Ultrastructural changes in moist chamber corneas. E. M. Schaeffer"

Size: px

Start display at page:

Download "Ultrastructural changes in moist chamber corneas. E. M. Schaeffer""

Marsha Palmer
5 years ago
Views:

1 Ultrastructural changes in moist chamber corneas E. M. Schaeffer" Crossly normal human corneas received through the Iowa Eye Bank and stored in cold moist chambers for 12, 24, 36, 72, and 96 hours were studied with the electron microscope. Significant changes observed were: shrinkage of the nuclei, ground cytoplasm, and cell membranes; small vacuole formation from swollen and distorted mitochondria; large vacuole formation from cytoplasmic spaces near cell membranes; agranular clumping of the nucleoplasm; breakdown and disappearance of nuclear membranes; degeneration bodies from mitochondria; and an increase in perinuclear ribonucleoprotein granules and endoplasmic reticulum about the epithelial nuclei. Morphological changes considered to be irreoersible by earlier workers ivere first noted at 24 hours. These changes progressed as the time of storage increased. T,,his study was undertaken to show what fine cellular changes occur in the corneas of donor eyes stored for use in penetrating Jceratoplasties. Special attention was given to the nuclei and mitochondria in hope that some information, on a morphological basis, could be gained regarding the viability of these cells. Materials and methods Crossly normal eyes received through the Iowa Eye Bank, and all enucleated within one hour after death, were kept in Eye Bank specimen jars containing cotton wet with saline and stored at 4 C. for 12, 24, 36, 72, and 96 hours. Two grossly normal corneas were obtained from eyes immediately following enucleation for retinoblastomas. All corneal specimens were removed and pre- Paper No. 13 from the Neurosensory Center of the Department of Neurology and Ophthalmology, supported by Program-Project Grant B-3354 of the National Institute of Neurological Diseases and Blindness. Department of Ophthalmology, State University of Iowa, Iowa City, Iowa. pared in the same manner. Central corneal buttons were removed by use of a 6 mm. trephine. These specimens were placed immediately in cold (4 C.) 1 per cent osmic acid and phosphate buffer for Wz to 2 hours. After fixation, the specimens were cut into 1 by 2 mm. pieces and rapidly dehydrated through a series of graded alcohols. Embedding was carried out in Araldite." Sectioning was done with glass knives on the Porter- Blum Microtome. All sections were stained for 20 minutes in a filtered saturated solution of lead acetate. Viewing was done on an RCA EMU3F electron microscope. Observations Epithelium. The architecture of the normal epithelium (Figs. 1 and 2) reveals a compact arrangement of cells with typical interdigitating processes and desmosomes. Mitochondria are mainly of the filamentous type but some oval forms are present. Mitochondria in the more superficial layers are oriented in a circular fashion around the cell nuclei, whereas in the basal layer they are found about their inner and outer poles. Although the more superficial cells "Ciba A. R. L. Limited, Cambridge, England. 272

2 Volume 2 Number 3 Ultrastriictural changes in moist chamber corneas 273 D Fig. 1. For legend see page 275. Fig. 2. For legend see page 275.

3 274 Schaeffer Investigative Ophthalmoiogy June 1963 N t> v»t Fig. 3. For legend see opposite page. I * '», >* %. J^fc/* I p Fig. 4. For legend see opposite page.

Volume 2 Number 3 Ultrastructural changes in moist chamber corneas 275 stain more darkly than the basal cells, the nucleoplasm of all epithelial cells appears homogeneous and granular.

4 Volume 2 Number 3 Ultrastructural changes in moist chamber corneas 275 stain more darkly than the basal cells, the nucleoplasm of all epithelial cells appears homogeneous and granular. The relative scarcity of vesiculated endoplasmic reticulum is impressive at all levels of epithelium. When ribonucleoprotein granules are seen in any number, they are generally scattered and not concentrated in any one location within the cell. Cytoplasmic tonofibrils are present at all levels. Fig. 3 is representative of epithelium stored for 12 hours. The gross architecture is still compact, but the nucleoplasm is forming definite aggregations especially near the nuclear membrane. Mitochondria are sparse and there are a few notable areas of vesicular endoplasmic reticulum about the nuclei. Most striking is the prominence of RNP granules about the nuclei. Twenty-four-hour epithelium (Fig. 4) reveals still more densely aggregated nucleoplasm and more vesiculated endoplasmic reticulum. The few mitochondria present are for the most part swollen and have lost their cristae. The intercellular connections show early separation although the desmosomes remain fully attached. Fig. 5 shows perinuclear activity at 36 hours. The nuclear membrane at many points is indistinct and suggests that the nucleoplasm is spilling out into the cytoplasm. Near these areas can be found a high concentration of endoplasmic reticulum. The mitochondria are swollen and disorganized. The epithelium at 3 days (Fig. 6) shows shrinkage and separation of the intercellular membranes. Yet many of the desmosomes are still attached. The superficial cells show mainly a filamentous component to the cytoplasm and many small vacuoles. At 4 days (Fig. 7) many of the desmosomes have pulled apart. The nuclear membranes are poorly defined and the granular pattern of the nucleoplasm is greatly disorganized. RNP granules surround the nuclei and follow their indentations. The mitochondria are poorly defined and around many are spaces in the cytoplasm. Stroma. The architecture of the normal stromal cells (Fig. 8) changes rapidly during storage. By 12 hours (Fig. 9) the nucleoplasm has lost its homogeneous granularity. The cell membranes are thin and the cytoplasm contains empty spaces. Fig. 10 shows a shrunken stromal cell at 36 hours. Cytoplasmic spaces are present and the mitochondria are wavy. Later stages of storage (Figs. 11 and 12) reveal nuclei nearly devoid of nucleoplasm, mitochondrial disorganization, and loss of cytoplasmic substance. Endothelium. Normal endothelial architecture is represented in Fig. 13. These cells in the immediately fixed state are flatly arranged and their organelles are Fig. 1. Normal epithelium (x5,000). Note compact relationship of densely staining wing cells (WC) and basal cells (BC) by firm apposition of interdigitating processes (IP) and desmosomes (D). These nuclei (N) contain relatively homogeneous nucleoplasm and are surrounded by many mitochondria (M). Basement membrane (BM). Bowman's layer (BL). Fig. 2. Normal epithelium (xl4,800). Note clearly defined double-walled nuclear membrane (NM), cytoplasmic tonofibriles (CT), mitochondria (M), endoplasmic reticulum (ER) and interdigitating processes (IP). Fig. 3. Twelve-hour epithelium (xl4,000). Normal appearing interdigitating processes (IP). Note scarcity of mitochondria, prominent RNP granules (P), and dense aggregations of nucleoplasm (N) inside nuclear membrane. Fig. 4. Twenty-four-hour epithelium (xl3 ; 200). Darkly staining surface epithelial cells show dense clumping of nucleoplasm (N), prominence of particular and vesicular RNP (P), swollen and disorganized mitochondria (M), and some early separation of interdigitating processes (IP).

276 Schaeffer Investigative Ophthalmology June 1963 evenly distributed throughout the cytoplasm. The nuclei are homogeneous. Changes occur early in this layer of stored corneas.

The nucleoplasm becomes clumped and the nuclear membranes are generally indistinct after 36 hours. In late stages (Figs.

Discussion Observations made on corneas fixed immediately after enucleation in this study generally concurred with those previously described by Jakus.

5 276 Schaeffer Investigative Ophthalmology June 1963 evenly distributed throughout the cytoplasm. The nuclei are homogeneous. Changes occur early in this layer of stored corneas. Swelling of the mitochondria and formation of spaces within the cytoplasm are commonly noted after 12 to 24 hours (Figs. 14 and 15). The nucleoplasm becomes clumped and the nuclear membranes are generally indistinct after 36 hours. In late stages (Figs. 16-1S) large vacuoles are present, the cells appear shrunken, and degeneration bodies are noted. Discussion Observations made on corneas fixed immediately after enucleation in this study generally concurred with those previously described by Jakus. 1 " 2 The changes observed in this series were by no means constant for all cells at the various intervals of fixation; however, when evaluating the findings of several hundred micrographs a series of obvious progressive changes was noted. These more obvious and most frequent changes were pointed out in the figures of this paper. Gross architectural changes of the cells as a whole consisted of shrinkage and vacuole formation. These alterations were progressive and related to time in storage. The basic ground cytoplasmic pattern changed with time from a homogeneous granular structure to one of localized areas of cytoplasmic spaces and other areas of dense, more compact agranular aggregates. This sequence of events was most readily observed in the endothelium. Nuclear changes were marked and relatively constant for all layers. The homogeneous granular nucleoplasm became distorted into dense agranular aggregates. In late stages the nucleoplasm diminished in amount and the normally clearly defined double-walled nuclear membranes became indistinct and often had broken down. The interesting observation of the presence of large amounts of RNP granules and rough-surfaced endoplasmic reticulum in close proximity to the nuclear membranes of epithelial cells of stored corneas, and not of immediately fixed corneas, suggested that a transporting mechanism was involved during autolysis in these regions, i.e., a carrying away of nuclear particles Fig. 5. Thirty-six-hour epithelium (xl7,400). Further clumping of nucleoplasm (N) and breakdown of nuclear membrane (NM) with concentration of endoplasmic reticulum near these sites (ER). Disorganized mitochondria (M), Fig. 6. Seventy-two-hour epithelium (x5,900). Surface excresences (SE) remaining as old intercellular attachments. Cytoplasmic separation (S) and vacuolation (V). Nuclear shrinkage (N). Separation of interdigitating processes (IP). Maintenance of desmosomal connections (D). Inset (x25,700): Vacuoles (V). Disorganized mitochondrion (M). Fig. 7. Ninety-six-hour epithelium (xl6,500). Shrunken, indented nucleus (N) with poorly defined nuclear membrane. Accumulation of RNP granules at nuclear indentations (I). Separation of interdigitating membranes (IP). Note poorly defined mitochondria (M) with surrounding spaces (S).

6 Volume 2 UUrastntctural changes in moist chamber corneas 277 Number 3 Fig. 6. For legend see opposite page. b -IP i I M m. I M Fig. 7. For legend see opposite page.

7 278 Schaeffcr Investigative Ophthalmology June 1963 M. N ER 10 Fig. 8. For legend sec opposite page. Fig. 10. For legend see opposite page. V N CM M ER II ' N Fig. 9. For legend see opposite page. Fig. 11. For legend see opposite page.

Volume 2 Number 3 Ultrastructural changes in moist chamber corneas 279 Fig. 12. Ninety-six-hour stromal cell (*9,200). Shrunken cell with disorganized cytoplasm (C) and nearly empty nucleus (N). Fig. 8.

Cellular membrane (CM). Note scarcity of mitochondria and endoplasmic reticulum. Fig. 10. Thirty-six-hour stromal cell (xj.4,200). Nucleus (N). Mitochondrion (M). Cytoplasmic spaces (S). Fig. 11.

We were unable to find particles resembling nueleoplasm within endoplasmic reticulum connected to the nuclear membranes, although this was strongly suggested in many sections.

8 Volume 2 Number 3 Ultrastructural changes in moist chamber corneas 279 Fig. 12. Ninety-six-hour stromal cell (*9,200). Shrunken cell with disorganized cytoplasm (C) and nearly empty nucleus (N). Fig. 8. Normal stromal cell (x22,500). Nucleus (N). Mitochondria (M). Endoplasmic reticulum (ER). Fig. 9. Twelve-hour stromal cell (*18,500). Early clumping of nueleoplasm (N). Cytoplasmic space (S). Cellular membrane (CM). Note scarcity of mitochondria and endoplasmic reticulum. Fig. 10. Thirty-six-hour stromal cell (xj.4,200). Nucleus (N). Mitochondrion (M). Cytoplasmic spaces (S). Fig. 11. Seventy-two-hour stromal cell (*24,000). Nucleus nearly devoid of nueleoplasm (N). Swollen mitochondria (M). Endoplasmic reticulum (ER). by the endoplasmic reticulum. We were unable to find particles resembling nueleoplasm within endoplasmic reticulum connected to the nuclear membranes, although this was strongly suggested in many sections. Mitochondrial changes were similar to those described by others' 5 in starvation, toxic, and postmortem experiments with animals. In stored epithelial and stromal cells mitochondria were less frequent than in normal tissues. Swelling of the mitochondria occurred early. The internal structure was distorted and eventually lost, leaving small vacuoles in the cytoplasm. In some the outer membranes remained as shrunken structures surrounded by cytoplasmic spaces. In the endothelium we were unable to observe large vacuoles arising from mitochondria. Here large vacuoles appeared to form as cytoplasmic spaces containing debris near intercellular membranes, or around the nuclei; whereas many endothelial mitochondria, after initial swelling, became compact and changed to dense degeneration bodies. Gansler and Rouiller 1 pointed out in their starvation experiments with rat liver that mitochondrial swelling was reversible, but that swelling with a loss of cristae and subsequent vacuole formation or formation of degeneration bodies was irreversible. On these grounds it would appear that irreversible changes in the mitochondria of the corneas in our study occurred at different times for the different layers. In the epithelium loss of mitochondrial cristae and early vacuole formation was noted as early as 24 hours. In the stroma these changes and the presence of degeneration bodies was noted at 24 to 36 hours. In the endothelium early mitochondrial swelling with or without loss of cristae was followed by mitochondrial shrinkage, and only after 96 hours were degeneration bodies observed. It should be pointed out again that these changes were not constant for all cells at the various intervals of fixation. There is, therefore, a variation in degree of mito-

$/ " " \ wi»<i-,k"n M -IM 15 Fig. 14. For legend see opposite page.$

9 280 Schaeffer Investigative Ophthalmology June 1963 Fig. 13. For legend see opposite page. / " " \ wi»<i-,k"n M -IM 15 Fig. 14. For legend see opposite page. Fig. 15. For legend see opposite page.

Volume 2 Number 3 Ultrastructural changes in moist chamber corneas 281 P ' -'", NM -M 17 Fig. 13. Normal endothelium (xl4,800). Nucleus (N). Descemet's membrane (DM). Mitochondria (M). Fig. 14.

10 Volume 2 Number 3 Ultrastructural changes in moist chamber corneas 281 P ' -'", NM -M 17 Fig. 13. Normal endothelium (xl4,800). Nucleus (N). Descemet's membrane (DM). Mitochondria (M). Fig. 14. Twelve-hour endothelium (xl6,500). Cytoplasmic separation (S). Intercellular membrane (IM). Mitochondria (M). Descemet's membrane (DM). Fig. 15. Twenty-four-hour endothelium (x5,300). Nucleus (N). Swollen mitochondria (M). Intercellular membrane (IM). Descemet's membrane (DM). Fig. 16. Thirty-six-hour endothelium (x47,000). Nucleus (IV}. Swollen mitochondria (M). RNP granules (P)- Fig. 17. Seventy-two-hour endothelium Swollen mitochondrion (M). Cytoplasmic separation (S). Indistinct nuclear membrane (NM). Descemet's membrane (DM). Fig. 18. Ninety-six-hour endothelium (x35,400). Vacuole (V). Degeneration body (DB). Compact mitochondrion (M). Descemet's membrane (DM). 18 OM

11 282 Schaeffer j Ophthalmology June 1963 chondrial change between the different cellular layers and within individual cells of stored corneas. In general, we have observed irreversible changes as early as 24 hours of storage, and the longer these tissues are stored the greater these changes become. REFERENCES 1. Jakus, M. A.: The fine structure of trie human cornea, in Smelser, G. K., editor: The Structure of the eye, New York, 1961, Academic Press, Inc., pp Jakus, M. A.: Further observations on the fine structure of the cornea, INVEST. OPHTH. 1: , Gansler, H., and Rouiller, C: Physiologic and pathologic changes in mitochondria, Schweiz. Ztschr. allg. Path. 19: , Takaki, T. et al.: Electron microscopic studies of vacuolar degeneration, presented at the Fifth International Congress for Electron Microscopy, Philadelphia, Pa., Ito, S.: Post-mortem changes of the plasma membrane, presented at the Fifth International Congress for Electron Microscopy, Philadelphia, Pa., 1962.

Contains ribosomes attached to the endoplasmic reticulum. Genetic material consists of linear chromosomes. Diameter of the cell is 1 m

Contains ribosomes attached to the endoplasmic reticulum. Genetic material consists of linear chromosomes. Diameter of the cell is 1 m 1. (a) Complete each box in the table, which compares a prokaryotic and a eukaryotic cell, with a tick if the statement is correct or a cross if it is incorrect. Prokaryotic cell Eukaryotic cell Contains