ON LIVER NECROSIS AND CIRRHOSIS PRO- DUCED EXPERINENTALLY BY COAL TAR.

JAMES DAVIDSON. From the Depurtment of Pathology, University of Edinburgh.

(PLATES XXXII1.-XXXV.)

CIRRHOSIS of the liver has frequently been produced in animals by experimental methods. Papers on this subject have been published recently by Opie who experimented with chloroform and cultures of S. coli, Gye and Purdy with colloidal silica, Jaff6 with amyl alcohol and phenylhydrazine, Pearce with h8molytic immune sera, Wells with Witte’s peptone, and Findlay with manganese chloride.

The investigation here described was undertaken because in experi- ments where coal tar had been applied to the skin of rabbits’ ears to produce cancer, it was found that in several of the animals cirrhosis of the liver had supervened.

Rabbits are not altogether satisfactory for experiments on cirrhosis. Reagents which produce the disease in them fail to have the same effect on clogs, and from the spontaneous cirrhosis which occurs they would seem to be peculiarly susceptible t o the disease. The common coccidiosis must also be excluded.

Great care was taken in the selection of rabbits for these experi- ments. They were all free from coccidiosis and only one of the series showed an incidental infection (pseudo-tuberculosis). Coal tar, the pix Ziquida of the hospital dispensary, was applied to the external surface of both ears or was dissolved in ether and given as a subcutaneous injection.

Tar was applied to the ears a t intervals varying from a few clays to a month. The animals lost weight though they continued to feed as usual. If the applications were a t short intervals the loss of weight was continuous : it varied, however, considerably in different animals. The average loss was about 5 per cent. of the total weight per month: the greatest total loss recorded was 34 per cent. When the applica- tions were made a t longer intervals, the weight fell after each dose but subsequently rose slightly. Subcutaneous injections of the ether solution had a similar ‘action: controls injected with ether alone showed no loss of weight. A combined application and injection did not cause specially severe symptoms.

The effect on the liver varied according to the individual suscepti- bility of the animal and to the method of application.

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622 J DAVZDSON

(a) Acute necrosis.-The following is an example which illustrates acute necrosis and degeneration :-

The rabbit weighed 2730 grms. Tar was applied to both ears three times at intervals of 4 and 8 days respectively. On the 19th day after the first application the animal died and a post-mortem examination was done immedi- ately. The peritoneal cavity contained a small amount of clear fluid. The mesenteric vessels were slightly congested. The liver (wt. 131 grms.) showed an extremely mottled appearance (fig. 1). There were large pale yellowish areas of varied size, amongst which were interspersed smaller dark brown areas of more normal liver tissue. The whole surface was covered with petechial hzemorrhages. There was nothing to note naked-eye in the spleen or kidneys. Both lungo, more especially the right, showed small hsmorrhagic and Congested areas particularly towards the margins of the lobes. The urine contained albumen and a large quantity of leucin and tyrosin, but no diacetic acid acetone or sugar.

LlficroscopicaZZy the liver sections showed large areas of necrotic tissue where the outlines of the cells were very indistinct. In other areas the cells were much swollen and were undergoing degeneration. Numbers of polymorphonuclear leucocytes were present amongst the necrotic tissue.

This animal showed the most extensive acute necrotic changes of the liver in the whole series.

The most acute change observed was that of a coagulation necrosis of liver cells and this was present sometimes over large areas. The necrotic cells stained a very bright pink with eosin. There was a faintly staining irregular shrunken fragmented nucleus in a certain number of the cells (figs. 3 and 4), in others the nucleus had entirely disappeared. I n areas alongside the necrotic patches the liver cells showed extensive hydropic vacuolation and the majority of the cells which had survived necrosis were in this condition.

I n some other cases where acute changes were present, alongside the areas of necrosis were collections of liver cells which showed in the acidophil cytoplasm deposits of a substance staining a dark purplish colour. This substance formed a coarse meshwork in the cells similar to that seen in the hyaline degeneration (fig. 5) described by Mallory in human alcoholic cirrhosis. The necrotic areas were sharply defined : there was no gradual change from these to areas of healthy tissue: immediately adjacent to cells showing complete necrosis were cells hardly affected a t all.

There was an infiltration of small round mononucleated cells and polynuclear leucocytes in the affected areas and in the necrotic area there were scattered fragments of chromatin (fig. 8). This cellular infiltration. was present in the most markedly degenerated areas round the vicinity of the central veins. The liver cells immediately adjacent to the portal tracts were the healthiest, and sometimes there remained only a single column of these healthy cells (fig. 7). The cells of the middle and central zones were markedly degenerated and necrotic, more especially those of the central zone of the lobule.

There was little evidence of bile pigment; where it was seen, it

TAR AND LIVER NECROSZS 623

was situated amongst the remains of the degenerated liver cells and therefore would be in the bile canaliculi. Bile was absent in the ducts of the portal tracts. Multiple areas of hczmowhage were present throughout the necrotic and degenerating tissue.

(b) Subacute changes.-Another example in which the effect produced differs in degree, and which may be described as subacute, is the following:-

Rabbit, wt. 2350 grms. Tar was applied to both ears eight times, namely on the 2nd, 3rd, 7th, sth, loth, 15th and 20th days after the first application. The animal was killed on the 23rd day, the weight being 2880 grms.-a loss of 70 grms., i.e. 3 per cent. The post-mortem showed no abnormal naked-eye appearances. The liver and spleen were of average size. The surface of the liver was smooth and dark red in colour and appeared healthy on section: no chronic perihepatitis : no ascitic fluid. Microscopical examination showed that the normal definition of the lobular arrangement of the liver cells was lost. The cells were swollen and their outlines could hardly be distinguished.

I n the earlier or less acute stages of degeneration, the cells became swollen and their cytoplasm stained a paler red than usual with eosin and was of a homogeneous appearance. The outlines of the cells could still be seen but only with difficulty. Where large areas showed this change, the columnar arrangement of the cells could not be defined: the swollen cells encroached on the blood sinusoids and these were no longer visible. I n certain cells vacuoles were beginning to appear as more palely staining round areas (fig. 6) : these in time became almost colourless, but staining with Mallory’s fibrous-tissue stain revealed dark or pale blue contents. As the vacuoles increased in size, they coalesced and pushed the nucleus to the side of the cell. The result was that in many cases there were areas consisting of cells so vacuolated that the cellular outline could not be defined. Finally the large vacuoles contained no stainable material or only a few threads or granules which stained with the acid dyes (fig. 4). All these cases were examined in the fresh condition for fat but this was found only in very small quantity and entirely out of proportion to the amount of vacuolation present. This vacuolation was of the nature of a hydropic degeneration and the contents became gradually more and more watery.

The nuclei in these damaged cells showed various stages and types of degeneration. The majority of them were well-defined in outline and had a definite nucleolus. The chromatin consisted of very firm threads or granules, or was entirely absent, only the sharply defined nuclear outline remaining. Karyolysis and karyorrhexis were present and many of the cells showed nuclei which were much swollen and vacuolated. The Xupffer cells were swollen and had conspicuous nuclei. There was early proliferation of fibroblasts and it was frequently difficult to distinguish these from the Kupffer cells. The

The portal tracts showed no change.

There was no indication of glycogen being present.

624 /. DAYIDSON

portal tracts in certain areas showed an infiltration of small round mononucleated cells and along the margin of the tract these were seen to be extending amongst the adjacent liver cells.

(c) Chronic changes.-cases which survived the treatment for a considerable time and in which there were regeneration of liver cells and hyperplasia of the stroma'form a group which may well be compared with chronic atrophic cirrhosis in the human subject. The following is nn example :-

Rabbit, wt. 2520 grms. Tar was applied to both ears and again 17 days later (wt. 2130, a loss of 11 per cent.). A small papilloma had developed on the back of the left ear. Tar was again applied on the 28th day (wt. 2000 grms.), on the 44th (wt. 1850 grms.) and again on the 78th day (wt. 1900 grms.). On the 82nd day the animal died: it had lost 25 per cent. of its weight.

For comparison, details of another case are given, in which the rabbit received subcutaneous injections of tar dissolved in ether.

Rabbit, wt. 1700 grms. A dose of 0'5 C.C. tar in 0'5 C.C. ether was given on each of eight occasions, i.e. after 12 days (wt. 1700), 22 days (wt. 1600), 36 days (wt. 1360), 40 days (wt. 1270), 49 days (wt. 1250), 57 days (wt. 1220) and 64 days (wt. 1270). The animal died on the 69th day after the first injection. The rise in weight a t the end of the period was doubtless due to accumulation of ascitic Huid.

Post - morten both animals showed the characteristic changes of chronic interstitial hepatitis. To the naked eye the liver showed a characteristic granularity of its surface with irregular thickening of its capsule (fig. 2). There was much ascitic fluid.

Nicroscopically the tissue was divided into islands separated from each other by broad bands of fibrous tissue. These islands represented the original lobules in which necrotic degeneration had taken place and in some areas was still progressing. Accompanying this, there was also a process of regeneration in which the cells were large and in a state of active mitosis: the regenerated cells were found in these islands a short distance from the periphery. The columns of liver cells formed as a result of the regeneration were not arranged in definite lobules round a central vein (fig. 9) but appeared to grow in any direction. The individual cells formed by this regeneration were larger than the average normal liver cell, more rounded in outline and stained more faintly with eosin. In the area of regeneration the blood sinusoids were dilated and tortuous and green plugs of bile pigment were present in the canaliculi. The areas of degeneration and regeneration were side by side: the two processes were progressing simultaneously and in some areas the degeneration was affecting the regenerated cells.

I n the chronic cases another interesting change of structure was observed. The new liver tissue had the arrangement of a tubular adenornu. The cells closely resembled normal liver cells in cytoplasm and in the form of the nucleus : they were slightly undersized and were arranged in a single layer lining a cavity and forming definite tubular acini. The development of this could be followed step by step from a niass of cells in which there was no arrangement to the complete adenomatous tubule. Mitosis was frequent in these cells, and in the lumen of the tubules a pale, greenish brown pigment was present (fig. 10). Glyiin has described a case of cirrhosis of the liver which

JOURNAL OF PATHOLOGY.-VOL. XXVIII. PLATE XXXIII.

FIG. 1.-Liver : pale areas of acute necrosis and areas of hemorrhage.

FIG. 2.-Nodular appearance and chronic perihepatitis typical of atrophic cirrhosis.

JOURNAL OF PATHOLOGY.--VOL. XXVIII. PLATE XXXZV.

FIG. 3. -Area of acute necrosis : homo- geneous appearance of liver cells and disappearance of nuclei. x 80.

FIG. 4.-Advanced hydropic vacuolation. x 160.

FIG. 5.-Degeneration similar to that found FIG. 6.-Vacuoles in liver cells : gradual in alcoholic cirrhosis. x 390. disappearance of the contents. x 120.

FIG. 7.-Remains of healthy liver near a portal tract, the remainder degenerated.

FIG. 8.-CelIular infiltration round a central vein amongst necrotic tissue.

JOURNAL OF PATHOLOGY.-VOL. XXVIII. PLATE XXXV.

FIG. %-Island of regenerated liver sur- FIG. 10. -Adenomatous forniation of the rounded by the original degenerating regenerating liver tissue. x 160. liver cells. x 60.

FIGS. 11 and 12.-Regenerated liver tissue surrounded by well-formed fibrous tissue of typical atrophic cirrhosis. x 47.

FIGS. 13 and 14.-Regenerating bile ducts coalescing with columns of newly-formed liver cells. x 240.

TAR AND LIVER NECROSIS

showed adenocarcinoma and adenoma which closely resembled rabbit tumour. We have here an experimental demonstration of possible connection between cirrhosis and tumonr of the liver.

Hyperplasia of the fibrous tissue could be demonstrated in

625

the the

all stages from the earliest appearance of fibroblasts to that of well-formed broad fibrous tissue bands similar to those seen in atrophic cirrhosis in the human subject (figs. 11 and 12). There were numerous collections of small mononucleated cells lymphocytes and undifferentiated con- nective tissue cells chiefly in the portal tracts in the region of the bile ducts; they were occasionally abundant in areas where the fibrous tissue was beginning to appear between the columns of a degenerating lobule.

As already noted in describing the subacute changes, the Kupfw cells were swollen and had conspicuous nuclei and here also they seemed to be relatively resistant to the irritant.

The bile ducts in the portal tracts showed distinct proliferation. The cells were oval or polyhedral, the cytoplasm homogeneous and the nuclei large. At first they were arranged in irregular groups: later duct formation appeared and the cells acquired the ordinary cuboidal shape (fig. 14). I n some animals the proliferation of bile ducts was a very marked feature : they could be traced extending t o the islands of regenerated tissue. I n some cases it appeared as if liver cells had been formed by a proliferation of the cells of the bile duct a t its termination, but the possibility that this was merely a coalescence of the new bile ducts with the liver lobule could not be altogether excluded (figs. 13 and 14).

Reviewing these observations it is clear that in coal tar there is a substance which acts as an irritant on the glandular cells of the liver and produces changes closely resembling those seen in the so-called acute, subacute and chronic " atrophies " of the human subject. This has an important bearing on the relation to each other of the various types of atrophy, as it suggests that a single irritant may conceivably be responsible for the whole series: further investigation is still required to determine whether the changes are due to a singIe specific substance in the tar or to the combined action of several ingredients.

COSCLUSIOXS. I. Coal tar applied externally t o the ears of rabbits or injected

subcutaneously in ethereal solution causes necrosis of liver cells. 11. The acute changes are similar to those found in acute yellow

atrophy in the human subject. The chronic changes, including regeneration of the liver, closely resemble those of typical atrophic cirrhosis.

111. These observations suggest that the changes observed in acute yellow atrophy, subacute yellow atrophy and atrophic cirrhosis of the liver are different stages in one process.

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626 J. DAVIDSON

IV. The regenerated tissue is still susceptible to the irritant. V. The arrangement of the cells in the regenerated tissue is

irregular and in some instances adenomatous growth occurs.

I am much indebted to Professor Lorrain Smith for his kind assistance, advice and criticism in carrying out this work.

The experimental work of the investigation was carried out during six months’ leave of absence under the Carnegie Research Scheme for members of the University staff. A grant for research expenses was also obtained from the Moray Fund of Edinburgh University. A preliminary note appeared in this r7~~?.nal , 1923, vol. xxvi. p. 127.

FINDLAY . . . . . . . GLYNN . . , . . . . . GYE AND PURDY . . . . . J A F F ~ . . . . . . . . . MALLORY . . . . . . . OPIE . . . . . . . . . PEARCE . . . . . . . .

WELLS . . . . . . . .

11 . . . . .

,, . . . . . . . .

REFERENCES. Brit. Journ. Exper. Path., 1924, vol. v. p. 92. Brit. Ned. Journ., 191 I , vol. ii. p, 1192. Brit. Journ. Exper. Path., 1922, vol. iii. pp. 75,86. Ibid., 1924, vol. v. p. 238. Frankfurt Zeit. f. Path., 1920, vol. xxiv. p. 241. Principles of Path, Histology, 1914, p. 504. Journ. Exper. Med., 1910, vol. xii. p. 367. Journ. Bxper. Med, 1906, vol. viii. p. 64. Journ. Med. Research, 1906, vol. xv. p. 99. Trans. Chicago Path. Soc., 1903, vol. v. p. 240.