical changes after collagen cross-linking in eyes with keratoconus?-A pilot study[END_REF] measurements including corneal hysteresis (CH) or corneal resistance factor (CRF), because concentric anterior pressure is applied by these indentation methods causing interlamellar sliding movement [START_REF] Elsheikh | Numerical study of the effect of corneal layered structure on ocular biomechanics[END_REF] , which is not influenced significantly by crosslinking as shown by the present study.

In conclusion, our study has found that there is no significant effect of crosslinking on interlamellar cohesion which is upheld mainly by interlacing collagen lamellae and the proteoglycans. Αmylase-treated samples with digestion of proteoglycans had a reduction in interlamellar cohesive force by 31.5% corroborating the significant role of the proteoglycans in interlamellar cohesion. Our results explain the absence of significant changes in tonometry and ORA measurements after corneal crosslinking and might help to further elucidate the pathogenesis of progressive keratoconus and post-LASIK ectasia.

FIGURES

Figure 1

 1 Figure 1 Cohesive force measurements of (A) riboflavin/UVA crosslinked and (B) α-amylase treated anterior porcine corneal flaps being torn apart at 200 µm depth by the clamps of a biomaterial testing machine. Note the yellowish tint of the crosslinked samples due to the photosensitizer riboflavin and the increased transparency of the αamylase treated sample.

Figure 2

 2 Figure 2 Representative load-extension profiles of (A) riboflavin/UVA crosslinked and (B) αamylase treated specimens. Note the characteristic spikes corresponding to interlamellar tears and the significantly reduced tearing load in the α-amylase treated specimens.

Figure 3

 3 Figure 3 Histologic section of the cleaving plane in A control (PAS stain, x 400), B crosslinked, and C amylase-treated samples (H&E, x400). In all groups, torn lamellae could be seen. The tissue in the crosslinked group (B) looked more compact than the control corneas (A). The tissue in the α-amylase-treated sample (C) was much less tight, the collagen fibrils appeared wavy and the cell nuclei less basophilic.