Regeneration of corneal endothelial cells following keratoplasty in rats with bullous keratopathy

• Introduction
• Methods
• Results
• Discussion

Abstract

Purpose: Little is known about the behavior of the endothelial cell (EC) layer following keratoplasty. In vitro experiments suggested that the peripheral endothelium might have a higher regenerative capacity than the central endothelium, and some authors hypothesized that endothelial progenitor cells are present in the limbal area. Thus, we analyzed the corneal endothelial regenerative capacity in vivo in a rat model of bullous keratopathy using either bullous central grafts or bullous peripheral recipient corneas to analyze differences in EC regeneration depending on central versus peripheral cell origin.

Methods: Bullous keratopathy was induced in Lewis rats with an intracameral injection of benzalkonium chloride (0.05%; BAK). Three days later, the central area of the bullous cornea was excised and used as a bullous graft, transplanted to a healthy, green fluorescent protein (GFP)-transgeneic Lewis receipient rat (group ‘bullous graft’). In return, the mentioned rat eye with the bullous keratopathy received a healthy GFP-transgeneic corneal transplant (group ‘bullous host’). A subgroup of these animals received a healthy, excentrically trephined including parts of the limbus (group ‘bullous host, limbo-keratoplasty’). The grafts were monitored clinically for 7 weeks. Subsequently, the mean EC density was calculated on corneal whole mounts with Alizarin Red S staining. GFP was analyzed with confocal microscopy to determine EC origin. Untreated fellow eyes served as controls.

Results: BAK injection led to a significant decrease in the mean EC density with subsequent bullous keratopathy. In the control eyes, the mean EC density was 3,744 cells/mm² in the center and 2,811 cells/ mm² in the periphery. In eyes with bullous keratopathy, only 233 cells/mm² in the center and 622 cells/mm² in the periphery were observed three days after BAK-injection. Bullous transplants in the group ‘bullous graft’ cleared, and GFP-positive cells were detected on the transplant. In contrast, no GFP-positive ECs were detected on the host cornea in the groups ‘bullous host’.

Conclusions: ECs from the peripheral cornea have the ability to cross the graft border and compensate for the pathologically altered/absent endothelium on the graft. In contrast, ECs derived from the central cornea remain central on the graft and do not replace or regenerate peripheral ECs in our model of bullous keratopathy.

The corneal endothelial cell (EC) layer is crucial for corneal clarity: If the corneal EC density falls below a critical threshold, the barrier and pump function of the endothelium cannot be maintained, and the cornea becomes edematous and presents as bullous keratopathy. This is a major indication of orthotopically placed penetrating keratoplasty, or posterior lamellar keratoplasty [1,2]. However, even after corneal transplantation, which has a good prognosis for most low-risk indications [3,4], corneal decompensation due to chronic EC loss can occur and might lead to transplant failure [5]. Even though the EC layer is important for corneal clarity and thus visual acuity, little is known about the endothelial reactions following corneal transplantation.

In rodents and humans, some degree of reparative mechanisms are present in the EC layer. Even though human EC rarely divide physiologically, they have the ability to migrate and enlarge to compensate for smaller defects in the EC layer [6]. Ex vivo experiments suggest that the location of ECs might play a role in their regenerative capacity: Different groups have demonstrated that ECs obtained from the corneal periphery compared to the center have a higher regenerative capacity in vitro [7-9], and the presence of endothelial precursor cells in the corneal periphery or limbal area has been proposed. These data obtained in vitro support the hypothesis that the peripheral and central endothelium might differ in their regenerative capacity.

To further clarify this issue and its relevance in vivo, we analyzed central and peripheral endothelial regeneration following keratoplasty in a rat corneal transplant model. EC density was reduced before keratoplasty by treatment of either the corneal graft or the host with benzalkonium chloride (BAK), leading to bullous keratopathy. Subsequently, either a BAK-damaged corneal button was grafted to a healthy recipient rat or an untreated transplant was transplanted into a BAK-damaged recipient cornea. We also evaluated EC regeneration using untreated limbocorneal grafts in BAK-treated recipients to assess the possible role of limbal ECs in repopulating the damaged peripheral recipient endothelium. Transplantations were performed between green fluorescent protein (GFP) transgenic and wild-type rats to monitor EC origin aside from cell density and morphology. An immune response was excluded by syngeneic grafting.

Ex vivo experiments have indicated that peripheral ECs might have a higher capacity to proliferate or compensate for endothelial wounds, and have even led to the hypothesis that EC precursor cells are located in the corneal periphery or limbal area: As early as 1998, Bednarz et al. cultured human corneal ECs and detected different characteristics of ECs depending on their position on the human cornea. Cells isolated from the central part of the cornea showed cell enlargement but no cell division to cover defects in the monolayer. In contrast, ECs isolated from the peripheral part of the cornea seemed not as tightly packed, and space was filled due to cell division rather than cell enlargement [13]. Later, Mimura et al. examined healing responses to scrape wounds of whole ex vivo cultured human corneas. The researchers noticed that human cultured ECs from the peripheral area retained higher replication competence compared to central cells, regardless of donor age [9]. The observation of cells with increased regenerative capacity finally led to the hypothesis of endothelial precursors in the posterior limbal area, from where these cells might migrate to the endothelial periphery and, perhaps, to wounded areas of the corneal endothelium when needed [8,14,15].

It was our goal to explore potential differences in the regenerative capacity of the central and peripheral corneal endothelium in vivo. Therefore, we used a syngeneic rat keratoplasty model. Our own recent experiments (data not shown) confirmed that syngeneic transplantation between Lewis rats using GFP-positive donors and GFP-negative littermates did not lead to rejection of the graft, and the host and graft endothelium remained intact. To study corneal endothelial regeneration, EC damage was induced by BAK, resulting in bullous keratopathy in either donors or recipients.

BAK is toxic for ECs [16] and has been used to experimentally induce bullous keratopathy by intracameral injection in rabbits [17-19]. Recently, the bullous keratopathy model has also been adapted for use in mice [20]. Yang et al. produced bullous keratopathy in one eye of a male rabbit, and the graft from a female rabbit was transplanted. Four months later, the detection frequency of the sex chromatin stained with acetic orcein was similar between the cells in the graft and in the bullous host cornea, indicating that the cells in the host had migrated from the graft [18]. In contrast, Nakahori et al. performed exchange keratoplasties between rabbits with damaged graft endothelium and damaged host endothelium. The authors found that an EC layer was apparent on previously damaged grafts after 4 weeks, while the former host endothelium did not recover [19]. These results are similar to our own observations, even though their experiments did not show whether the regenerated cells on the graft were host derived or equaled recovered graft endothelium.

In humans, little is known about the fate of donor or host-derived ECs following transplantation. A distinct marker is needed to distinguish donor and recipient cells. Some studies have used fluorescence in situ hybridization of sex chromosomes on corneal graft explants in a sex-mismatched situation between the original donor and the host to analyze whether the ECs on the graft are graft or host derived. The first study found no donor ECs 1–30 years after the original keratoplasty (in ten out of these 14 cases endothelial failure/rejection was the reason for graft explantation) [21]. In contrast, Lagali et al. demonstrated that 26 out of 35 grafts retained graft ECs up to 3 years after transplantation in varying percentages. Donor cells were reported to be distributed throughout the corneal sections in a seemingly random fashion [22]. Thus, although these experiments showed that graft ECs in humans can either remain intact or be replaced by the host endothelium (reasons for either reaction are not clear thus far), these experiments cannot answer the question whether graft endothelium can repopulate the host cornea just as well [23] since the peripheral host cornea is not accessible for fluorescence in situ hybridization in living humans.

In our set of experiments, GFP expression allowed us to monitor the cell origin of the graft and the host cornea. Transplantation of a BAK-damaged graft into a healthy host resulted in replacement of the damaged graft endothelium by GFP-positive host ECs. Previously, we have shown that when transplanting an EC-free graft (mechanical EC removal), the healthy peripheral rat corneal endothelium is capable of repopulating this transplant. When performing allogeneic rat keratoplasties, the immune reaction leads to EC loss of the graft, and then is likewise followed by a host-derived EC repopulation [12,24]. All these results consistently demonstrate that the peripheral rat endothelium has the potency to recover the EC layer of a graft devoid of or with a strongly damaged endothelium.

Although proliferation was not specifically assessed again in the current study, immunohistochemical staining in our previous study clearly showed that EC regeneration in the rat keratoplasty model is based on not only cell migration but also EC proliferation [12].

Interestingly, when the exchange keratoplasty was performed (transplanting a healthy graft into a host with a damaged endothelium, which equals the common clinical implication of keratoplasty for bullous keratopathy), the graft endothelium did not replace the peripheral host ECs in our rat model. This might be due to the higher regenerative capacity of peripheral Ecs, and might support the hypothesis of peripheral endothelial precursor cells. However, peripherally trephined transplants including a limbal portion did not change this result. This observation does not necessarily refute the hypothesis of the increased peripheral regenerative capacity of ECs. There are several possibilities why these transplants did not show increased EC regeneration. First, the transplants were placed centrally due to technical reasons in rat keratoplasty, and whether limbal areas transplanted to a different area of the cornea function unchanged is unknown. We observed that the epithelium (data not shown) of the graft was rapidly replaced by the host epithelium, directed from the periphery to the center, just as in grafts without a limbal portion. This failure to preserve the epithelial regenerative capacity of the transplanted limbal area in this rat keratoplasty model might also be true for the endothelial regenerative capacity of the peripheral endothelium. Second, EC regeneration might be directional, and the regeneration of transplanted limbal area might still be directed toward the center and thus not extend onto the host cornea. This hypothesis is emphasized by the work of He et al., which suggested a continuous slow centripetal migration of ECs throughout life [25].

Taken together, our in vivo experiments in the rat indicate that EC repopulation of damaged corneal grafts occurs by the host, while the repopulation of damaged peripheral ECs by a graft is not as easily accomplished. This further supports the hypothesis hat there are differences between central and peripheral corneal ECs in vivo.