STIRLING, M.D., SC.D., F.R.S.R, Regius Professor of the Institutes

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1 ON THE EPITHELIUM OF THE CORNEA. By WM. STIRLING, M.D., SC.D., F.R.S.R, Regius Professor of the Institutes of Medicine, and D AVID SKINNER, M.A., Student of Medicine in the Universty of Aberdeen. (PI. XIII.) [From the Physiological Laboratory in the University of Aberdeen.] OUR attention was directed to a peculiar appearance observable in the corneal epithelium of the rabbit, which we were led to investigate. The cornea was that of an albino rabbit which had been hardened in a mixture of chromic acid and spirit, usually employed for hardening the retina. Sections were then made and stained with various dyes. On examining a vertical section of a cornea which has been stained with picro-carmine and mounted in glycerine, we get such a view as is shown in Fig. 1, P1. xiii., where a represents the epithelium covering the anterior surface of the cornea proper, b; c the posterior elastic lamina. The picro-carmine has stained the nuclei of all the cells red, their substance yellow, and the cornea proper and elastic lamina red. We wish to direct especial attention to the layers of cells that cover the anterior surface of the cornea. The usual arrangement exists, i.e. the lowest cells resting on the cornea are more or less columnar, whilst the cells above them are more irregular, and on the surface they gradually pass into squames. The staining with picrocarmine, however, brings a peculiarity of this lower layer of columnar cells very clearly into view. As seen in situ the general mass of each cell is yellow and the nucleus red, but the base of each cell has attached to it a clear narrow band, hem or stripe, highly refractive and occupying from ;- A of cell, according to the height of the cell, for they vary in height (d, Fig. 1). In unstained preparations one gets at first the notion that this is due to an anterior elastic lamina, such as occurs in the cornea of many animals. That this clear refractive band is not an anterior elastic lamina is shown by the following facts: 1st. The posterior elastic lamina is stained red by the picro-carmine, whilst this band is stained yellow. 2nd. On examining it more closely it is seen not to belong to the cornea proper, but to the bases of the lowest layer of cells, each of wbich has a clear hem; and it is

2 336 W. STIRLIVG AND D. SKINIER. simply by these clear hems being placed end to end that the appearance of a hand is produced. Further, in this stripe there are to be seen fine lines mapping it out into small blocks, each corresponding in breadth to the base of a cell. The clear stripe which has so mnarked an affinity for the picric acid clearly belongs to the cells, and has nothing to do with an anterior elastic lamina, which is absent in the rabbit as it is in some other animals, e.g. cat. Fig. 1 d is very instructive, for it shows a part of the corneal epithelium detached from the cornea proper, each cell showing its clear hem. We have described this stripe as it is seen in a section of the cornea of the albino rabbit, but it is not peculiar to this animal, for we have found it in the corneal epithelium of the ordinary coloured rabbit also. It does not require even picro-carmine to bring out the stripe clearly, for if the sections are placed for twenty-four hours in one-quarter per cent. osmic acid, after hardening in chromic acid and spirit, the stripe stands out clearly. This method of putting sections hardened in chromic acid for some time into osmic acid afterwards, is a most invaluable one, many fine details being then observable which were formerly only indistinctly seen. We have not observed this stripe so nmarkedly on hardening the cornea merely in dilute chromic acid or in picric acid. The columnar cells forming the lowest layer are not all of the same height, nor of the same size, as generally represented. This is well brought out in Fig. 2, a and b, and Fig. 2c shows the bases of these cells, and here we note that they are not all of the same size at the base. We now proceeded to isolate the corneal epithelium, and this we did by placing it for a day or two in iodised serum, which rapidly d;ssolves the cement and the cells float free. The lowest columnar cells are always slightly expanded at their bases, and the clear band is not so marked as after hardening. Their appearance is shown in Fig. 2 a, taken from a preparation isolated by iodised serum. From the middle layers a few prickle cells are always obtained of various forms, and Fig. 2 b shows the difference in the height of these cells. From above this layer we have succeeded in isolating smaller cells-digitate cells, whose teeth fit in between and grasp the apices of several columnar cells. WVe shall allude to these more fully presently. Prickle cells can also be easily isolated, Fig. 2 e. We have also examined the epithelium of the ox and sheep. In the ox the number of layers of epithelium is muclh greater and the cells of the lowest layer are always much taller than the similar

3 EPITHELIUM OF CORNEA. 337 epithelium from the rat, rabbit or cat. This is seen at once in vertical sections, nor have we found the clear stripe so marked here as in the sheep or rabbit. Fig. 3 gives a view of a section of the cornea of the ox hardened in a similar way, but certain of the appearances however are peculiar and apt to lead one into error, unless controlled by examining other preparations. Every here and there groups of short broad cells (b) are to be observed, occurring between groups of tall cells (a). This is not a normal appearance but due to the swelling out of those at b compressing those at a, and thus giving rise to this peculiar appearance, which we have also seen in the rabbit's cornea. It is easy both in the cornea of the sheep and ox to detect several layers of prickle cells, such as occur in stratified epithelium generally. We find that Rollett* has also noticed in the lowest layer of cells of the cornea of some animals a basal 'hem' or 'border'. The method of preparation which we have indicated causes it to stand out most pronouncedly in the cornea of the rabbit. Rollett figures such a cell from the frog's cornea. He says, "The expanded basal borders of the cells are so applied to each other, or are so superimposed upon one another by their thin edges, that the borders of the several cells seen collectively in situ make a bright stria which forms the line of demarcation between the epithelium and the corneal tissuet." This is also the view we take of the matter, for the base of each cell is always expanded somewhat and broader than the part immediately above the base (Fig. 4, a and b). We now proceeded to isolate the cells from the cornea of the ox by means of iodized serum. Fig. 4, a and b, shows the lowest layer of columnar cells. They are very remarkable on account of their being so elongated; they are as elongated as any epithelium in the body. Invariably their base is flat as described by Prof. Cleland + and always expanded as we have figured it, resting directly on the cornea and sending no process into it. The upper end is pointed or may be slightly branched. The oval nucleus is generally situated above the middle of the cell, which is there slightly swollen out as indicated in the figure. The clear hem is very delicate and not nearly so pronounced as in the rabbit. Above these cells Prof. Cleland in 1867 described cells of peculiar shape, to which he gave the name 'digi- * Rollett, article Cornea in Stricker's Histology, English Edition, p t L.c. p $ On the Epithelium of the Cornea of the Ox, Journal of Anat. and Physiology, Vol. ii. P. 362.

4 338 W. STIRLING AND D. SKINNER. EPITHELIUM OF CORNEA. tate'. "They are of irregular shape, about twice as broad as the columnar epithelium, but by no means so elongated." He describes them as " rounded and even in outline at the superficial extremity," (but they are not all so (Fig. 4 c), though some are,) and "jagged at the other, and send in processes or 'digitations', which may be three or four in number, and which appear to fit in between the tapering points of the columnar cells." We have directed especial attention to these peculiar 'digitate' cells, for their existence has been denied by Rollett*, but there cannot be the slightest doubt of their existence, and in fact we have observed them in situ in vertical sections of the cornea of the ox, especially where the cement substance and outer part of each cell has become stained yellow, as not unfrequently happens in tissues kept for a long time in chromic acid. They present much the appearance of a molar tooth with its fangs, dipping in between the apices of the columnar cells, and Fig. 4 b shows how they are placed, each cell being in relation with the apices of several columnar cells. In the figure one cell shows three processes, another four. We have already indicated that we have succeeded in isolating similar 'digitate' cells from the cornea of the rabbit. We have, therefore, confirmed Prof. Cleland's observation, and are at a loss to know how they could have escaped Rollett's observation, so easy is it to isolate them. Above this layer of digitate cells we have a stratum of smaller cells some of which are 'digitate' (Fig. 4 d), and there is a gradual transition between them and small prickle cells (which occur in very large numbers in the cornea of the ox and sheep), and gradually as we pass upwards we come to the large flat squames on the surface. EXPLANATION OF FIGURES, PLATE XIII. Fig. 1. V. S. cornea of albino rabbit, hardened in a mixture of chromic acid aind spirit; a, corneal epithelium; b, cornea proper; c, elastic lamina, posterior epithelium removed; d, clear hem on corneal epithelium. Fig. 2. a, various forms of cells from lowest layer of columnar epithelium isolated by iodised serum; b, similar cells; c, bases of a and b; d, ' digitate' cells from higher layer; e, prickle cells further up towards surface of cornea. Fig. 3. Peculiar appearance sometimes seen in vertical sections of cornea of ox; a, tall compressed cells; b, similar cells swollen out and compressing a. Fig. 4. Corneal epithelial cells of ox isolated by iodised serum; a and b, lowest layer of cells; b. apex grasped by fangs of a digitate cell; c, ' digitate' cells; d, smaller digoitate cells higher up; e, prickle cells. * Op. c. p. 424.

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