Amniotic Membrane Transplantation for Persistent Epithelial Defects and Ulceration due to Pseudomonas Keratitis in a Rabbit Model

Abstract Purpose The use of amniotic membrane has been suggested in the treatment of infectious keratitis for its intrinsic anti-infective properties probably mediated by its anti-inflammatory effects. The aim of this study was to investigate the effect of amniotic membrane transplantation (AMT) along with ciprofloxacin to cure the primary stages of Pseudomonas keratitis. Methods In total, 28 rabbits were selected and divided in four groups as follows: group 1 as control, group 2 with amniotic membrane, group 3 with ciprofloxacin, and group 4 with amniotic membrane combined with ciprofloxacin. About 0.05 cc suspension of Pseudomonas aeruginosa, 27853 ATCC was injected into corneal stroma. Results The results showed groups of AMT, AMT + ciprofloxacin, and ciprofloxacin had 0% perforation while the control group had 85.6%. Average infiltration of 5.5 mm was observed in ciprofloxacin group, 5 mm in AMT + ciprofloxacin group, 24 mm in AMT group, and finally 23.75 mm for control. Amniotic membrane showed to be effective in prevention of cornea perforation as well as remission of Pseudomonas keratitis. There was no significant difference between ciprofloxacin groups in comparison with ciprofloxacin + AMT group. However, regarding the anti-inflammatory effect, the process of improvement of inflammation in ciprofloxacin + AMT group was faster. Conclusion Transplantation of amniotic membrane in the primary stages of Pseudomonas keratitis treatment remarkably prevents the disease and it can be used to control its process.


INTRODUCTION
Human amniotic membrane (AM) forms the inner wall of the membranous sac that surrounds and protects the embryo during gestation. It consists of a single layer of ectodermally derived columnar epithelial cells attached to a basement membrane with an underlying layer of mesenchyme. [1] Amniotic membrane transplantation (AMT) is widely used in various ocular surface diseases such as neurotrophic keratitis and persistent epithelial defects, [2,3] band keratopathy, [4] bullous keratopathy, [5,6] after excimer laser photorefractive keratectomy, [7,8] after the excision of a conjunctival mass, [9,10] pterygium, [11,12] ocular surface reconstruction in symblepharon, [13,14] acute chemical injury, [15,16] and a chronic limbal deficiency. [17,18] When used as a graft (epithelial side up), AM is expected to become incorporated in the recipient tissue. If it is used as a patch (epithelial side down), it works as a biological bandage affording a cover for a limited duration or a combination of these. The use of AM has been also suggested in the treatment of infectious keratitis because of its intrinsic anti-infective properties probably mediated by its anti-inflammatory effects and because AM may act as a long-term drug delivery system. [19][20][21] The antimicrobial effects of AM have been demonstrated against several species such as Escherichia coli, Group A Streptococci, Pseudomonas aeruginosa, and Staphylococcus aureus. [22,23] AM graft for epithelial reformation has been employed in order to eradicate the P. aeruginosa infection of keratitis and have exhibited desirable outcomes. [24] The aim of the current study was to investigate the effect of AMT along with ciprofloxacin to cure the primary stages of Pseudomonas keratitis. In total, 28 male rabbits with an average weight of 1.5-2 kg were selected. The AM was prepared according to Song and Kim method. [25] Human placenta was obtained after an elective caesarean section in a woman who was seronegative for human immunodeficiency virus, hepatitis B, C, and syphilis. Under a lamellar flow hood, the placenta was first washed free of blood clots with sterile saline. The inner AM was separated from the rest of the chorine by blunt dissection and flattened onto a nitrocellulose membrane. [26] The membrane with the filter was then washed three times with phosphate buffered saline (PBS) containing 50 μg/ml penicillin, 50 μg/ml streptomycin, and 2.5 μg/ml amphotericin B and put in M199 culture for 24 hr with antibiotic solution including streptomycin, cloxacillin, ceftriaxone, and amphotericin B, and finally packed in pieces of 1.5×1.5 in three of sterilized nylon and stored in -80ºC in freezer. Twenty-eight rabbits were divided into four groups as follows: group 1 as control group [ Figure 1A], group 2 as AM [ Figure 1-B], group 3 as ciprofloxacin [ Figure 1C], and group 4 as AM combined with ciprofloxacin [ Figure 1D].
The rabbits were anesthetized with intramuscular injection of ketamine hydrochloride (30 mg/kg) and xylazine hydrochloride (5 mg/kg) and then a drop of tetracaine HCL 0.5% was applied to the rabbits' right eye. About 0.05 cc suspension of P. aeruginosa 27853 ATCC was injected into corneal stroma with a sterile 30 G needle connected to a micro-syringe, using an operating microscope. The experimental keratitis was allowed to proceed untreated for 20 hr. There This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms. was no interference in the control group. In groups 3 and 4, the AM in pieces of 1.5×1.5 cm transplanted to the entire corneal surface by eight interrupted 10.0 nylon sutures. On the first day, ciprofloxacin drop was injected to groups 2 and 4 every 30 min.
On the second day to seventh day every 2 hr, the results were registered in aspect of perforation in cornea and the amount of infiltration by the use of image J 1 software.

RESULTS
Results were registered as clinical reports on the first, third, and seventh day [ Table 1]. During the first 20 hr, after injecting P. aeruginosa, a white opacity appeared in all rabbits. Rabbits had corneal ulcers and on the second day, the conjunctiva was markedly hyperemic in four groups. At the end of the first week, Hypopyon formation was noticed in five eyes in the AMT group. Corneal perforation was noticed in four cases in the control group but in no case in other groups.
The results showed that the cornea had infiltration in which central part, an area with the size of 6 mm had descemeto cell and progress toward causing perforation in cornea control group (A: Control group). On the other hand, AMT group conjunctiva inflammation showed to be less than control group and in cornea examination the amount of infiltration showed to be 24 mm (B: AMT group). For ciprofloxacin group, ciprofloxacin sediment was clearly visible (C: ciprofloxacin) and finally in the group with ciprofloxacin + AMT as well as the ciprofloxacin group the decrease of opacity cornea was quite visible (D: AMT + ciprofloxacin).
The clinical results of four groups in examination of Pseudomonas keratitis are also shown in Table  2. These results showed that AMT + ciprofloxacin group had 0% perforation and the control group had 85.7%. Average infiltrations were 5 mm in AMT + ciprofloxacin groups and 23.75 mm in control.
The result showed that the AMT, ciprofloxacin, and AMT + ciprofloxacin were effective on perforation and infiltration on all groups compared to the control group.
In addition, there was no significant difference between ciprofloxacin group and ciprofloxacin combination with AM. In the same way, there was no significant difference between membrane amniotic group and ciprofloxacin compared with combined group of ciprofloxacin with membrane amniotic.

DISCUSSION
Few studies have investigated the effect of AMT in the surgical treatment of severe infectious keratitis with corneal ulceration or perforation. [19,23,27] The main advantages of AMT in the treatment of bacterial keratitis that we observed are the epithelial bandage properties, which allowed early use of topical steroids; the anti-inflammatory and anti-scarring effects of the AM, the promotion of epithelialization, and the possible benefits of a direct antimicrobial role of the AM. [28] These studies found AMT to be effective in treating neurotrophic ulcer, inflammatory corneal ulcer, bullous keratopathy, inflammatory or noninflammatory scleral ulcer and as an adjuvant treatment of pterygium excision. The basement membrane of an AM promotes epithelial growth and differentiation, reinforces the adhesion of basal epithelial cells and prevents epithelial apoptosis. The stroma matrix suppresses TGFb signaling, proliferation, and myofibroblastic differentiation of normal human corneal and limbal fibroblasts and thus, inhibiting the unwanted production of extracellular matrix and scarring. [6] These properties have made the AM an ideal reconstructive substrate for repairing persistent epithelial defects and corneal ulcers, [2] conjunctival defect, [9,10] chemical or thermal injury, [15,29] and limbal cell deficiency. [16,17] The application of AM in the treatment of corneal perforation and scleromalacia has been also reported. [30,31] The human AM possesses anti-inflammatory, antifibrotic, and antiangiogenic properties, and these attributes make it ideal for ocular surface reconstruction procedures. [32,33] In addition, the AM also has antimicrobial properties due partly to its anti-inflammatory effects, and also due to secretion of elfin and secretory leucocyte proteinase inhibitor, both of which have antimicrobial actions and act as components of the innate immune system. [34,35] It also contains cystatin E, an analogue of cysteine proteinase inhibitors, which has complementary antiviral properties. [36] Furthermore, AM transplantation (AMT) is reserved for cases of postinfectious ulcers after an appropriate period of anti-infective treatment when clinical signs are improving. [37] This is because the anti-infective properties of AM are nonspecific and not considered to be potent enough to be effective in acute infective keratitis. This is the reason behind the concept  Average infiltration 5 mm and without epithelial defect and scar *Findings in the first 20 hours only the first day for all groups **AMT, amniotic membrane transplantation of fortifying AM with antimicrobial drugs to make it a viable therapeutic modality in the setting of active infections of the cornea. Antibioticimpregnated medical devices such as catheters, bone and cardiac implants have been in use for over a decade. [22,38] Various studies have shown the potential of such an approach, for example, in vascular surgery and arthroplasty, where they appear effective in reducing the risk of bloodstream infections or in limiting deep wound infections. [39,40] AM is effective in remission of Pseudomonas keratitis and prevention of Table 2. Clinical outcomes of perforation and infiltration in all four groups of Pseudomonas keratitis

AMT* AMT + ciprofloxacin ciprofloxacin Control
Perforation (0%) 0 (0%) 0 (0%) 0 (85.7%) 6 Infiltration 24 mm 5 mm 5.5 mm 23.75 mm Significant difference compared to control group (P-value) P < 0.5 P < 0.5 P < 0.5 -*AMT, amniotic membrane transplantation cornea perforation and controlling as well as anti-Pseudomonas effects. There was no difference between ciprofloxacin group in comparison with ciprofloxacin + AMT group. However, regarding anti-inflammatory effects, the process of improvement of inflammation in ciprofloxacin + AMT group was faster. During this research, we came to conclusion that transplantation of AM in the primary stages of Pseudomonas keratitis treatment remarkably prevents the disease and it can be used to control its process.