FormalPara Key Summary Points

The lack of a fresh donor and the disadvantages of conventional keratoplasty affect the treatment of corneal perforation and therefore require us to design a modified surgical procedure.

In this prospective interventional case series, we demonstrate that double lamellar keratoplasty is an effective surgery for off-center and small corneal perforations.

All treated eyes remained transparent, visual acuity was improved, and no immune rejection or recurrence was detected during the follow-up period.

Our modified surgery could reduce the risk of postoperative adverse events and achieve satisfactory outcomes.

Digital Features

This article is published with digital features, including videos, to facilitate understanding of the article. To view digital features for this article go to https://doi.org/10.6084/m9.figshare.21647177.

Introduction

Corneal perforation is considered a severe and final phase of corneal ulcers secondary to various etiologies [1]. The integrity of the globe structure is destroyed, which leads to profound vision loss and serious complications, such as secondary glaucoma, endophthalmitis, and globe tissue prolapse [2]. Emergency and appropriate treatments are required to restore the integrity of ocular anatomy and prevent complications. Medical therapies, such as antimicrobials, aqueous suppressants, anti-collagenases, and anti-inflammatory agents, are crucial during the initial administration. However, in some cases with long-standing perforations, the perforated area is plugged with the iris and inhibits re-epithelialization [3, 4]. Hence, a variety of surgical methods have been introduced to intervene in this process, including amniotic membrane transplantation, tenon patch graft, conjunctival flap transplantation, tissue adhesives, collagen cross-linking with photoactivated riboflavin, and corneal transplantation with autologous or exogenous grafts [5,6,7,8,9,10].

Treatment selection mainly depends on the etiology and characteristics of the perforation, such as size, location, and underlying primary corneal disease status [11]. Currently, one of the most common treatment options is penetrating keratoplasty (PK), which allows for complete removal of lesions and requires viable donors to replace full-thickness corneas [12]. However, PK is more likely to induce postoperative complications, such as allograft rejection, epitheliopathy, glaucoma, and non-immunological failure [13,14,15]. Moreover, a lack of fresh corneal donors, a requisite for PK, remains a non-negligible issue in many countries, especially in developing countries [16, 17]. Therefore, accumulating studies have focused on lamellar keratoplasty (LK), which is regarded as an accessible way to patch perforations and reinforce the thinned and necrotic stroma. LK is preferred and desirable over PK with the inherent advantage of fewer intraocular complications and lower risk of endothelial rejection [17, 18]. However, due to the coverage of partial corneal thickness, it is prone to double anterior chamber or pseudo-anterior chamber, persistent interface fluid, and interlamellar neovascularization, which results in turbidity and affects corneal transparency [14, 15, 19]. The disadvantages of these traditional surgical methods urged us to introduce a modify surgery.

In the present study, we report a series of patients who underwent double lamellar keratoplasty for corneal perforation. We have reviewed the surgical techniques, anatomical and functional results, and complications of treatment in this group of patients. Clinical effect was assessed to evaluate the efficacy and safety of our novel techniques as an available choice for corneal perforation.

Methods

Research Ethics

Fifteen eyes were selected from 15 consecutive patients who were enrolled in this prospective study. There were nine males (60.00%) and six females (40.00%) with an average age of 50.73 ± 19.89 (range, 9–84) years. All patients were examined and treated in the Department of Ophthalmology, Fujian Medical University Union Hospital, Fuzhou, Fujian, China, from June 2019 to December 2020. The study was conducted in compliance with the tenets of the Declaration of Helsinki and was approved by the Ethics Committee of Fujian Medical University Union Hospital (2019KY132). This study was also preregistered at http://www.chictr.org.cn (study number ChiCTR2100044371). All patients provided detailed written informed consent. Double lamellar keratoplasty was performed by the same experienced surgeon (JZH) in all cases.

Patients

Inclusion criteria were patients with corneal perforation that could not be treated with medical therapy, those who also had iris prolapse and shallow anterior chambers, those whose perforated center was outside the 5 mm diameter of the central cornea, and those whose perforated diameter was within 1 mm. Exclusion criteria were patients with primary collagen vascular disease, which could have caused severe corneal lytic-related lesions, such as rheumatoid arthritis, Sjogren's syndrome, and Wegener's granulomatosis.

Preoperative Examination

Preoperatively, demographic characteristics and medical history were collected, and etiology, size, and location of perforation were also recorded. Diagnosis was based on clinical manifestations and laboratory tests, and all patients received standardized treatment according to the etiology. Every patient underwent a comprehensive ophthalmological evaluation, including best-corrected visual acuity (BCVA), slit-lamp examination, and anterior segment optical coherence tomography (AS-OCT, Casia; Japan).

Surgical Procedure

Briefly, the key steps of the surgical procedure were as follows (Fig. 1): after topical and retrobulbar anesthesia was administered, conjunctival peritomy was performed in the corresponding range of the ulcer. According to the location and size of the perforation, a trephine was used to make an off-center mark, including ulcer lesions, pannus, and relatively healthy lateral corneal tissue, which was slightly larger than the perforation hole in diameter and further prepared for posterior graft. The recipient bed was carefully made along the mark, and the necrotic tissues and pannus around the perforation were removed. After removal of the superficial exudate, the prolapsed iris was returned to the anterior chamber with an iris spatula. Isolation of the posterior graft in the prepared relatively healthy lateral corneal stroma using microscalpel and microforceps. The perforation hole was patched with this approximately 100-um-thick graft using interrupted 10-0 nylon sutures. After the donor corneal graft was taken from glycerol (for resuscitation and rehydration), a matched outline of donor cornea as anterior lamellar graft was prepared using corneal trephine and microscissors, approximately 0.25 mm larger than the recipient bed in diameter. The anterior lamellar graft was fixed to the recipient bed using intermittent 10-0 nylon sutures. Sterilized air was injected into the anterior chamber to separate the synechia and prevent postoperative re-adhesion and interface fluid. Finally, a bandage contact lens was used to promote corneal epithelial repair and relieve pain. Details of the operations are reported in Video 1.

Video 1: The key steps of the surgical procedure (MP4 20450 kb)

Fig. 1
figure 1

Surgical procedures for double lamellar keratoplasty. a Corneal perforation was shown. b Corneal ulcer was removed. c A relatively healthy lamellar graft was separated and matched with the perforated size. d Corneal perforation was placed by the recipient graft. e The anterior lamellar graft from a donor was transplanted

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Postoperative Care and Follow-Up

Postoperatively, all patients received active treatment for their primary disease and a regimen of dexamethasone and tobramycin (except for fungal and viral keratitis) four times per day for 2 weeks, with progressive taper down over the next 3 months. Subsequently, we prescribed fluorometholone and tacrolimus twice per day for 6 months, continued them on a slowly tapering dose once a day, and discontinued them completely at approximately 1 year. The selective suture of the anterior lamellar graft was removed 3 months after the operation and completed at 12 months. If the intraocular pressure (IOP) was high, systemic or topical anti-glaucoma medications were applied.

Postoperative reexaminations were scheduled once every 2 weeks for 1 month and then once a month for 2 months and every 3 months for 1 year. Moreover, BCVA, tonometry, slit-lamp examination, AS-OCT, specular microscopy (Nidek; Japan), and in vivo confocal microscopy (IVCM, Heidelberg; German) were performed and complications recorded during the follow-up period.

Statistical Analysis

Data analysis was performed using SPSS 23.0 software (IBM, New York, USA). All results were expressed as mean ± SD for quantitative variables and as counts and percentages for categorical variables. Paired Student's t-test was used to compare pre- and post-operation with a p-value < 0.05 considered statistically significant.

Results

Patient Information

A total of 15 eyes from 15 subjects with different etiologies that induced corneal perforation that met the criteria mentioned above and underwent double lamellar keratoplasty were included in this study. Clinical characteristics of the patients are summarized in Table 1 and supplementary Table 1.

Table 1 Clinical related characteristics of the patients underwent double lamellar keratoplasty (n = 15)
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The causes of corneal perforation were classified as infectious or non-infectious. In 11 patients, corneal perforation was secondary to infectious keratitis (6 due to bacterial keratitis, 3 due to herpes simplex keratitis, and 2 due to fungal keratitis), 4 cases were secondary to non-infectious diseases (2 due to phlyctenular keratoconjunctivitis, 1 due to Mooren’s ulcer, and 1 due to neuroparalytic keratitis). Mean preoperative visual acuity improved from 1.44 log MAR (SD = 0.413) to 1.06 log MAR (SD = 0.417, p < 0.001, by paired t-test, supplemental Fig. 1) at the final follow-up examination. In short, visual acuity was improved in 14 patients (93.3%), while 1 patient had no sense of light before surgery.

The anterior chambers of all subjects were formed, and all grafts were completely transparent. The mean time of integrated re-epithelization was 5.57 ± 1.56 (range, 3–8) days, and the grafts became transparent without edema in an average of 9.64 ± 2.27 (range, 6–14) days. The average hospitalization duration was 8.71 ± 3.36 (range, 4–17) days. Participants were followed for up to 30 months with a mean follow-up period of 21.14 ± 5.25 months.

Graft and Safety Evaluation

Pre- and postoperative slit-lamp microscopy images were obtained from two patients. Figure 2a, f exhibits the corneal ulcers and perforations of these patients before keratoplasty. The anterior graft is transparent without edema, and the posterior graft can be seen approximately 7 days after the operation as shown in Fig. 2b, g. The whole corneal grafts remained transparent and were nearly similar to the surrounding normal cornea during the follow-up at 3 months (Fig. 2c, h), and all sutures were removed at 6 months (Fig. 2d, i). All donor grafts and recipients matched well, and corneal grafts remained transparent at 12 months (Fig. 2e, j).

Fig. 2
figure 2

Representative slit-lamp microscopy images of pre- and postoperative patients. a, f Corneal ulcers with perforation shown. b, g Seven days after the surgery, double lamellar grafts could be noticed. c, f Three months after the surgery, the whole corneal grafts kept transparency, and all the grafts matched well. d, i Six months after surgery, the sutures of the anterior grafts were removed completely. e, j Twelve months after the surgery, corneal grafts remained transparent

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AS-OCT was used to obtain corneal cross-sectional photographs to evaluate the depth and width of the corneal lesions. Figure 3a reveals the presence of corneal perforation and iridocorneal touch as well as an iris plug in the corneal perforation site. After the surgery, the perforation healed without leakage and was covered with a well-attached graft. In addition, the double lamellar structure and localized corneal edema could be easily identified in the early postoperative period (Fig. 3b–d). With graft edema relieved and corneal epithelium restored, the cornea became transparent in long-term follow-up. AS-OCT demonstrated formation of a smooth posterior surface of the cornea and localized thinning in the perforated area (Fig. 3e, f). Mean corneal thickness in the center of the perforated area was 807.28 ± 227.11 μm, 635.83 ± 254.34 μm, 589.98 ± 234.89 μm, 539.98 ± 214.74 μm, and 501.13 ± 189.33 μm at 7 days, 1 month, 3 months, 6 months, and 12 months after surgery respectively.

Fig. 3
figure 3

Representative anterior segment optical coherence tomography (AS-OCT) images after double lamellar keratoplasty. a The corneal perforation and iris prolapse could be seen preoperatively. b, c Postoperative AS-OCT shows double lamellar structure and localized corneal edema of the same eye at 7 days and 3 months after the operation, respectively. d, e Postoperative AS-OCT demonstrates the formation of a smooth posterior surface of the cornea and localized thinning in the previously perforated area of the same eye at 6 months and 12 months after surgery, respectively

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Non-contact specular microscopy showed that the central area of the corneal perforation lacked endothelial cells (Fig. 4a). Abnormal and disorganized swollen endothelium, such as polymegethism (coefficient of variation of cell size) and pleomorphism, can be detected around the perforation site (Fig. 4b). In addition, the periphery regions of the perforation showed a normal hexagonal monolayer structure (Fig. 4c). Central corneal endothelial cell density was within the normal range after surgery.

Fig. 4
figure 4

Representative specular microscopy images obtained 12 months after double lamellar keratoplasty. a The central area of corneal perforation lacks cell structure. b Around the perforation site, abnormal and disorganized swollen endothelium are seen. c The periphery area of perforation shows a normal hexagonal monolayer structure

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IVCM is an indispensable tool for assessing corneal architecture and physiology at a dynamic cellular level [20]. One patient who underwent IVCM 24 months after keratoplasty showed a graft had already been epithelialized, with compact organized cells in a normal epithelial cell layer (Fig. 5a). Many thin linear nerve fibers were observed at the level of the Bowman layer (Fig. 5b). However, there were some dendritic cells accompanied by sub-basal plexus nerve and slight subepithelial stromal haze with highly reflective microdots (Fig. 5c). In the mid-stroma, normal but slightly disordered keratocyte nuclei were observed in the corneal graft (Fig. 5d). In addition, obvious corneal stromal nerves and dark lines produced by stromal folds were observed in the corneal graft bed (Fig. 5e). At the donor-recipient interface, hyperreflective particles and mild haze were seen (Fig. 5f). Figure 5g shows abnormally swollen endothelial cells in the perforated boundary. Normal appearance of the endothelial cell layer was observed around the transplanted area (Fig. 5h).

Fig. 5
figure 5

Representative in vivo confocal microscopy (IVCM) images obtained 24 months after double lamellar keratoplasty. a IVCM shows a normal corneal epithelial cell layer. b At the level of Bowman’s layer, sub-basal nerve plexus and some Langerhans cells are observed. c Subepithelial stromal haze with some highly reflective microdots exist. d Normal keratocyte nuclei are observed in the corneal graft. e Corneal stromal nerves (long arrow) and stromal folds (short arrow) are shown in the corneal graft bed. f At the level of the donor-recipient interface, hyperreflective particles and mild haze can be noticed. g, h The appearance of corneal endothelial structure of corneal perforated area (g) and normal cornea (h) are visible. Bar, 100 μm (original magnification ×300)

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Complications

During the follow-up period, no allograft autolysis, immunological rejection, primary disease recurrence, or corneal re-perforation was observed in any patient.

Discussion

Corneal perforation can lead to severe visual loss and secondary intraocular complications, resulting in poor prognosis. The principal goal of therapy is to seal the perforation to reconstruct the anterior chamber and rebuild the anatomical integrity of the eyeball [2]. Over the last few decades, conjunctival flaps, lamellar scleral graft, tenon patch graft, tissue adhesives, and amniotic membrane transplantation have been regarded as accessible interventions to restore the ocular surface in a timely manner without donor cornea [2, 7, 13, 20]. However, each of these interventions has certain disadvantages [21]. Whereas autologous tissue is both cost-effective and easily accessible, the lack of tectonic support is a major limitation [22]. Tissue adhesives are good alternatives for tectonic support, but surface irregularity and tissue reaction are potential limitations [23]. The availability, cost, and poor tectonic support of the amniotic membrane restrict its use [12]. The most important reason is that they are non-optical surgical methods that can affect visual outcomes and may induce the risk of deterioration or recurrence [24].

In our study, double lamellar keratoplasty successfully treated 15 eyes in 15 patients with corneal perforation. All postoperative patients recovered anatomical and functional integrity of the eyeball. At the end of the follow-up period, corneal grafts remained transparent. The visual acuity of all patients improved to varying degrees. No significant aqueous leakage or immune rejection was observed during follow-up in this study.

Based on our follow-up results, the novel operation could recover integrity of the eye globe and allow acceptable visual rehabilitation. After surgery, we used AS-OCT to measure corneal thickness and assess stability of the cornea and graft. Because of the deficiency of corneal endothelium in the perforated area, the width was slightly thinner than the normal range, and internal malposition was common. Pre-designed graft of the appropriate size was used to prevent poor positioning of graft-host junction.

From the IVCM examination, normal corneal epithelial cells, sub-basal nerve plexus, and corneal stromal cells were observed, indicating double lamellar keratoplasty could restore the structural integrity of the cornea after surgery. In addition, some highly reflective particles and mild haze were observed at the donor-recipient interface. These particles are hypothesized to be necrotic and aggregated host corneal endothelial cells (CEnCs) [25]. It is gratifying to note that cornea edema and corneal endothelial decompensation did not occur during the 12-month follow-up. The IVCM images showed that counts and hexagonal structure of CEnCs around the perforated area were normal or near normal. Specular microscopy also revealed similar results.

Given the above results, the success of our modified surgery is closely related to the following issues: first, previous literature strongly suggests that the anterior stroma, because of the composition of proteoglycans and anteroposterior branching, has deferential behavior compared with the posterior stroma. The specific architecture of the anterior stroma is responsible for its stability in maintaining stromal morphology and corneal curvature intact [26]. Second, double lamellar keratoplasty is more inclined to be applied in patients with a smaller diameter (< 1 mm) of corneal perforation. We surmised that the small scale of the recipient posterior graft would act as a scaffold for residual CEnCs to migrate, which may serve as a compensatory response for the maintenance of stromal dehydration and corneal clarity. The changes of CEnCs around the perforation from specular microscopy and IVCM may provide some evidence to verify our speculation. Third, the sub-basal nerve of Bowman’s layer and stromal nerve were observed 24 months after surgery by IVCM. This might be due to the hypothesis that our modified surgery could make sub-basal nerves travel directly from the host plexus to the donor cornea, while building suitable surroundings for keratocytes to migrate and form orderly fibers [27], like PK and LK [28], which improve nerve generation, re-epithelialization, and re-endothelialization.

Some advantages exist in our sample of patients with corneal perforation who underwent double lamellar keratoplasty compared with conventional therapeutic keratoplasty. Due to its compact structural and functional features, anterior stroma could be more conducive to preventing the aqueous humor entering into the interlayer and stroma and further alleviate interlamellar-related complications, such as interlamellar neovascularization and opacification, which could be seen in LK [3, 19]. Moreover, compared to PK, the posterior graft came from the healthy stroma of the recipient. It retained an immune barrier to reduce the contact between the recipient’s intraocular tissues and the donor cornea and also formed a scaffold that promotes endothelial cell migration, thereby reducing allograft rejection and endothelial decompensation [13, 15].

Nevertheless, our study has some limitations. Deeper sutures fixing the posterior graft will not be removed postoperatively, so residual sutures might cause astigmatism to affect vision quality. Hence, our surgery is inclined to be applied to eccentric perforations to reduce the impact. Moreover, most of the procedure was performed freehand, and the clinical results may be largely associated with the experience of the surgeons.

Looking forward, the current study will benefit from a longer follow-up time and larger sample sizes. In addition, it is recommended to establish control groups, such as PK, sole LK, or amniotic membrane transplantation, to further validate the benefits of our modified surgery.

Conclusion

In summary, performing double lamellar keratoplasty is effective and safe in reconstructing the corneal anatomical structure and achieving satisfactory clinical outcomes for corneal perforation in the absence of fresh donor corneas. We recommend this surgery to treat the corneal perforation away from the pupil center, and the size of the corneal perforation is < 1 mm in diameter. To further evaluate the prognosis of the procedure, more efforts are needed to provide more robust and reliable clinical evidence to support the application of double lamellar keratoplasty.