On inflammation

Chapter I

Inflammation, the pivot, according to the late Professor Bennett, upon which medical philosophy has revolved in all ages, is a com¬ prehensive term which embraces a number of changes in the elements which compose animal tissues. The nature and sequence of these changes are ill understood, and are consequently subjects of unending controversy. And when it is considered that the normal structure of the tissues in which the changes take place, is just as much subject of controversy as the inflammatory changes which take place in them, we cannot wonder that the nature of inflammation continues to divide successive generations and the different contemporary schools of pathology. In beginning the study of inflammation, it becomes therefore absolutely necessary to consider first how much it is possible to know of the healthy tissues.

Investigations into the nature of connective tissue having led me to different conclusions from those which have been hitherto taught, I applied them to an experimental study of the nature of inflammatory change, and I believe I have found in them a key to the perplexing difficulties which meet any one who endeavours to reconcile the conflicting observations of the schools of Strieker, Cohnheim, and other contemporary writers. In this number, I shall therefore, as a fit introduction to the subject, endeavour to explain what I believe to be the general plan on which all the connective tissues are formed. As the studies to which I have referred have been largely made on the cornea, and as most of the observations by myself and others to which I shall after¬ wards refer, have been made on the inflamed cornea, it is con¬ venient that I begin by a study of that structure. I will then

A show that it can he considered as typical of the other forms of the tissue.

The substance proper of the cornea may, like other forms of fibrillary tissue, but not so readily as some of them, be resolved into exceedingly fine fibrillse, whose diameter it is impossible to measure with accuracy.

In a cornea which has been sealed on an object-glass in aqueous humour, and in which a few incisions have been previously made in different directions, there may, in some instances, after twenty-four to forty-eight hours, be observed, lying free between the edges of the incisions, long, even, straight-contoured bands of various length. Their breadth in the frog approaches the diameter of a human red blood-corpuscle, and they are all of equal breadth and thickness.

In the edges of the incisions, similar bands may be seen lying parallel to each other, and occasionally they may be seen in uncut portions of the cornea. A cornea similarly treated, and sealed up in a mixture of gold solution1 .and acetic acid, occasionally shows the same appearances.

Considering the fibrilla as unity, I shall for convenience apply the term primary bundle to these bands.

Tig. 1 represents two of a group which were examined in aqueous humour.

In sections of the cornea treated in gold, and in teased preparations variously ob¬ tained, broader bands of fibrillary substance can be seen more or less perfectly isolated.

These correspond to the so-called “ bundles " of fibrillary tissue, the existence of the definite element which I have designated as a “primary bundle" not having been recognised. The “bundle" of authors I shall distinguish as a secondary bundle. It is composed of a number of primary bundles applied parallel to each other.

The anterior surface of a cornea carefully examined in aqueous humour, may be frequently seen to be intersected by clefts. They are recognisable by the distinct double contour of their borders, and their detection is facilitated by examining a frog’s cornea in blood serum which contains a considerable number of blood-corpuscles. The red corpuscles, in virtue of some attraction or current, find

1 The gold solution here and afterwards referred to is prepared by dissolving 1 gramme. (15 grains) of chloride of gold in 200 cent. cub. (7 ounces) of distil] ed water.

Fig. 1. — Primary bundles from a frog’s cornea in aqueous humour.

— Hartnack, Obj. 8, Oc. 3.

their way into the clefts, and, being drawn into the narrowing channel, they are seen at some parts of it as long narrow bodies.

On careful examination, it can be sometimes seen that even at the narrowest parts the superficial epithelium does not bridge the cleft, but dips into it over each edge. The clefts are wider nearer the limbus, and run obliquely into the substance of the cornea, occasionally diverging as they approach its centre. When once seen, they need never afterwards be confounded with the nerves of the cornea. They are more easily recognised in inflamed preparations.

These clefts indicate a further arrangement of the fibrillary substance into tracts or compartments, which, if the phraseology of “ bundles ” is to be adhered to, might be designated tertiary bundles.

Horizontal layers of cornea substance, thicker than the lamella} seen under the microscope in a vertical section, may be detached from a cornea which has been for some days in weak acetic acid. Those, I believe, have a relation to layers which actually exist, because in the same animals the relative number of lamellae so obtainable is approximatively the same, and because, as I shall subsequently show, they correspond to continuous layers of flat cells in the substance of the cornea, which, we are justified by all analogy in assuming, cover free surfaces.

When a cornea is placed 15 to 25 minutes in solution of chloride of gold, and is then allowed to remain exposed to a bright light for several days in distilled water, made faintly sour by adding a few drops of acetic acid, there will usually be seen interspersed through its substance large nuclei, surrounded by a dark purple granular deposit, which is continued in straight projections of varying breadth in all directions, their anastomosis forming a network.

This appearance is believed by many histologists1 to coincide with a system of branched anastomosing cells, the dark purple deposit representing the cell and its processes. The constant presence of a nucleus in the centre of the mass undoubtedly ren¬ ders the interpretation a plausible one. But it is nothing better than a hypothesis. If any one chooses to say that the presence of a nucleus in the central points of a network which varies exceed¬ ingly even in animals of the same species, is no proof that the net¬ work stands in the relation of a cell to the nucleus, the objection is unanswerable. All that the gold preparations have shown is, that the constancy with which the metallic deposit has taken place in the same general form indicates a structural peculiarity

1 See, for example, Rollett, Strieker’s Handbook p. 1101 (German edition).

mm

m of the cornea, and the uniform presence of a nucleus in the central points indicates that there is a cell there.

To ascertain definitely the nature of the cell of which the nucleus is so easily made visible, it is necessary to have recourse to other methods.

By examining the cornea of a frog or a transverse section of a larger cornea made with a sharp razor some hours after death, with an immersion lens, it is possible to see the same large nucleus which is visible in gold preparations, and immediately surrounding it a /

mass of branched protoplasm which by its ap- \

pearance and contours differs from the metallic \

gold deposit. Fig. 2 shows such a cell and j

processes from the ox cornea. ''

In a similar section sealed in aqueous ox^omeaT Fresh6 Mag^soo humour the cell and its processes become still diametersmore sharply defined. Fig. 3 shows a stellate cell from a sheep’s cornea after being sealed twenty hours.

W

Fig. 3. — Stellate cell from sheep’s cornea, twenty hours sealed in aq. hum. — Hartnack, Obj. 8, Oc. 3.

If a cornea has been subjected before the death of the animal to irritation sufficient to produce distension by serous effusion, and the excised cornea is placed for twenty-four hours in a solution of one part osmic acid in 200 water, transverse sections made with¬ out further preparation, and examined in glycerine, frequently show the stellate cells and their anastomosing processes. If the section is stained by logwood, the nucleus is also visible. It is then seen

Fig. 4. — Stellate cells with anastomosing processes, from a rabbit’s cornea. Osmic acicl

preparation. — Hartnack, Obj. 8, Oc. 3.

that some of the anastomosing processes are of great length. Fig. 4 shows a long anastomosing process connecting two cells in an osmic acid preparation of a rabbit’s cornea. The other processes are not shown. The length in the figure is the apparent length seen under the microscope as nearly as could be judged by the eye.

To demonstrate the branched cells of the cornea, I have lately practised a more satisfactory method than any other previously known to me, the object aimed at being to inject the interfascic¬ ular spaces by gold solution from the carotid. When sufficient pressure is maintained for a few minutes after the eyeball has become tense, the solution penetrates the cornea from the con¬ junctival vessels. I have then usually placed the excised eye¬ ball entire in gold solution for about an hour, and at intervals made small incisions at different parts of the corneal surface, and then placed the eye, still entire, for twenty-four hours in faintly acid water. When the operation has been successful, such a cornea presents under the microscope an appearance that will strike a histologist who sees it for the first time with astonishment. Long, straight, anastomosing fibres cross the field transversely and obliquely in all directions. These fibres have all the objective characters by which we distinguish elastic fibres, and the stellate cell-protoplasma, shrunk by the solution, is seen adherent to the central points of the network. Such preparations as I have hitherto obtained were examined in glycerine, in which unfortu¬ nately after a very short time the fibres cease to be visible.

In one of my preparations I cut the cornea in small fragments Avith a knife, and then succeeded in obtaining isolated cells with parts of the processes of various lengths still adherent.

My best preparations of this series were from the cornea of young rabbits.

In studying the nuclei of the different corneal cells, it is to be borne in mind that the stellate nucleus can always be recognised by its size. It is larger than the nucleus of any other cell Avliich is found in the cornea, and is the only cellular element that has been usually seen. The so-called cornea-corpuscle of authors is, as can be seen by their figures, nothing but this nucleus surrounded by amorphous deposit in the interfascicular spaces.

In a frog’s cornea, sealed in a mixture of gold solution and acetic acid, very delicate spindle elements are sometimes visible, but they are seen much more satisfactorily in a cornea injected from the carotid by gold solution. They can be readily recognised in the same field as the stellate cells, and in a good preparation their number is very striking. They lie in parallel chains, distant from each other by the breadth of a primary bundle. Two can fre-

B quently be seen lying side by side in the same interfascicular space. The small size of the nucleus — smaller than any nucleus that has been hitherto recognised — and the disposition of the scant protoplasma in spindle shape, distinguish them from the stellate cells. In a frog’s cornea injected by gold, it sometimes happens that the spindle

cells processes and nuclei are visible, whilst no- ~ m - sP

thing of the stellate cells but the nuclei is visible. ^

Larger spindle nuclei and cells are seen in the tracts of the nerves, and between the larger cornea bundles. The above methods are alone reliable for demonstration of the spindle cells. They are essentially distinct from the stellate cells, with fig. 5.— From cornea

t i P iii of a fr°s inJ‘ected hy

which thev can never be confounded when they gold solution from the

^ aorta. Sp, spindle cell

have been once seen. and nucleus, st, nuc-

mi . . . . pi • n lens of a stellate cell. —

I he size and relative position of the spindle Hartnack, obj 8, oc. 3. cells towards each other and to the stellate nucleus is shown in Fig. 5. The recognition of spindle cells in the cornea, apart from its interest, looked at from a purely histological point of view, is of great importance, as we shall afterwards see in studying the inflamed cornea.

In inflamed tissue they are shown conspicuously by nearly every method of investigation usually employed, and they have been confounded with white blood corpuscles on the one hand, and with products of “pro¬ liferation ” on the other.

Fig. 6. — Spindle elements in muscular

These spindle cells of the cornea fibre of fr°g- Gold and acetic acid mix-

x # ture. — Hartnack. Obj. 10 (immersion),

correspond in size and position oc. 3.

relatively to each other, and to the primary bundles with similar spindle elements in muscular fibres, which I described in a pre¬ vious paper in this Journal (September 1874), as will be evident from a comparison of Fig. 5 with Fig. 6.

In the previous paper on muscle, being then ignorant of the existence of these fine spindle elements in connective tissue, I termed them, in my description of Fig. 6, “ longitudinal fibres of elastic network,” and in the text I spoke of them as “ fine fibrillse which present oval swellings at short intervals.” I also stated that they communicate with a transverse network, which encloses the primary bundles in its meshes. Subsequent investigations have shown me that I was mistaken in regard to this supposed communication between the two sets of fibres. The longitudinal elements seen in Tig. 6 are chains of minute spindle cells, which lie between the primary bundles of muscle, and are distinct both from the muscle fibrillse and from the transverse network.1

When the temperature produced by the solution of caustic potash in an equal weight of distilled water falls to 107°-105° Fahr., a cornea, or part of a cornea, placed in the solution at that temperature becomes completely dissolved, with the exception of masses of cells, which can be seen isolated or in connexion with each other, when a part of the altered corneal mass is examined in a drop of the solution.

For a more complete account of these cells, the reader is re¬ ferred to the paper in which I described them, in the Pro¬ ceedings of the Royal Society, FTo. 155, 1874. In the Plate which accompanies that paper, figures are given, showing the relative

Fig. 7. — Flat cells from ox cornea by potash solution. — Hartnack, Obj. 8, Oc. 3.

position of the cells to each other. Fig. 7 shows the size and form of some of these cells.

In sealed aqueous humour prepara¬ tions in which the cornea has been care¬ fully cut obliquely, the same cells are seen isolated singly and in small masses, as is shown in Fig. 8. They are then seen as hyaline bodies, with a slightly projecting, faintly-tinged nucleus. In fig. 8. isolated flat ceils from a preparation in which they are visible,

obliquely cut sheep cornea two days 1 x d

sealed in aq. hum.— iiartnack, obj. they can usually be traced from the

8 Oc. 3. xj d

obliquely cut surfaces a certain dis¬ tance into the structure. Sometimes, but much more rarely, a preparation is obtained, in which their position relatively to the bundles is distinct. Such a preparation is represented in Fig. 9. It is then seen that the long narrow cells are applied to the surfaces

1 I take this opportunity of correcting a misapprehension which I find some readers of my previous paper have fallen into, through my neglecting to state that the acetic acid which I used was not glacial acetic acid, hut the weaker preparation known in England in commerce as “ concentrated.”

Fig. 9. — From frog’s cornea sealed in aq. hum. Long narrow cells seen lying on the primary bundles, a, Cell, b, Part of primary bundle from ’which the cell has disappeared. — Hartnack, Obj. 8, Oc. 3.

of the primary bundles, and that their edges are in contact after the manner of an epithelium, although, as will be seen from the figure, some of the cells become disintegrated, and disappear during the manipulation. In gold pre¬ parations, which have been pre¬ pared by placing the whole bulb for one to one and a half hour in the solution, removing the surface epithelium and making incisions in the cornea, and finally excising it and placing it in acidulated water, the polygonal and rounded cells can be seen to form layers in the substance of the cornea. Successful preparations of these cells by gold in the uninflamed cornea are very rarely obtained. In a very large number of cornese treated in different ways by gold, I have only seen them three or four times. If the cornea has been irritated for a few hours, they are more frequently seen in gold preparations.

It is to be noted that, although layers of the polygonal or rounded cells of the ordinary epithelial type can be seen of various sizes in the cornea, both in potash and aqueous humour preparations, patches can be isolated which are continuous with the long narrow cells that cover the primary bundles.

It was shown some years ago by Schweigger-Seidel, that there are cells in the cornea which show the dark lines of epithelium when treated by nitrate of silver. He was for an unusually long time in the minority of one, in which Carlyle says all truth begins. But the demonstration of the fact is not very difficult. Of all the animals on which I have operated, with a view to the demonstra¬ tion of this epithelium, the cornea of the mouse is the best adapted, and gives most frequent success. The cornea is placed, unwashed, in solution of 1 part nitrate of silver in 200 or 400 water for one to three minutes, dipped for a second in salt solution of the same strength, and then exposed to sunlight in glycerine. The pre¬ parations which succeed best are those in which the ground sub¬ stance is only slightly stained, being seen as faint brown islands scattered over a white field, instead of the darker and more abun¬ dant stained ground substance of ordinary silver-stained cornese. The dark epithelial lines can be seen traversing the white ground¬ work, generally appearing to be interrupted when they come in contact with a stained patch of ground substance, and this usually prevents the completion of the visible boundaries of the cell outline. The interruption is, however, only apparent, the black line being lost in the dark mass which surrounds it, as may be seen when a patch is only slightly stained. The line can then be fre¬ quently traced over and beyond a dark island, completing the cell-contour.

big. 10 shows part of a successfully stained epithelial surface in the interior of a mouse's cornea, in which this continuation of the

Fig. 10. — Part of a layer of flat (epithelial) cells in the substance of a mouse’s cornea Silver preparation. — Hartnack, Obj. 7, Oc. 3.

dark lines over the stained islands is in some parts visible. In the preparation there is another similar layer below the one repre¬ sented, cornea substance intervening.

It is important to note that the cells indicated by the dark lines in Fig. 10 belong to a class of larger size than those of the layers of polygonal cells seen in the mouse’s cornea in gold preparations.

The ordinary appearance presented by a cornea which has been treated by nitrate of silver, solid or in solution, is represented in pig. ii.

In such a preparation the silver has completely permeated the substance of the fibrillary bundles. Wherever there is an un¬ coloured space, wTe know that the ground substance is wanting to an extent sufficient to be entirely out of focus. In other circum¬ stances these spaces may be found filled with a black albuminate of silver. Sometimes a large unmistakable nucleus of the stellate cell can be seen in each space/ establishing the relation between the cell and the /space. But the space is larger than the cell, does not correspond to it accurately in outline, and the colourless space-processes are very much broader than the processes of the stellate cell. It is evident that the silver permeating the fibrillary substance with ease, either does not readily find its way into the spaces, or if it does, that the spaces are bounded by a structure that does not stain by nitrate of silver. We have seen that it is possible to show the dark lines of epithelial cells in the walls of the spaces ; and it is thus clear that either the substance of the cells does not stain, or that in such preparations the silver does not penetrate them. The fact of the dark lines being present would seem to show that there is a comparative difficulty of stain¬ ing as regards the cells.1 But that the epithelial layers in the

Fig. 11. — Section of a rabbit’s cornea, stained by nitrate of silver.

a , Lymphatic vessel enclosing nerve.

Line between the epithelium.

c, Continuity of space with vessel.

d, Ground substance.

e, Space in which the stellate cell lies.

cornea form an obstacle to the passage of silver solution is shown by preparations obtained by placing the bulb entire in the solu¬ tion for a few minutes, and subsequently removing the upper from the under layers of cornea substance. It is then often found that the silver solution has diffused itself equably through the whole

1 Analogous cells can be stained in the subcutaneous tissue by an interstitial injection of silver solution, as can be shown by thus injecting the subcutaneous tissue of a mouse’s back immediately after it has been killed.

of the fibrillary substance of one layer, but has not penetrated at all to the layer below it ; from which I infer that a layer of flat cells in the cornea, and the delicate sub-cellular membrane on which such cells always lie, present an obstacle to the easy pas¬ sage of fluid.

The demonstration of layers of flat cells in the cornea, and of an epithelium covering the spaces, shows that Recklinghausen was mistaken in supposing that the colourless stellate spaces and connecting processes are lymph canals (saft-kanalchen), channelled out of a substance which cements the fibrillary bundles. Each stellate space is formed by the superposition of lamellae, one over the other, the surfaces being at points corresponding to the situa¬ tion of the stellate cells, removed from each other to an extent sufficient to enable the distance to be appreciated under the micro¬ scope. In a silver cornea where the staining is pronounced, the distinction of the surfaces where they are in contact is lost to the eye. Thus, a “ space ” simply shows that two membranes, which are at other points in close contact, have at one point left a gap between them, and it is in these gaps that the stellate cells are to be found.

I have previously shown that the canals in which the nerves lie are lined by an epithelium, and are in reality large lymphatic channels, and, as will be seen by referring to Fig. 11, there are points in the lymphatic where the colourless spaces adjoining communicate with the uncoloured outline of the vessel. At these points it is certain that the fibrillary or ground substance fails, but it does not follow that the uncoloured breach in the outline of the wall of the vessel represents the extent of the actual free communication. That there is, however, a free communication at these points, between the lymph channel and the spaces, can be satisfactorily made out by watching the movements of bacteria in a cornea which has been placed in conditions favourable to their development. It can be seen that they move in the large lymph channel, but that at certain points one oscillating rod after another leaves the vessel and enters an adjoining space, and that always at the same point. When, as we shall afterwards see, white blood-corpuscles enter the inflamed cornea, they are sometimes found in considerable numbers in these lymph channels between the nerve and the Avail of the channel, and it can then be seen that they make their way first into those spaces which are next the vessel. Whatever the nature of the- communication may be — and it may not be more than a slight aperture at the angle of junction of the epithelium of the vessel — it is sufficient to permit the passage not only of fluid, but of minute solid particles between the lymph channel and the spaces.

The structure of the cornea, as shown by the above histological methods, can be described shortly as follows : — The fibrillary or ground substance consists of parallel bands, whose breadth is nearly the diameter of a human red blood-corpuscle, and whose thickness is somewhat less than their breadth. These bands, which I have described for the first time, and which I call primary bundles, are covered by narrow, elongated flattened cells, after the manner of an epithelium. Injected masses can be made to pass between the primary bundles, the spaces into which they pass being the corneal tubes of Bowman. These tubes are not, therefore, canals, with a complete unbroken wall, but are the spaces left between the bundles, on account of the somewhat cylindrical or bevelled form of their borders. The walls of a corneal tube are formed by parts of the surface of several con¬ tiguous bundles.

The primary bundles are collected in larger bundles, and on the surface of these larger bundles are found layers of small flattened polygonal cells. The junction of the cells of the primary bundles with those of the secondary bundles shows that there is a con¬ tinuous free surface for the passage of fluid from the sur¬ face of the layer into the corneal tubes between the primary bundles.

The secondary bundles are combined so as to form large flat¬ tened tracts, which are separated into distinct compartments by a membranous investment, which is covered by epitlielial-like cells. If it were necessary to give these tracts a distinctive name, the term tertiary bundles might be applied to them. These are further arranged in layers, which can be appreciated by the naked eye, and the surfaces of which are covered by the larger cells shown in Fig. 10.

The flat cells of the cornea being thus arranged in layers, may fulfil all the functions which are usually attributed to the epi¬ thelium of a serous membrane.

Between the primary bundles are chains of very small spindle cells. They are entitled to the name of cell, notwithstanding their small size, because they are composed of a well-marked nucleus, surrounded by a small quantity of protoplasm. The processes of each cell anastomose with those next it.

In spaces left by the separation from each other of the laminae.

formed by the bundles, are the large stellate cells, from the borders of which radiate in every direction, but mostly transversely and obliquely, a great number of transverse processes. The nucleus of this cell has formed the basis of the notions generally taught regarding the corneal cells. This nucleus and the surrounding spaces, filled with dark gold or silver deposit, constitute the appearances erroneously figured in the text-books as the corneal cell.

The processes of the stellate cells pass between the bundles of the upper and lower laminae ; and one result of this interlacing is

the binding together of the bundles and laminae. The anastomosing fibres pene¬ trate between the flat cells at the angles constituted by their points of junction.

The bundles are thus woven into a tex¬ ture by the stellate fibres. In Fig. 12 I sleep's1 co™a?”eIiSeine' 10“ p« llave represented an appearance seen in oTjt8,sao1c.rutl0n--Hartnack’ a section of a cornea, sealed up in a

solution of 1 part of common salt and 10 of water, which illustrates this relation between the stellate cells and the fibrillary tissue.

The circular apertures which are seen to form a margin round the shrunken cell, and under one of which a fibre dips to reappear through another, I have seen only in preparations thus sealed up in strong salt solution.

For the sake of readers who endeavour to reconcile my views with those expounded in the text-books, it is necessary to explain that the recognition of primary bundles, the fiat cells which cover them, the spindle cells which lie between them, and the layers of small polygonal cells, has been brought forward for the first time in the papers I have lately published. The distinction between the large epithelium seen in silver preparations and the stellate cells, as different anatomical elements, is also new, and was first brought forward in the same papers. The establishment of these anato¬ mical facts has been the result of several new histological methods, the wider application of which will, I believe, add much to our knowledge of the intimate structure of the tissues. Of these the chief are the isolation of cells by saturated solution of potash, the application of which will be further minutely described in the January number of the Quarterly Journal of Microscopical Science , in a paper on the Structure of Cartilage ; the detection of elements in situ , which have hitherto escaped notice by simply sealing fresh