Reversibility of Ultrastructural Freeze-Thaw-Induced Injury

Horn, Diane L. Van

doi:10.1001/archopht.1972.01000020424012

Cited by 17 publications

(1 citation statement)

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“…Specular microscopy89 and scanning electron microscopy'0 were not employed at this initial stage, but more recently there have been reports of the appearance of the endothelium in preserved tissue using transmission and scanning electron microscopy. [11][12][13] The purpose of this report is to describe morphological changes in the endothelium of cryopreserved corneae using specular microscopy and scanning electron microscopy. Endothelial function is also to be assessed by the measurement of comeal thickness maintenance in a perfusion system.…”

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Assessment and interpretation of corneal endothelial cell morphology and function following cryopreservation.

Madden¹,

Easty²

1982

British Journal of Ophthalmology

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SUMMARY Scanning electron microscopy, specular microscopy, and staining techniques were used in a short-term study of the standard method of cryopreservation. Correlation of the techniques revealed that the endothelium had an atypical morphology, showed apparent evidence of latent cell damage, and lacked function.A successful and simple technique for comeal cryopreservation continues to hold out the hope for indefinite storage and easy transport over long distances. It would enable setting up a tissue bank which could be used for the close matching of donor and recipient by typing techniques"2 and allow the use of tissue in regions where donor material is unavailable. Early investigations of preservation techniques3 have led to the development of the present clinical method.7 The studies employed vital staining, endothelial culture, and keratoplasty in animals to assess survival and function. Specular microscopy89 and scanning electron microscopy'0 were not employed at this initial stage, but more recently there have been reports of the appearance of the endothelium in preserved tissue using transmission and scanning electron microscopy. [11][12][13] The purpose of this report is to describe morphological changes in the endothelium of cryopreserved corneae using specular microscopy and scanning electron microscopy. Endothelial function is also to be assessed by the measurement of comeal thickness maintenance in a perfusion system. Materials and methodsDutch rabbits were killed with pentobarbitone. The eyes were immediately enucleated and precooled to +4°C along with the preservation solutions of dimethyl sulphoxide and sucrose in albumin or serum. Freezing to -196°C and thawing were carried out according to the method of Capella and Kaufman' by the prescribed cooling box, " or by a method designed to reduce mechanical trauma. In the second method corneae were mounted on specular microscopy rings and frozen endothelium-side-up in 5 ml solution in plastic scintillation vials. Freezing and thawing profiles were suitably adjusted for the different thermal properties of the plastic vial. Any subsequent staining or fixation procedure was undertaken with the comeae still mounted.Nitroblue tetrazolium was used to demonstrate viable cells after 3 hours incubation. After fixation for scanning electron microscopy (SEM) with glutaraldehyde and osmium tetraoxide'°the specimens were viewed at 9-5 kV on a Cambridge S4 microscope. Post-thaw incubation prior to SEM was carried out at 35°C in glutathione bicarbonate ringer (GBR at pH 7.4). ' This solution was also used for perfusion of mounted cornea on the specular microscope. Specular microscopy was used to assess comeal function by serial thickness measurements. The average of 3 readings from endothelium to the stromal/epithelium interface was taken. The microscope was also used to assess cell morphology. ResultsImmediately after the freeze/thaw cycle nitroblue tetrazolium staining demonstrated, at best, 70% endothelial survival as shown by dye precipitation resulting from ret...

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