Fig. 4.1
Anteroposterior section of keratoconic corneal button obtained after transplant surgery. The section has been stained using Celltracker green which stains the cytoplasm of all living cells green and counter immunolabelled with an antibody to Integrin α3β1, which labels the basal epithelium. Many of the histopathological features of keratoconus are visible in this tissue section including epithelial hypertrophy, epithelial basement membrane irregularities, breaks in Bowman’s layer, stromal fibrosis and central corneal thinning. Bar = 100 μm
The incidence of recurrence of keratoconus is reported to be rare, occurring at a rate of 5.4–11.7 % and with a latency of 17.9–21.0 years post-PK [71, 72], and at 1.6 % with a dramatic shorter latency period of 5 years for post-DALK eyes [73, 74]. Such reported incidences of recurrence of keratoconus in grafted corneal tissue may provide important clues as to the pathogenesis of keratoconus .
There are three speculated mechanisms for the recurrence of keratoconus:
- (a)
The host route: Recurrence of the host’s disease in the graft, such that the cells from the remaining host cornea induce the grafted tissue to develop keratoconic pathology. This seems to be the most accepted mechanism due to the slow progression of recurrence post-PK, as the initial appearance of keratoconus takes decades to develop and similarly the recurrence of the disease in the grafted tissue takes two decades to resurface. It is speculated that the remaining abnormal keratocytes from the host continue to migrate and infiltrate the donor cornea [75, 76] gradually replacing the donor keratocytes and producing abnormal collagen. Or alternatively, the residual basal epithelial cells may secrete proteolytic or autolytic enzymes that lead to loss of collagen fibres [73, 77, 78]. The speculated host route was further supported by the dramatic shorter time frame of recurrence (3–4 years) for post-DALK [74, 79]. DALK is thought to provide less possibility of graft rejection by retaining the recipient stromal bed; however, the earlier onset of recurrence could be the result of either increased host keratocyte invasion in to the donor tissue and/or donor tissue weakening due to the excision of the donor Descemet’s membrane prior to transplant [73]. In opposition to this theory however, is the fact that recurrence has been reported when a corneal button size of greater than 8 mm was used to minimise the volume of host cornea that remained in the tissue bed that could effectively lead to recurrence [71]. Also, the ability of significant numbers of abnormal host keratocytes to infiltrate the donor tissue and lead to keratoconus manifestation is debateable. Thus, there might be other mechanisms that lead to the recurrence.
- (b)
The donor route: Transmission of undiagnosed keratoconus from the donor cornea [80]. One case [81] report described using the two corneas of a donor which were grafted on an advanced keratoconic eye and a corneal leukoma eye. Both keratoconic and non-keratoconic recipient eyes eventually developed reported keratoconus post-transplantation. This was assumed to be likely due to the presence of as yet undetected forme fruste keratoconus in the donor tissue that eventuated once grafted to the recipients. This report could serve as evidence for a possible donor transmission route; however, there are no studies known that routinely follow the fate of contralateral grafts from the same donor to establish if this occurs on a regular basis. Also, presumption that the donor tissue must have had forme fruste keratoconus is completely unproven. Furthermore, one case [69] reported recurrence of keratoconus 7 years after the initial graft for keratoconus; however, the fellow donor cornea appears to be normal 12 years post-transplantation. It is speculated that this could still occur due to donor transmission if the donor exhibited unilateral keratoconus [75] or the second graft is yet to develop keratoconus. Certainly long-term follow-up analysis will be needed in order to determine the likelihood of this mechanism in majority of cases of recurrent keratoconus. It does seem to be clear though that the recurrence of keratoconus via donor transmission is not the sole mechanism responsible.
- (c)
Mechanical trauma such as contact lens wearing or eye rubbing can cause damage to the sutured cornea and failure of the wound healing process leading to apoptosis and a weakened graft cornea [82, 83]. However, it is difficult to isolate mechanical trauma as the major route [79] for the recurrence during latency period as there are plenty of reported recurrences with no evidence of eye rubbing or contact lens wearing. Nevertheless, mechanical trauma is most likely to at least play the role of ‘second-hit’ [83].
No doubt each of the three speculated mechanisms of recurrence of keratoconus in grafted corneal tissue can explain the development of disease in certain isolated cases, but none appear to be the major underlying mechanism for the recurrence, especially when the recurrence is rare.
Interestingly, the classical signs of keratoconus such as corneal thinning and epithelial incursion are not unique only for post-transplantation for keratoconus but also for other indications [84]. This has raised doubt whether the observation of recurrence has sometimes been confused with failed graft due to decompensation. Perhaps the clues lie away from the central graft and towards the peripheral region where the graft–host junction is sited. At the graft–host junction, lamellar disruption and thinning were observed [85–87], and stress test studies show that the graft–host junction remains weak even after the wound appears to have healed [77]. Adding to that, the epithelium at graft–host junction is much thickened (Fig. 4.2). Thus, corneal decompensation may be due to the increased physical stress at the graft–host junction during wound healing between the weakened keratoconus host and the normal thickness graft [84].
Fig. 4.2
An anteroposterior section of the junction between the host tissue and a corneal graft (GHJ) many years after the original operation. The graft tissue including some of the host tissue was removed during a regraft operation and processed for immunohistochemistry. Many of the histological features which may represent the recurrence of keratoconus are present in the graft material including epithelial oedema, breaks in Bowman’s layer, stromal thinning and folds, folded stromal lamellae, stromal ectasia, thinning in central cornea, lipid thickening of Descemet’s membrane and endothelial atrophy are visible. Bar = 100 μm
In addition to wound healing, impaired nerve innervation post-transplantation will likely lead to epithelial complications later on [88]. Being a primarily central corneal disease, the peripheral innervation in keratoconic corneas appears to be intact [13]. In terms of nerve re-innervation post-PK , despite the sub-basal nerve fibre density being significantly reduced [89], patients had higher nerve fibre density and greater nerve branch density than patients who underwent PK for other indications. This might partially explain the lower rate of recurrence after PK for keratoconus at around 11.7 % [72] than other stromal dystrophies at 21 % [90]. Taken together, the weakened graft–host junction, impairment in nerve re-innervation and mechanical insults such as eye rubbing and contact lens wearing can all cause damage which might lead to apoptosis indicating a fourth speculated mechanism of abnormal wound healing [85].
4.7 A Role for Inflammation in Keratoconus
Keratoconus has long been categorised as a non-inflammatory corneal dystrophy due to the lack of neovascularisation and cellular infiltration [91, 92]. However, there has been increasing evidence that may refute this concept [17, 93]. In keratoconus with acute corneal hydrops, dendritic cells (presumed leukocytes) [94] were observed clinically with in vivo confocal microscopy. Recently, studies using tear fluids of keratoconic patients revealed elevated levels of interleukin-6 (IL-6), tumour necrosis factor-α (TNF-α) and matrix metalloproteinase (MMP)-9 [95–100]. The expression of other factors such as MMP-1, -3, -7, -13, IL-4, -5, -6, -8, TNF-β [97] and tissue plasminogen activator (t-PA) [17] was also elevated, whereas lactoferrin and IgA [101, 102] were reduced in keratoconus. Tears from keratoconic patients associated with eye rubbing and contact lens wearing showed an increase not only in MMP13, IL-6, -10 and TNF-α, but also in the intercellular adhesion molecule (ICAM) -1 and vascular cell adhesion molecule (VCAM) -1 that are known to be important for inflammatory response [95, 103, 104]. Tear fluid of pseudokeratoconus caused by ocular rosacea [105] showed significantly elevated levels of IL1 and MMP9 [106, 107].
4.7.1 Immunohistochemical Evidence of Inflammation
The central cornea has a resident population of epithelial and stromal dendritic cells (DC), which function as antigen-presenting cells (APCs) . Although the corneal periphery contains mature and immature resident bone marrow-derived CD11c + DC, the central cornea is endowed exclusively with immature and precursor DC, both in the epithelium and the stroma, wherein Langerhans cells and monocytic DC reside, respectively [108–111]. In addition to the DC, macrophages are present in the posterior corneal stroma [108, 109].
Immunohistochemical labelling in central cornea of keratoconus showed CD11b+ macrophages, CD45+ leucocytes and HLA-DR MHC-II receptor on APCs localised in increasing number in epithelium and stroma (Fig. 4.3) [22, 112]. The density of APCs decreased from the paracentral towards the central part of the cornea [111]. Dendritic cells in keratoconus had the appearance of brighter bodies and shorter dendrites than those found in inflamed corneas [111]. Another macrophage marker, CD68, seemed to provide conflicting findings given its presence in epithelial basement membrane [17, 112, 113].
Fig. 4.3
A section of keratoconic tissue labelled with CD11b (green) shows macrophages present in the basal layers of the epithelium. The same section is also shown counterstained with DAPI (blue) which stains the nuclei of all the cells of epithelium and keratocytes within the stroma and CD11b highlighted with arrows to show the localisation of the macrophages within the epithelium. Bar = 100 μm
In hydrops-associated keratoconus, extensive presence of CD11b+ macrophages have been observed. Though the number of leukocytes and APCs appeared reduced when associated with hydrops, langerin+ DC numbers were similar with and without hydrops [22]. A huge elevation of numbers of leucocytes were noticed in the stroma, epithelium and even the endothelium in hydrops-associated keratoconus with subsequent neovascularisation [22]. (The authors find that neovascularisation in sections of keratoconic tissue is best identified using antibodies to Von Willebrand Factor (Fig. 4.4))
Fig. 4.4
A section of keratoconic tissue is shown with extensive neovascularisation as revealed by labelling using antibodies to Von Willebrand Factor . Bar = 100 μm
This suggested a chronic inflammatory process with recruitment of inflammatory cells [22]; however, the signs of inflammation appeared to attenuate in hydrops-associated keratoconus indicating that inflammation is not specifically associated with the occurrence of hydrops [22]. Thus, we believe inflammation in keratoconus is likely to be a chronic process rather than an acute occurrence.
4.8 Pathology Informing Pathogenesis
Modern histological techniques can be effectively used to determine pathological changes occurring within the matrix and cells in keratoconic corneal tissue. However, the real question to be answered is whether investigation of pathological changes within the tissue can add to the analysis of the pathogenesis of the disease or are we simply looking at the effect of disease progression. Many of the pathological phenomena that occur in keratoconus (described earlier) mirror tissue remodelling processes in that changes in cell activation, enzyme secretion matrix removal, deposition and remodelling are all involved in corneal repair. This has led to the proposal that progression of keratoconus is in fact due to aberrant repair mechanisms which continuously try to repair the matrix loss in keratoconus but instead compound that loss further. The beginning of this destructive cycle may begin with an external environmental trigger and afterwards be fuelled by aberrant internal processes. This is further supported when we combine molecular studies with the histological data. Recently, in our laboratory we were able to show that cells isolated from keratoconic corneas expressed the main repair modulating molecules at levels higher than normal corneas and equated to the levels found in a normal wounded cornea. However when the keratoconic corneas were supplied with a secondary insult they were unable to mount an adequate repair response [114].
In summary, modern histopathology can successfully combine with molecular techniques to uncover the elusive pathogenesis of keratoconus .
Declaration
Trevor Sherwin, Salim Ismail, I-Ping Loh and Jennifer Jane McGhee declare that they have no conflict of interest.
All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2000. Informed consent was obtained from all patients for being included in the study.
No animal studies were carried out by the authors for this article
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