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The PALM Technique: histological findings of masked phototherapeutic keratectomy on rabbit corneas
© Katsanevaki et al; licensee BioMed Central Ltd. 2003
Received: 17 December 2002
Accepted: 19 February 2003
Published: 19 February 2003
To compare the corneal healing response between conventional and phototherapeutic keratectomy through a masking agent, in rabbit corneas.
24 adult rabbits underwent phototherapeutic keratectomy. Animals were divided in two groups: 12 received photoablation through a masking agent (PALM gel) and the remaining 12 received conventional phototherapeutic keratectomy of equal depth and served as control. Light and transmission electron microscopy was performed in specimens of both groups obtained: immediately after, four hours, one week, one, three and six months after treatment.
Reepitheliazation was complete within five days in all eyes. Light and transmission electron microscopy did not reveal any differences of the healing process in the experimental eyes compared to the controls.
Photoablation through the PALM technique did not result any evident alterations of the reepithelisation and stromal healing process.
The excimer laser ablation is currently widely used primarily to correct refractive errors by altering the anterior curvature of the cornea. Due to its potential for accurate ablation of anterior stromal corneal lesions its initial clinical utilization was removal of diseased corneal tissue in a procedure called photo therapeutic keratectomy (PTK). In 1985 Serdareviz et al proposed the first therapeutic use of the excimer laser to treat an experimental Candida infectious keratitis . Despite the lack of controlled clinical trials comparing PTK with other treatment modalities such as superficial keratectomy or lamellar keratoplasty, the US Food and Drug Administration (FDA) identified PTK as an alternative therapeutic approach for the treatment of superficial corneal and epithelial membrane dystrophies, irregular corneal surfaces and corneal scars and opacities.
The major inherent obstacle of PTK for the management of corneal surface irregularities is that through photoablation these irregularities are reproduced deeper within the stroma.
Masking techniques refer to photorefractive procedures utilizing various masking means or so called modulators to protect flatter corneal areas while steeper areas are excised with the excimer laser.
Kornmehl et al  have shown that an ideal masking agent should have moderate viscosity (between that of saline and 1% carboxymethylcellulose) and concluded that very viscous fluids would not cover irregular surfaces uniformly whereas fluids of inadequate viscosity would run off quickly exposing both peaks and valleys thus resulting in irregular surfaces after ablation.
Numerous previous investigators have used different masking agents as methylcellulose [3–6] and sodium hyaluronate [5, 7, 8] at various concentrations or performed transepithelial (so that the epithelium acts as a natural masking agent) treatments [5, 6, 9, 10] and reported the beneficial effect of masked PTK. Fasano et al  reported that the ideal masking agent should have the same ablation rate to that of the cornea, be biocompatible and adhere well to the cornea.
These properties have been met with the use of collagen solutions as modulators. Their applicability as masking agents for corneal photoablation was examined in experimental models of animal and cadaver eyes (Scott et al: Collagen modulator fluids for use during photoablation keratectomy: ARVO 1992, Margaritis et al. Properties of a new two component gel material as an aid in PRK corneal remodeling: ARVO 1994) [11–13] as well as in limited clinical trials of partially sighted eyes (Pallikaris et al: Photoablated lenticular modulator PALM technique: A report of ten cases: ARVO 1998) [14, 15].
Photo-Ablatable Lenticular Modulator (PALM) technique refers to the use of a modified collagen gel solution for the photorefractive correction of corneal surface irregularities . The PALM gel similarly to other collagen modulators [12, 13, 15] is thermo reversible. The gel is in liquid state when heated to solidify to a firm gel as its temperature lowers. Its use for masking purposes requires its application onto the corneal stoma at a temperature of 49°C where it can be molded to form a stable lenticule that serves as the final masking agent.
At temperatures of 40° to 53°C, the collagen individual fibril bonds are broken and the collagen denatures to its parent gelatinous form. When the tissue temperature drops to below 40°C, these bonds can reunite with possible rearrangement of the matrix but without any cellular damage . Under this consideration the thermal contact of the 49°C preheated PALM gel onto the bare corneal stroma for the in situ molding of the lenticule, would not be expected to induce any irreversible changes of the corneal collagen chains. However its sequential use as masking agent for PTK could affect the laser-tissue interaction and probably the corneal healing response to irradiation.
In the current study we examined the possible implication of the PALM gel when used as described above, to the corneal wound healing on rabbit corneas.
We examined the reepithelization process of 12 rabbit eyes after PTK through PALM gel lenticules formed in situ. We used a moving slit delivery system in a 5 mm ablation zone confined by means of a metal iris diaphragm. An equal group of rabbit eyes received conventional PTK of the same depth and treatment zone and served as control.
The PALM gel as a masking agent
The gel used, as modulator is a soluble mixture of purified type A gelatin from porcine skin and a polysaccharide, type III carrageenan K in nanopure water . Both components are commercially available for laboratory use (Sigma Chemical Co, St Louis, Mo).
Being thermoreversible, the gel can be stored at room temperature in solid form. When heated to 49°C it liquefies to a high viscosity fluid that solidifies within minutes when exposed to room temperature. When still in liquid form the gel can be molded by means of a rigid contact lens to form a stable lenticule that its upper surface will reproduce the inner surface of the molding lens.
A total of 24 adult New Zealand white rabbits (average weight 4–5 Kg) were anaesthetized by intramuscular injection of ketamine hydrochloride (40 mg/kg) and xylazine hydrochloride (7 mg/kg) mixture. All experimental procedures were carried out in accordance with the Guiding Principles in the Care and Use of Animals (DHEW Publication, NIH 80–23) and were approved from the University's of Crete ethics committee.
One drop of 0.5% proparacaine hydrochloride (Alcon, Ft Worth, TX) was instilled into each eye and the corneas were deepithelized with the use of a rotating brush. We treated one eye of each animal. 12 were treated with PTK through PALM gel (experimental eyes) and the remaining 12 received conventional treatment and served as controls. The laser system used in all eyes was the Aesculap Meditec Mel 60 (Karl-Zeiss, Jena, Germany), with laser fluence of 180 mJ/cm2 pulse rate at 20 Hz, in PTK mode.
Experimental eyes received PTK through PALM gel lenticules formed in situ onto deepithelized corneas as described above. In order to shorten the time of the surgical procedures we obtained the thinner possible lenticules using as molds the "best fitting" contact lenses for each operative eye. Molds used in the current study had inner radii of curvature ranging from 8.25 to 9 mm. After the removal of the mold, an iris diaphragm having an inner aperture of 5 mm was centrated over the pupil onto the upper surface of the lenticule to define the ablation zone. The gel lenticules were ablated through with irradiation in PTK mode. As soon as fluorescence faded each experimental eye received an additional 60-micron deep PTK.
The control eyes were submitted to a phototherapeutic keratectomy of 5 mm diameter ablation zone (confined by the same iris diaphragm), depth of 60 μm, on deepithelised cornea without the use of any masking agent.
Postoperative treatment of all treated eyes included daily application of combined tobramycin (0.3%), dexamethasone (0.1%) ointment (Alcon Ft Worth, TX) until reepithelisation was complete. No topical steroids were used subsequently in the study.
The reepithelization process was examined daily with staining of the epithelial defect area using 2% fluorescein dye and a hand held slit-lamp. Corneal clarity was recorded using a predetermined 5-scale grading of haze .
Two animals of each group (experimental and control) were sacrificed with an overdose of penthobarbital sodium through the marginal ear vein at each of the following postoperative intervals: immediately after treatment, 4 hours after treatment, 1 week and at one, three and six postoperative months.
Immediately after enucleation the eyes were fixed using 2.5% gludaraldehyde in 0.1 mol/l cacodylate buffer (pH 7.4). After short prefixation the corneas were excised and placed in the same fresh fixative overnight. The tissue samples were post fixated in 2% osmium tetroxide in 0.1 M cacodylate buffer (Ph 7.4) for 2 hours at 4°C, dehydrated in a series of alcohol and propylene oxide and embedded in epoxy resin.
1-μm sections were stained with trichrome stain and were processed for light microscopic examination whereas ultra thin sections were stained with uranyl acetate and lead citrate and were examined with transmit ion electron microscopy.
Reepithelisation was complete (no epithelial defect was observed with fluorescein staining) within five days in all treated eyes (within 3 days in two control and one experimental eye, 4 days in three experimental and one control eye and on the fifth postoperative day to the rest of the treated eyes). No persistent defects or recurrent erosions were recorded.
Corneal haze was evident from the first week in all treated eyes ranging from grade 1 to grade 2.
Corneal haze reached its maximum on the first postoperative month ranging from grade 2 to 3 (grade 3 in one experimental eye).
All treated eyes were clear or had minimal haze by the third month (grade 1 in one experimental and two control eyes), which was resolved in all eyes by the sixth postoperative month.
Six months postoperatively, we didn't find any significant morphological difference compared to the three-month interval obtained specimens, neither in the experimental nor in the control group.
Application of a heated agent on denuded cornea or even ablation through a chemical agent could affect laser-tissue interaction, corneal reepithelization or stromal healing response after photo ablation.
We used rabbits to examine the probable effect of the PALM gel if used as a masking agent for PTK since stromal healing is very similar in rabbits and humans .
The healing course proved similar in the experimental and control eyes. No specimens obtained at any postoperative interval had any sign of necrosis or macrophages infiltration.
The simultaneous reepithelization as well as the similar histological findings between experimental specimens and controls is indicative of negligible gel's impact on corneal healing response. The newly synthesized extracellular matrix presented as foam layer at the early specimens is a common finding reported after photo refractive keratectomy or even simple epithelial scrape injury in a number of previous studies [21–24] and is supposed to manifest the healing response of rabbit corneas [21, 22, 24].
The epithelial hyperplasia observed on the seventh postoperative day was more intense than that reported in the literature [21, 24, 25]. The possibility of the gel's implication to this response is minimized since it was also observed in the control eyes. We assume that this finding is related to the ablation profile of our treatments (a rather deep keratectomy with sharp edges), which would justify an intense epithelial healing response .
The extrusion of electron dense fibro-granular material into the descemet's membrane is also a common finding observed in a number of previous studies [20, 21, 27–29] and has been attributed to acoustic shock waves by irradiation  or the distraction of the epithelial integrity after trauma . The presence of this finding in only one of our specimens may be related to the homogenous and smooth removal of the corneal epithelium with the rotating brush.
In conclusion the use of the PALM gel did not seem to seriously affect the healing process after PTK on rabbit corneas as compared to controls. A larger number of animals would allow for statistical analysis of pathologic findings such as activated fibroblasts by depth and thickness of newly formed extracellular matrix between experimental and control eyes. Furthermore, tenascin and fibronectin staining of the specimens would allow for better understanding of the corneal healing process.
The use of new generation laser systems that offer phototherapeutic ablation mode with transition zones are expected to minimize epithelial hyperplasia and corneal stroma healing response. The major remaining drawback of the PALM as well as of similar PTK techniques  in order to obtain an optimal refractive result is the accurate centration and placement of the molding lens for the proper formation of the lenticule before irradiation.
The study was supported by a research grant (August 1999) from the LASIK Institute (LASIK Institute, 750 Washington Street, box 450, Boston Massachusetts 02111, USA).
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