|Year : 1982 | Volume
| Issue : 4 | Page : 337-340
Histochemical changes in corneal endothelium at different temperatures for variable storage of time
TN Agrawal, D Srivastava, SC Gupta
State Institute of Ophthalmology, M.L.N. Medical College, Allahabad, India
T N Agrawal
State Institute of Ophthalmology, M.L.N. Medical College, Allahabad
Source of Support: None, Conflict of Interest: None
|How to cite this article:|
Agrawal T N, Srivastava D, Gupta S C. Histochemical changes in corneal endothelium at different temperatures for variable storage of time. Indian J Ophthalmol 1982;30:337-40
|How to cite this URL:|
Agrawal T N, Srivastava D, Gupta S C. Histochemical changes in corneal endothelium at different temperatures for variable storage of time. Indian J Ophthalmol [serial online] 1982 [cited 2020 Apr 7];30:337-40. Available from: http://www.ijo.in/text.asp?1982/30/4/337/29466
Normal functioning endothelium is essential for successful corneal transplantation. Corneal hydration and transparency of the donor's eye depend largely upon temperature and duration of the storage.
Lactic dehydrogenase was located in the soluble components of the cytoplasm of cells and succinic dehydrogenase in the mitochondrial. Paranitroblue tetrazolium has frequently been used as an indicator of cell life or death.
Most of the work done so far pertains to the effect of temperature either at 4° c or below. The present study was undertaken with a view to study the histochemical changes that occur in corneal endothelium at different temperatures for variable durations of storage in experimental animals to assess the endothelial viability.
| Materials and methods|| |
Succinic dehydrogenase (SDH) activity of corneal endothelium was studied in 18 healthy albino rabbits with 36 eyes. They were divided in three groups of 6 rabbits each. The eyes were enuceated and eye balls were placed with corneas upwards in eye bank specimen containers with a layer of cotton soaked in saline solution at its bottom.
The eyes of the first group of animals were kept at 4°c and second group at 20°c for 4, 12 & 24 hours while that of the third group at 37°c for 2, 4 & 12 hours. Unstored corneas were studied as control from two rabbits with four eyes.
The S.D.H. activities of the corneal endothelium was studied by incubating the corneas in the media desired by Baum.
Phosphate buffer (ph 7.4) 3.0c.c.
Sodium succinate (0.2M) 0.5c.c.
0.1 percent para nitrobluetelra zolium 0.5c.c.
The corneas were incubated in toto in above solution for 2 hours at 37'c. After that time the PNBT reduction as evident by blue stain, was seen in the endothelium.
The corneas were then fixed in Carnoy's solution for 30 minutes and transferred to 70% alcohol for 5-10mts. After that four radial slits were made by scalpel in full thickness corneas to flaten out preparation. This was mounted in glycerine jelly.
Those cells which showed deposition of formazan granules were taken as damaged cells. For quantitative analysis of the damaged cells, 100 cells in six different microscopic fields were studied under high power. The percentage of damaged cells was calculated by taking the average of all the six values.
| Observations|| |
Flat mount preparations of control cornea after staining with para-nitro blue tetrazolium and sodium succinate showed no granular formazan formations. All cells were well preserved.
| Discussion and conclusion|| |
Various oxidative enzymes have been used to evaluate endothelial cell viability. In present study enzyme succinic dehydrogenase was studied because of their importance in cellular matabolism.
The observations made in the present study indicates that rabbit corneal endothelium stored at 4° C for four hours showed no demonstrable changes in flat preparations. At the same temperature after storage of 24 hours, no formazan granules were visible but cellular oedema was more marked. However Rena carrillo concluded in his studies that eyes stored at 4°C for 24 hours showed reductase activity about 25 to 30 per cent cells.
In present study when corneal endothelium was stored at 20°C for 12 hours no cellular damage was observed except the oedema of cells but after 24 hours of storage, cells showed marked cellular oedema and formazan granules deposition in 20% endothelial cells. When endothelium was stored at 37°C for 4 hours marked cellular oedema with formazan granules deposition in 25% endothelial cells was present but the endothelium which was studied after storage of 12 hours showed marked cellular oedema and peeling of cells at places. Formazan granules deposition was found in 40% of endothelial cells.
Rena-Carrillo (1964) noted in his studies formazan granules preferentially around nucleus of cells. However we found these granules deposition in cells without any preference.
It was found that peeling of endothelial cells in sheets at places at 4°c stored for 96 hours but we found peeling of endothelial cells at 37°c after storage of 12 hours.
These observations clearly indicate that the alteration in the cells go parallel to increase of storage timing and rise of temperature.
| References|| |
Cogan, D. and Kuwabera, T., 1960 J. Histochem. Cytochem.8: 380
Pea., se, A.G.E. : 1970 Histochemistry-The retical and Applied, 2nd ed., 582, Churchill. London.
Baum, J. L : 1963, Arch. Ophthalmol 70 : 59
Rena-Carrillo, J. and Polack, F.M, 1964 Arch. Ophthalmol., 72:811.
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[Table - 1], [Table - 2], [Table - 3], [Table - 4]