Year : 1969 | Volume
: 17 | Issue : 3 | Page : 81--90
Adenwalla oration-1969- Cornea-the real window to the outside world
Dr. Rajendra Prasad Centre for Ophth. Sciences, All India Institute of Medical Sciences, New Delhi-16, India
L P Agarwal
Dr. Rajendra Prasad Centre for Ophth. Sciences, All India Institute of Medical Sciences, New Delhi-16
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Agarwal L P. Adenwalla oration-1969- Cornea-the real window to the outside world.Indian J Ophthalmol 1969;17:81-90
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Agarwal L P. Adenwalla oration-1969- Cornea-the real window to the outside world. Indian J Ophthalmol [serial online] 1969 [cited 2021 Apr 21 ];17:81-90
Available from: https://www.ijo.in/text.asp?1969/17/3/81/38518
Mr. President, respected teachers, promising young pioneers and Colleagues:
Let me at the outset express my deep sense of gratitude at the warm invitation offered to me by the Scientific Committee to give this oration. When I was first approached for this, I felt reluctant because I feel that many more young pioneers should be honoured for such orations if we at any time want to produce NOBEL laureates from this country. Whenever some of us are honoured abroad, a spate of emotional and sentimental reactions are visible on the horizon which come like a galaxy of stars in the dark sky of the night, it seems dawn is not far off but perhaps academically and in reactional maturity we are still in polar regions and if we do not change our attitude, dark clouds will never be cleared and the silver lining will never be visible.
India, at the moment, is passing through an age of gerontocracy where the revolutionary voice of the young is turned down as an immature exhibitionism and the voice of the old as sensible experience. While, on the contrary, what we need is to consider the voice of the young as passionate realism with a desire to uplift the country from the doldrums. In all processes of developments and self-reliance, risks have to be taken and we should start right now. It is not the equipment, not perhaps the illusory facilities or even the lure of money that obstructs our progress. If we believe in this we are giving a very low premium to our exuberant knowledgeable restless youths. Let us loosen the stranglehold of the so called old and experienced scientists and bureaucrats on the young scientists and see the galvanisation and results. Let these people be not frustrated and our country will be happy for this. Much effort will then not be required to keep our `brain' here and thus "brain drain" will be forgotten.
The craze to visit foreign lands and get recognition there will also end, if our young dynamic patriots know that such will not be considered as "Mecca" before salvation, and we become honest in the assessment of our own work at all levels including postgraduate examinations.
Many an eminent ophthalmologists have delivered this oration before me and many will - my only hope is that there will be a change of heart and young pioneers will be given an opportunity. It is not age and patronage only which should be respected, it is work and the excellence of work alone which needs to be recognized. I may add, let we of the older generation permit the spirit of youth to develop into restless human spirit, ever on the march towards the conquest of new horizons.
Coming to the subject proper 1 submit that a transparent healthy cornea is the real window through which an individual sees the dynamic marvels of the outside world since the start of life on earth. It is not the site of perception, relay, or interpretation of the various visual stimuli, but unless the light impulse in its proper character, wave length, and intensity is allowed to be refracted through the all important cornea, no perception or interpretation by visual cell stations can be made of the beautiful fantasy created by nature and man. The same may be said for the lens, but, when it is hindering the light rays to pass, it can be comparatively easily removed, while the same to be clone for cornea needs keratoplasty, which is far from satisfactory as vet.
The cornea once damaged by any pathological process, which has affected the Bowman's membrane or deeper, leaves a permanent mark of varying grades ranging from nebular, or macular, to leucomatous opacities. It is this stigma of a corneal opacity in the central seeing part, that leaves the person handicapped to a greater or lesser extent although in the recent years keratoplasty has provided a replacement of the damaged cornea by a more clear `real window', yet it has many a limitations, which will be improved by the progress of science and with the passage of time. However, we cannot neglect or forget the continued efforts in exploration for the mechanism of maintenance of perfect transparency of the cornea by the body. Constantly more information is being added to the processes of dialysis, the Donnan equilibrium, the "binding" of cation, and the active transport in the cornea, which either singly, or more likely collectively contribute in preserving the precious transparencies of our real windows to the outside world'. Keeping apace with space exploration, our medical science is also unravelling many a mystery of biology, which in other words were and are our ignorance.
From the clinical stand point, the damage to the corneal clarity is the cause of more visual incapacitation and even amblyopia or squint than any other single cause, specially in countries like ours where trachoma, small pox, untreated or poorly treated conjunctival or corneal inflammations, lack of hygiene and prevalence of ignorance, fads and quacks are the pests of the community. A survey of Delhi urban School Children has revealed that 0.7 per cent of the children have a corneal opacity and incapacitating visual function to a variable extent, while 5.74 per cent of Delhi and Haryana industrial workers have corneal opacity, and 6.77 per cent of Delhi viral population has corneal opacity. It is this appalling fact that has induced us in the last few years to probe deeper into the basis of maintaining the transparency of cornea by the body under the natural conditions.
The remarkable achievement of our body to maintain an optically transparent cornea is due to certain biological facts which characterize the cornea, such as:
It is almost an avascular tissue.It has two cellular limiting membranes, which constitute about 10 per cent of the entire tissue.The remaining 90 per cent is the stroma, and it contains mostly collagen and water.The characteristics of the collagen are probably not exactly the same as that of other avascular tissues, like cartilage.The cornea is bathed in fluid on either side - anteriorly by tear fluid and posteriorly by aqueous humour.
Though the cornea is in constant contact with fluid on both sides, it does not take fluids in any amount under normal conditions in the body. In fact it holds less water than what it is capable of, in vitro. Therefore, a high degree of dehydration maintained in the cornea, in vivo, has to be controlled by some mechanisms which draw water out of cornea. These mechanisms have to have a dynamic flexibility so as to keep the water content of the cornea more or less constant at all times, inspite of the changes in the osmotic pressure of its bathing fluids in the physiological or even most pathological states of the eye.
Earlier workers (Cogan and Kinsey, , ) believed that transparency and deturgescence of the cornea are maintained by the process of osmosis across its limiting membranes. This theory failed to explain certain important points, that is,
The osmotic pressure of tears and aqueous humour does not exceed that of stroma.At night, when no evaporation of tear fluid occurs, there no longer exists an effective osmotic force to facilitate the loss of water from the cornea and it should become turgid.Even when epithelium is denuded e.g. by trauma, no corneal edema may manifest.
Maurice  showed that epithelial and endothelial barriers are permeable to salts and water. He suggested that there must be some active process which is responsible for resisting the tendency of the stroma to swell. This active process is regulated by the two cellular layers-the epithelium or endothelium, or both, and it is solely dependant on the metabolism of the cells.
Taking the suggestion of an active transport, more evidence was given for the metabolic activities of the cornea in situ, which operate in maintaining its transparency (Davson,  ; Maurice,  ; Langham and Taylor,  ). Electron microscopic study of corneal endothelium lent full support to the presence of high metabolic activity required for an active transport (Smelser,  ; Kaye and Pappas,  ; Iwamoto and Smelser,  ). This study revealed the presence of many ovoid mitochondria with complex villous type crystae, large amount of granular rough surface component of the endoplasmic reticulum in direct continuity with the agranular vesicular reticulum and pinocytic vesicles in basal and apical cytoplasm. This evidence is consistent with the histochemical observations of enzymes participating in glycolysis, Kreb's cycle and Pentose shunt in endothelium, epithelium and stroma (Cogan and Kuwabara,  ;Morone and Manuelli,  ).
Having considered the role of the cellular limiting membranes of the cornea - the endothelium and epithelium, the investigative scientific attention was also focused on the stroma of the cornea. It consists of about 20 per cents of its weight by collagen lamellae. These lamellae are embedded as specific lattice pattern in the mucopolysaccharide gel suspension occupying almost all of the extracellular space available in the tissues (Maurice,  ). The nutrition of this avascular transparent tissue depends on the permeability of the mucopolysaccharidic gels for water and other metabolites. There is lot of experimental work on the mucoids of the stroma which provides strong evidence to the prominent role played by the polysaccharides in nutrition and maintaining the swelling pressure of the cornea (Stary,  ; Hedbys,  ). Leoven  speculated that the water-binding and light-transmitting properties of the cornea depend, at least in part, on the mucopolysaccharides which form a complex with collagen. Maurice  studying the birefringence of the cornea observed that all the swelling takes place in the ground substance while the collagen fibres remain unchanged.
With the advances in electronics, the transcorneal potentials were studied in greater detail, and this provided more proof to the existence of an active transport mechanism in the cornea. However, at present different authors consider either the epithelium or endothelium or both as the sites of active transport mechanism in the cornea. The suggestion that the corneal potential originates entirely from active transport has been made by Green  . He deduced this because the short circuit current (an index of active ion transport) remains unchanged after distortion of the excised cornea, that is, free of stress. Green  comparing the transcorneal, transepithelial, and transendothelial potentials, indicates that the transport system is located in the epithelium. The transepithelial potential is about 675'c of the total transcorneal potential, and this is likely to be due to a greater anion shunt across the epithelium. The author concludes that the transport characteristics of the cornea with regard to sodium and presumably to chloride are unquestionably located in the epithelium, with no active ion transport characteristics in the endothelium. More recently with the use of glass micro-electrodes, the electrical potential profile determined in isolated frog cornea shows that the stroma is always negative (upto minus 32 m v ) with respect to both endothelial and epithelial bathing solutions (Candia, Zandunaisky and Bajandas  ). A model excluding the epithelium as the location of a previously described chloride pump, indicates that the negativity of the stroma is consistent with the location of the pump only in the endothelium of the cornea.
Reviewing the whole situation, Harris  considered the maintenance of the normal corneal hydration in vivo as follows:
There is a certain structural rigidity which is imparted to the cornea with all layers intact and connected to the adjacent sclera which restricts swelling.There is an exchange of water and electrolytes, particularly across the endothelial surface, which because of the normal swelling tendency of the cornea would lead to corneal stromal hydration. This is balanced by the excretion of fluid from the cornea. The composition of the excreted fluid will vary with the state of hydration of the cornea and may be quite hypotonic when the cornea is hydrated.The dehydration of the cornea by evaporation is also a factor of maintenance of corneal hydration.The relatively slow movement of fluid through the cornea plus a continuing secretory activity on the part of the endothelium will account for the localized hydration which accompanies the isolated break in the endothelium or the epithelium.The epithelium does not appear to be involved in the excretion of fluid from the cornea.
With this background of many unsolved riddles involved in the energetics of the cornea and maintenance of its precious transparency, we explored further the respiratory metabolism of the cornea (Agarwal Bhuyan, Mishra and Ghanekar  ) studied the total cellular, extracellular and intracellular space of the goat cornea, and then investigated the effects of various metabolic inhibitors on the extracellular and intracellular water of the cornea (Agarwal, Bhuyan, Mishra and Ghanekar  to be published). Thereafter we undertook a qualitative analysis of mucopolysaccharides in normal corneas (Agarwal and Goswamy  ). These studies provided us the following information.
The experimental evidence obtained from the above studies demonstrate the presence of a potent cytochrome system in the cornea. This system which transfers electrons or activated hydrogen atoms from the metabolites to molecular oxygen, through a chain of electron carriers is referred to as the respiratory assembly or electron transport chain.
We found that cornea contains a potentially highly active metabolism. Demonstration of DPNH cytochrome -c- reductase and cytochrome oxidase systems in the epithelium, endothelium and stroma of cornea, established the presence of the conventional respiratory chain consisting of various cytochromes. Chemical energy liberated during the oxidation of various metabolites is conserved as the bond energy of ATP by the phosphorylation that accompanies respiration.
The study has been conducted to estimate the capacity of mitochondria from corneal layers to couple the respiration to phosphorylation of ADP to ATP, and this was compared with the heart muscle mitochondria.
From the results depicted in [Figure 1] it is interesting to note that in epithelium and stroma the oxygen consumption is initiated and accompanied by vigorous evolution of oxygen, whereas in endothelium and heart muscle there is only consumption of oxygen with absence of its evolution.
The overall respiration of mitochondrial preparations from corneal layers and heart muscle are represented in [Figure 2], which shows the mitochondrial respiration from endothelium is 2.5 times that of heart muscle, and the apparent value of respiration from mitochondrial preparation of stroma and epithelium (evolution + consumption) is nearly half of the heart muscle.
The phosphate oxygen ratios (P:O) obtained from various mitochondrial preparations vary from 2.5 to 3.0 [Table 1]. This means that 3 moles of ATP are synthesized during the consumption of one micro atom of oxygen when the substrate used is pyruvate.
It has been significant to note that the capacity of corneal endothelial mitochondria to synthesize ATP is nearly three times that of the heart muscle. This remarkable capacity provides a direct evidence that cornea is supplied with a rich source of energy, so very essential for the maintenance of its precious transparency. Corneal epithelial and stromal mitochondria are half as efficient as heart mitochondria in coupling ATP synthesis to respiration. This indicates that the metabolic activity of these layers is also significant, and play some role in keeping the cornea in natural deturgesced state. The reserve source of molecular oxygen in situ is thus made available to the avascular tissue of cornea, which has to spend the major part of its energy in maintaining its transparency besides the normal metabolic process.
Presence of a high activity of the Na + and K + activated ATPases in epithelium and endothelium suggest their role in active transport. These two layers also possess a highly active catalase. Endogenous source of peroxides, generated in situ, perhaps by enzyme systems like DPNH oxidase, glucose oxidase etc., available in these corneal layers enhance the importance of catalase. Therefore, cornea not only contains a highly active respiratory assembly which synthesizes ATP, but also a potent catalase - peroxide system which can serve as a reserve source.
From this work on the respiratory metabolism of the corneal layers, a theory of autoxidative respiratory mechanism has been postulated. Though the basic mechanism of respiration at cellular level is operated through the conventional respiratory chain, this avascular tissue is unique in that it is endowed with a potentially active catalase system and has an endogenous supply of peroxides. Thus it seems that the high energy demand of this tissue for maintaining its transparency and other metabolic functions is mostly met through the reserve source of in situ supply of molecular oxygen liberated by the hydrogen peroxide through the intervention of catalase.
The total cellular water in cornea was determined by using D 2 O (Deuterium oxide), as the tracer of water. The extracellular space in cornea was determined in terms of xylose. Intracellular space then was calculated by subtracting extra-cellular space from total space.
The extracellular space in cornea has been found to be 82 per cent, and this is in keeping with its anatomical structure. The concept that stromal lamellae embedded in mucopolysaccharidic gel constitutes the major portion of this tissue, is based on the observation that oedema of the cornea is caused by retention of water by the mucoid, whereas the hydration of collagen remains the same. A voluminous extracellular space thus can offer freedom of movement to the various nutrient substances, ions and water [Table 2].
It is interesting to find that under the influence of various metabolic inhibitors, the total corneal space is considerably increased, whereas the extracellular space is remarkably reduced. An increase in the total space signifies an increase in the hydration of the cornea. It is known that this increase is due to the high water binding capacity of the mucoid present in the extracellular space. Therefore, a decrease in extracellular space in presence of various inhibitors can take place, because this extracellular space gets occupied by the swollen mucoid [Table 3].
Inhibition of the corneal respiratory assembly and the normal spaces (as determined by deuterium oxide or xylose), caused by cyanide, 2:4 -dinitrophenol and azide, clearly shows that there is need of energy for maintaining the water balance.
Cyanide is a potent inhibitor of cytochrome oxidase, while 2:4 -dinitrophenol and azide are known to uncouple the oxidative phosphorylation. Cyanide causes more inhibition of the total space than that of the extracellular space. This suggests that cyanide acts at the subcellular level.
Ouabain (G-strophanthin), a cardiac glycoside, inhibits the process of active transport by inhibiting the Na + and K + activated ATPases of the epithelium and endothelium. In cornea Ouabain inhibition of the extracellular space is nearly 40 per cent less than that of the total space.
Fluoride and malonate inhibit the glycolytic process. They have no influence on the extracellular space, but their increasing the total space by 30 per cent shows that they act at the cellular level.
Increasing the strength of cyanide by 100 fold causes about 77 per cent inhibition of the water transport. Of all the inhibitors studied, cyanide is most potent, next in order come 2:4 - dinitrophenol and Ouabain.
The evidences obtained from the investigation of corneal spaces along with the effects of the inhibitors, lends full support to the presence of active transport of water or ions or any other substances across the cellular layers of the cornea.
With a very prominent role ascribed to the mucopolysaccharide for providing nutrition and in maintaining the swelling pressure of the cornea, and thus its transparency, our attention was also attracted to know little more about these mucopolysaccharides. We undertook a qualitative analysis of mucopolysaccharides in normal corneas, and compared the various fractions of mucopolysaccharides in goat, chicken and frog corneas. This fractionation was affected by Sephadex (A-50) Column chromatography with a gradient system of elution.
From this investigation we observed that cornea has two main anthrone-positive peaks, which correspond to the II and V peaks obtained by carbazole reaction (galactosaminoglycans), and the latter are usually six in number in the goat cornea. Whereas the chromatogram profiles of the galactosaminoglycans in normal corneas of goat, chicken and frog are characteristics, yet they differ from each other by the decrease, increase or absence of the low or high molecular weight fractions of these mucopolysaccharides. These profiles have provided a basis for comparison with the pathologic states of the cornea, and this investigation is underway to know more how the mucopolysaccharide fractions help maintain transparency or get altered when transparency is challenged by the cornea damage. On the other hand the individual peculiarities of goat, frog and chicken corneal galactosaminoglycans may be responsible for their variable water-binding capacity, or their difference in response to trauma, or else for a very specific antigenic make up responsible, possibly, for the graft reaction in keratoplasty.
Another inference drawn from this work is that when glycosoaminoglycans (kerathan sulphates) are less in the cornea, the cloudiness and antigenicity is low. This view has since been supported by Morisue and Toribe  who have reported that the more cloudy the cornea, the greater the decrease in total amount of mucopolysaccharides and that the keratosulphates show more decrease than chondroitin sulphates. They found the quantity of mucopolysaccharides in keratoconus to be normal. In parenchymatous keratitis, both keratosulphates and chondroitin sulphates are reduced, while in cloudy corneas due to systemic diseases e.g. measles and small pox etc., the keratosulphates are reduced markedly and chondroitin sulphate only slightly. More recently we have observed that keratocytes have more acid mucopolysaccharides and particularly keratan sulphates (47 per cent) and that its turnover within the cell is higher as compared to the extracellular keratan sulphate [Figure 3]. This might suggest that the keratocytes in the stroma of cornea play an important role in the up keep of adequate hydration for the maintenance of corneal transparency (Agarwal and Nag - to be published).
In view of the observations made here the inadequacies and the imperfections of our knowledge of corneal transparency is fully exposed especially the endogenous source of Hydrogen peroxide work on unravelling this is in progress and let us hope we will provide some answer in the near future. Sclera which has the same biochemical content is opaque while the cornea remains clear. What makes for this difference? Is it the metabolism, the avascularity, the arrangement of fibres or some intricate mechanism involving several processes-one needs to explore. I am reminded of the coal and the diamond. Both are carbon in pure form, one is black and opaque the other is shining and transparent. It is only the arrangement of molecules and atoms that makes for this difference. Does the difference between the sclera and the cornea bear the same analogy?
The more we know the more we realise how little do we know. I dedicate this oration to the upcoming youth in the hope that they will unravel many more facts about cornea which we of today are ignorant about.
Prof. R. K. Mishra and Miss Ghanekar from Biophysics department were my co-workers in some of this work and I am thankful for their help. My thanks and appreciation is also recorded for the good assistance in some aspects of the present investigations by Drs. K. C. Bhuyan, S. G. Nag and the others from our department. Finally I gratefully acknowledge and admire the great help afforded by Dr. Subhash Goswamy for some aspects of this work and in the preparation of this manuscript.
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