The specific architecture of the anterior stroma accounts for maintenance of corneal curvature

Abstract

Aim: To analyse the human corneal stroma in extreme hydration to discover if its structure is responsible for corneal stability.

Methods: Corneas in several hydration states were used: postmortem control corneas (PM; n=3), corneas left for 1 day in phosphate buffered saline (PBS; n=4), and corneas left for 1 day (n=4), 2 days (n=4), 3 days (n=2), and 4 days (n=4) in deionised water. All corneas were fixed under standardised conditions and processed for light and electron microscopy. In addition, two fresh corneas from the operating theatre were studied which were processed 6 months after storage in sodium cacodylate buffer.

Results: After 1 day in deionised water maximal stromal swelling was reached which did not change up to 4 days. The stroma of deionised water corneas (1400 microm) was much thicker than that of PBS corneas (650 microm) and PM corneas (450 microm). Deionised water treatment led to disappearance of all keratocytes leaving only remnants of nuclei and large interlamellar spaces. In these specimens the distance between the collagen fibres had increased significantly, but the diameter of the collagen fibres did not seem to be affected. A remarkable observation was that the most anterior part of the stroma (100-120 microm) in all deionised water specimens and those stored for 6 months in buffer was not swollen, indicating that the tightly interwoven anterior lamellae are resistant to extreme non-physiological hydration states.

Conclusions: The rigidity of the most anterior part of the corneal stroma in extreme hydration states points to an important role in maintenance of corneal curvature. Since a large part of this rigid anterior part of the stroma is either removed (PRK) or intersected (LASIK), it is possible that in the long run patients who underwent refractive surgery may be confronted with optical problems.

Figure 1

(Top) Electron micrograph of a cross section through the anterior stroma of the human cornea. Collagen lamellae are alternated and undulated. The different collagen bundles cross sectioned (*), longitudinally sectioned (arrowhead), and obliquely sectioned (x) alternate at irregular distances. (Bottom) Electron micrograph of the posterior stroma shows strictly alternating cross sectioned (*) and longitudinal sectioned (arrowhead) collagen bundles at more or less regular distances.

Figure 2

Light micrographs of semithin sections from corneas at different states of hydration. (A) Post mortem (PM); (B) phosphate buffered saline (PBS); (C) 1 day deionised water, and (D) 4 days deionised water. The magnification for all micrographs is the same. The most anterior stroma, above the broken line, is not affected by hydration (C and D, arrows). Most keratocytes in the mid and posterior stroma are shrunken or have disappeared and large interlaminar spaces (*) are left.

Figure 3

Light micrographs of the most anterior stroma in (A) post mortem (PM); (B) phosphate buffered saline (PBS), and (C) deionised water (H₂O). In (A) and (B) more keratocyte profiles are present (arrows) than in (C). The undulated collagen bundles (arrowheads) in (A) and (B) are similar; (C) is different and characterised by irregularly arranged grey structures.

Figure 4

Longitudinal collagen fibres of a PM cornea (A) and a PBS cornea (B). The irregular grey structures in Figure 3C after 1 day in deionised water represent amorphous extracellular matrix (C, arrows). Only occasionally keratocyte nuclei with a small rim of cytoplasm are observed in mid stroma. Treatment with deionised water does not allow well stained cross sectioned collagen fibres in the posterior stroma. Longitudinal collagen fibres (*) surrounded by amorphous matrix (arrow) can be visualised (E and F). All bars represent 0.5 µm.

Figure 5

Thickness of the corneal stroma after treatment with deionised water for 1, 2, 3 and 4 days. These groups are compared with PM and PBS treated tissue.