Injuries of the corneal endothelium

Examples of "endogenous" or direct and "endogenous" or indirect endothelial trauma are presented. Corneal contusion, McCannel suture, YAG laser iridectomy, congenital luxation of the lens, and chronic over wearing of contact lenses all cause traumatic changes. The etiology and effects of these changes are discussed. Specular microscopy of the corneal endothelium permits good clinical control of both the trauma and wound healing.     

Human corneal endothelium

Summary The infant cornea contains many densely packed cells with large nuclei and little cytoplasm. As the cornea grows during childhood, cell density decreases by almost 50% and the cells become correspondingly larger. The adult cornea maintains a relatively uniform density of hexagonal cells which retain a generally uniform size and appearance. In the aged cornea, however, a pleomorphism develops. The aged endothelial surface is covered by a mixture of enormous as well as very small cells.

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Zusammenfassung In dem Hornhautendothel des Kindes sind die Zellen dicht gedrängt; ihre Kerne sind groß und das Protoplasma ist gering. Während des Wachstums der kindlichen Hornhaut nimmt die Zelldichte um etwa 50% ab, während die Zellgröße entsprechend zunimmt. Das Hornhautendothel des Erwachsenen zeigt eine relativ einheitliche Zelldichte. Auch die Größe und Morphologie der Zellen ist in diesem Alter im allgemeinen überall gleich. Jedoch ist die senile Hornhaut durch einen Pleomorphismus charakterisiert und an der Oberfläche des Endothels findet sich ein Gemisch von sehr großen und sehr kleinen Zellen.

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Résumé L'endothélium cornéen infantile contient beaucoup de cellules en groupes compacts avec de larges noyaux et peu de cytoplasme. Pendant la croissance de l'endothélium la densité cellulaire diminue d'environ 50% et les cellules s'élargissent d'une façon correspondante. La cornée adulte conserve une densité relativement uniforme de cellules hexagonales. Il en est de même de leur dimension et de leur apparence générale. Cependant on constate un pléomorphisme dans la cornée âgée. La surface de l'endothélium est constituée chez des personnes de 65 ans et plus par un mélange de cellules énormes et très petites.

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This work was supported by U.S. Public Health Service Grants NB 05037 and NB 08210 from the National Institute of Neurological Diseases and Stroke, National Institutes of Health.     

自体血管内皮细胞移植替代角膜内皮层的实验研究

目的 探讨自体血管内皮细胞(VECs)移植替代角膜内皮层的可行性.方法 40只家兔随机分为4组.A组,家兔VECs培养移植于去后弹力层的白体角膜植片内表面;B组,角膜植片去后弹力层后移植回原植床;C组,角膜植片取下后移植回原植床;D组,家兔不做处理.术后观察VECs生长状况及角膜植片透明度,A型超声测其厚度.结果 A组角膜植片混浊,1周内最明显,后混浊减轻,2周可见前房.细胞生长良好,部分细胞形状不规则.B组角膜植片混浊且不改善.C、D两组角膜无混浊.结论 VECs可替代自体角膜内皮层的屏障功能,维持角膜的透明状态.     

Antimicrobials and the corneal endothelium

The effects of various intraocular antibiotics and preservatives on the rabbit corneal endothelium were evaluated using vital staining, light, scanning, and transmission electron microscopy after topical application of the drugs and up to 2 days following anterior chamber injection. Benzalkonium chloride consistently produced the most severe and probably irreversible damage. Some changes which persisted for 2 days were found with methicillin. Gentamicin produced a moderate amount of cellular edema which disappeared after 2 days. Minimal changes were produced by dexamethasone and amphotericin B. Although these results should not be directly interpolated to the human situation, the face that some of these agents did produce changes in the rabbit corneal endothelium, which is well known to be heartier than human endothelium, indicates that caution should be taken when one considers injecting antibiotics intraocularly.     

Clinical responses of the corneal endothelium

The corneal endothelium maintains corneal deturgescence and clarity by a pump-leak mechanism first described by David Maurice. This cell layer can be investigated clinically with specular microscopy, fluorophotometry, and pachymetry. We describe the clinical responses of the corneal endothelium to aging, drugs, glaucoma, contact lens wear, trauma, disease, and surgery.     

Regeneration of the human corneal endothelium

The ability of the human corneal endothelium to regenerate is studied with the scanning electron microscope through examples of corneal diseases and penetrating keratoplasties. This study does not lead to final conclusions on the possibilities of regeneration of the human corneal endothelium but allows us to say that:

regeneration occurs through size increasing and deformation of the remaining cells.

the increase in number and size of surface microvilli may simply indicate a state of cell activation.

the présence of two nuclei in one cell is probably obtained by amitotic division but no complete mitosis has been seen.

Displacement of endothelial cells is a real progression and the cell is able to overcome obstacles.

the fibroblastic transformation of the endothelial cells is present in man but this may simply represent the migrating form of the cells.

     

Proliferative capacity of the corneal endothelium

Corneal endothelium is the single layer of cells forming a boundary between the corneal stroma and anterior chamber. The barrier and "pump" functions of the endothelium are responsible for maintaining corneal transparency by regulating stromal hydration. Morphological studies have demonstrated an age-related decrease in endothelial cell density and indicate that the endothelium in vivo either does not proliferate at all or proliferates at a rate that does not keep pace with the rate of cell loss. Lack of a robust proliferative response to cell loss makes the endothelium, at best, a fragile tissue. As a result of excessive cell loss due to accidental or surgical trauma, dystrophy, or disease, the endothelium may no longer effectively act as a barrier to fluid flow from the aqueous humor to the stroma. This loss of function can cause corneal edema, decreased corneal clarity, and loss of visual acuity, thus requiring corneal transplantation to restore normal vision. Studies from this and other laboratories indicate that corneal endothelium in vivo DOES possess proliferative capacity, but is arrested in G1-phase of the cell cycle. It appears that several intrinsic and extrinsic factors together contribute to maintain the endothelium in a non-replicative state. Ex vivo studies comparing cell cycle kinetics in wounded endothelium of young (< 30 years old) and older donors ( > 50 years old) provide evidence that cells from older donors can enter and complete the cell cycle; however, the length of G1-phase appears to be longer and the cells require stronger mitogenic stimulation than cells from younger donors. In vivo conditions per se also contribute to maintenance of a non-replicative monolayer. Endothelial cells are apparently unable to respond to autocrine or paracrine stimulation even though they express mRNA and protein for a number of growth factors and their receptors. Exogenous transforming growth factor-beta (TGF-beta) and TGF-beta in aqueous humor suppress S-phase entry in cultured endothelial cells, suggesting that this cytokine could inhibit proliferation in vivo. In addition, cell-cell contact appears to inhibit endothelial cell proliferation during corneal development and to help maintain the mature endothelial monolayer in a non-proliferative state, in part, via the activity of p27kip1, a known G1-phase inhibitor. The fact that human corneal endothelium retains proliferative capacity has led to recent efforts to induce division and increase the density of these important cells. For example, recent studies have demonstrated that adult human corneal endothelial cells can be induced to grow in culture and then transplanted to recipient corneas ex vivo. The laboratory work that has been conducted up to now opens an exciting new door to the future. The time is right to apply the knowledge that has been gained regarding corneal endothelial cell proliferative capacity and regulation of its cell cycle to develop new therapies to treat patients at risk for vision loss due to low endothelial cells counts.     

Ultraviolet-B damages corneal endothelium

Direct in vivo observation of acute ultraviolet (UV)-induced corneal endothelial damage is not possible due to the more severe damage produced in the epithelium. In order to quantify damage and evaluate endothelial recovery an indirect method was used. Eyes of pigmented rabbits were irradiated with UV-B (290 to 320 nm) isolated from the output of a high-pressure 1000 W reflectorized Xenon arc lamp by a grating monochromator and appropriate filters. The peak wavelength of the radiation used was 305 nm, with a 18 nm bandwidth at half-maximum. Corneal thickness variations measured with a modified Zeiss (Oberkochen) pachometer were used to follow alterations in epithelial and endothelial function. Epithelial damage alone resulted in a maximum thickness increase of 13.5% within 24 hr with recovery within a further 24 hr. Greater increases in corneal thickness, in the absence of anterior uveal involvement, were taken to indicate endothelial damage, and reached maximum at 2 days, with recovery occurring in 7 days. The threshold for endothelial damage sufficient to disturb corneal deturgescence was 0.12 J X cm-2.     

角膜内皮基因转移

基因治疗的关键技术是基因转移。角膜内皮基因转移是角膜内皮基凶治疗的基础,其主要方法有病毒载体和非病毒载体介导法,他们各有其优缺点。对角膜内皮基因转移技术和载体的研究现状,及未来载体的研究方向进行综述。     

Intracameral thrombin and the corneal endothelium

We perfused the endothelia of isolated human corneas mounted in the specular microscope with BSS Plus containing 1,000-U/ml or 100-U/ml dilutions of two commercially available topical thrombin preparations. Corneas perfused with thrombin at 1,000 U/ml showed intracellular and intercellular vacuole formation and altered junctional complexes. As listed on the package inserts, the thrombin preparations contained preservatives and other additives that present a significant osmotic load in 1,000-U/ml preparations. Corneas perfused with 100-U/ml thrombin solutions showed a significant attenuation in their deswelling rate but no ultrastructural alterations. One available thrombin preparation when diluted to 100 U/ml had a glycine concentration associated with previous retinal electroretinography changes. Polyacrylamide gel electrophoresis of one manufacturer's thrombin solution showed multiple high and low molecular weight constituents. Analysis of particulate contamination showed one 100-U/ml thrombin preparation to have a large quantity of particulates. Although thrombin may be useful when applied topically as an aid in surgical hemostasis, its use intraocularly presents substantial concern regarding the preparation's purity, additives, contaminants, and adverse effects on ocular tissues.     

Vital staining of corneal endothelium

The efficacy and applicability of dual vital staining of human and porcine corneal endothelium with trypan blue and alizarin red S stains was investigated. Exposure of endothelial cells to different concentrations of these two dyes for varying periods of time revealed that 0.3% solution of trypan blue for 1 minute followed by 0.2% solution of alizarin red S (pH 4.2) for 1/2 to 1 minute was the most useful technique to assess the cell viability, micro to macro damage, cell division and cell counts, etc. Alizarin red S dye should not be used in in vivo evaluation, while trypan blue was very effective to evaluate the endothelial viability of donor cornea just prior to keratoplasty.