This is a preliminary study to assess the hen as an experimental animal model to train surgery of ICRS implantation and to future wound healing studies. In this study, we have performed the learning curve and described the clinical and optical outcomes of good follow up corneas after ICRS implantation. Many previous studies in humans have focused on the refractive, clinical, and optical events after ICRS implantation. To study the wound healing process, authors have used confocal microscopy, [17
] anterior segment optical coherence tomography, [18
] and scanning electron microscopy to analyze the explanted segments [12
]. However these techniques cannot detect the morphology of the cells implicated at each time of follow-up to determine if they are keratocytes, fibroblasts, and/or myofibroblasts. Some histological studies of human corneas that were removed for keratoplasty due to ICRS complications or refractive failure have been reported. However, these studies do not established a temporal sequence of events, and the number of eyes analyzed was small [27
]. For these reasons we thought it was necessary to develop an experimental animal model to study clinical, optical, and histological events together throughout the wound healing process after Ferrara ICRS implantation in good and complicated eyes.
The rabbit was used as an experimental model for wound healing after ICRS implantation, [7
] but there are differences between rabbits and humans in corneal anatomy and biomechanical properties, as well as in the healing process itself. Enucleated pig eyes have also been commonly used for surgical practice, but the difficulty in manipulating the living animal, along with the cost and the anatomical differences from human corneas also make them unsuitable as a model for training and long-term studies [29
]. All of these reasons led us to propose the chicken as an experimental animal model for developing the ICRS surgical implantation technique with the learning curve and studying the post-surgical clinical and optical responses. The chicken is ethically acceptable, cheap, easy to handle, and the corneal anatomy with respect to layers and the distribution and wound healing response are similar to that of humans [20
]. Nevertheless, our model has some drawbacks. First, the corneal dimensions are not exactly the same as those in humans. For this reason we had to create and adapt the surgical instruments for our animal model. Secondly, there are differences in the collagen arrangement [32
] and accommodative mechanism, [24
] although we believe that these would not interfere with the clinical and wound healing process.
The Ferrara ICRS chosen for this study all had the same size, arc, and thickness and were implanted at the same location in each cornea (inferotempotal quadrant) to make the measurements comparable between eyes. This location also coincide with the place where the ectasia is in the majority of the patients and where the majority of the segments are implanted [4
]. We selected the thinnest segment recommended for humans to follow the Ferrara rule of segment implantation: “The thickness of the implanted segment should not be more than 50% of the corneal thickness in the ring track and the incision depth should be preferably set at 80% of the corneal thickness” [14
]. The segment thickness was 150 µm, more than 50% of the hen central corneal thickness, but we estimated that it was less than 50% of the peripheral cornea thickness. We had difficulties in measuring the peripheral corneal thickness with ultrasound pachymetry due to the narrow anterior chamber of the hen. However, the measurements obtained were all more than 300 µm. Moreover, we implanted the segment at 71.2% of the corneal thickness (243.7 ± 24.0 µm) to avoid extrusions.
The incidence of complications in humans has been decreasing with more experience and especially with the use of the femtosecond laser [33
]. Too deep or too superficial stromal tunnels, with too deep or too superficial implantation of the segments, segments implanted closely to the incisions and aggresive, powerful or in bad direction maneuvers are related to high incidence of complications, especially in first surgeries. It is important to know how to insert instruments and the segments along the deep stroma where the resistence is lower.In most recent studies, no complications were reported or were reported only in small numbers such as 3.8% in the latest Ferrara publication [5
]. The incidence of complications in our study, 16% is similar to the first studies in humans [10
] and could be attributed, as in those studies, to the learning curve and particular34ly in our experiments, to the adaptation of the surgery to our animal model. We also have the same type of complications as those described in humans. Femtosecond laser to segment implantation is not avaliable in the majority of the centers, for this reason the use of an experimental animal model could be suitable to reduce the learning curve and the incidence of the first complications with manual technique.
Lamellar channel deposits have been reported as a complication in some human trials [4
]. Deposits are different from stromal haze. Stromal haze is described as a condition of mild anterior stroma opacification that occurs as a part of the normal wound healing response. Stromal haze has frequently been reported in the incision and surrounding the ICRS, and should be distinguished from intrastromal deposits that are only described around the segment.
Stromal deposits were reported in some studies from 7 days after surgery [22
]. We likewise found deposits in our study at that time point, but the incidence was higher (28.4%). However, in other studies the early deposits appeared at a mean of 1 month after surgery, [35
] and the authors stated that earlier deposits could be mistaken for haze around the channel. We disagree with this latter assessment because we were able to perceive little white speck-like deposits near the inner curvature that were different from the diffuse haze around the segment. The incidence of lamellar channel deposits in all studies increased over time. The incidence was related to the segment thickness and was inversely related to size of the channel where the segments were implanted [33
]. The incidence of deposits varies in different studies from 12.5% in femtosecond implanted segment studies to the majority of the eyes in first studies [7
]. However, in our animal model, 100% of the eyes presented intrastromal deposits at 3 months, a period that differed from other studies. Our animal model presented deposits located principally along the inner curvature of the segment at 1 and 2 months, which agrees with most long-term follow-up studies. However, we found that at 4 and 5 months, the deposits were mainly present along the external curvature of the segment and beneath it, locations that were not reported in other studies. This might be in part explained by differences in the morphology of the segments. In our case, using the Ferrara ICRS, the shape was triangular, but most wound healing studies after ICRS implantation were undertaken with hexagonal INTACS. Furthermore, these differences might be related to the involvement of more cells and the production of more new matrix during the wound healing process in the hen, which we believe might enable better understanding of the mechanism involved.
The severity of these deposits increased with time in all published studies [7
]. In rabbits, two types of deposits were described. One had an oil drop appearance and the other, which appeared later, had a crystalline appearance [7
]. In humans, different classifications have been described [17
]. The most widely used classification is that by Ruckhofer et al. who designated 4 grades of deposits based on a qualitative description [22
]. In our animal model, the evolution of deposits was slightly different. The deposits also increased over time from small and white in color at 1 month, to more confluent, larger and more yellow ones at 3 months. Later, they changed again to smaller, less confluent and white in color from 4 to 6 months, particularly those located under the segment. This could be related to the proliferation and differentiation of cells around the segment (date no shown in this manuscript).
Many studies in the literature have tried to describe the composition of these deposits, but it remains unknown. It is widely known that they are deposited in the empty space between the segment and the stroma where it is implanted. Most of the studies found new matrix synthesis, collagen disruption in addition to keratocytes, fibroblasts, or myofibroblasts [17
]. We agree with these findings without specify the type of cells with our preliminary analysis of hematoxilin eosin stained preparations. However, more studies about wound healing should be performed to understand the mechanism of this response specially in experimental animal models like the hen.
The refractive effects of ICRS have been widely studied in humans [5
] and all have shown statistically significant flattening of the cornea [4
]. This agrees with the expected change of curvature considering the mechanism of action of the ring segments described by Barraquer and Blavatslaya [4
]. ICRS implantation can reduce sphere from 0.43 to 5 D and cylinder measurements from 0.75 to 2.88 D. The mean keratometric change with ICRS varies depending on the author and ICRS type, with values ranging from 2.14 to 9.60 D after Ferrara Ring implantation.4 For the hens in our study, we found an increase in the hyperopic refraction of 2.85 D, which remained stable during the 6 months of follow-up. These results agree well with those described in humans. Moreover, central cornea is not affected.
In summary, with hen as an experimental animal model, a beginner surgeon could experiment and discover with his own hands the specifics tips of intrastromal corneal ring segment surgery; how to direct instruments and segments along the specific depth without being too superficial or too deep, how to rotate the segment to the semicircular channell, how to know the specific strength needed to drive the instruments or segments in order to minimize the number of complications and have a more successful surgery.
In this study, an experimental animal model of ICRS implantation has been assessed with its learning curve and characterized by clinical, biophysical, and preliminary biological parameters. The majority of the optical and clinical events described in hens correlate with those described in humans. The differences regarding incidence, location, and severity of deposits should be studied to better understand the wound healing mechanism.