Haematoxylin and eosin, and methylene-blue and eosin

Transcription

1 THE HISTOLOGICAL CHANGES PRODUCED BY THE INJECTION OF ADRENALIN CHLORIDE. BY W. B. DRUMMOND, M.B., C.M., F.R.C.P.E. (Four Figures in Text.) (From the Laboratory of the Royal College of Physicians, Edinburgh.) THE material upon which the present paper is based was furnished by a series of experiments performed by Dr Noel Paton in the course of an investigation of adrenalin glycosuria. The results of some of Dr Paton's observations have already been publishedw.)* The organs examined were removed from the animals immediately after death, and portions were fixed in corrosive sublimate solution and in formalin (10 I/0), passed carefully through graduated alcohols, cleared in cedar oil, impregnated with paraffin, and cut with the rockingmicrotome. Haematoxylin and eosin, and methylene-blue and eosin were the chief stains used. Special methods were made use of when indicated; for instance Weigert's method was used for the recognition of fibrin, and osmic acid and sudan for the recognition of fat. Some of the physiological effects which have been observed to follow injection of supra-renal extract have a bearing upon the present investigation. The most important of these is the rise of blood-pressure which follows injection. Oliver and Schafer (2) found that, when large doses were administered, the rise in blood-pressure might be so great as to cause extravasation of blood, especially in the liver. Respiration is also affected, as described by Oliver and Schiifer(2), there being shallowing or even temporary cessation of respiration, which passes off gtadually. Bleeding from the nostrils has been observed, and death may be preceded by convulsions resembling those of asphyxia(3). The changes in the urine are of great importance and interest. The discovery that glycosuria follows injection of adrenalin is attributed to Blum (1901)(4), whose observations were confirmed by Luelzer (1901)(5), and by Loftan (1902)(6). Albuminuria and even haematuria also occur when the doses are sufficiently great. The post-mortem appearances, as described by others, and as found 6 PH. XX[XI.

2 82 W. B. DRUMMOND. in these experiments, will be referred to under the several organs, but one or two points may be noticed here. The changes varied a good deal in degree. The most intense changes were found in those animals which died after a single dose. In such cases convulsions usually preceded death. Animals which were receiving smaller doses over a prolonged period sometimes quite unexpectedly had convulsions and died. Some of the post-mortem changes, such as ecchymoses in the abdominal wall, may have been produced by the convulsions. The changes actually found included the presence of a variable amount of fluid in the peritoneum, the pleura, and the pericardium; more or less congestion of the abdominal viscera, especially of the liver; and congestion of the lungs. The state of the lungs often left little doubt that asphyxia was the immediate cause of death. Histological changes. Pancreas. The theory that diabetes mellitus is in some way associated with lesions of the islands of Langerhans has been maintained in recent years by Opie(10) and other writers, and therefore the condition of the pancreas in experimental glycosuria is of special interest. Herter and Richards(7), in one of the two animals examined after death from adrenalin poisoning, found numerous focal necroses in the pancreas and degenerative changes in the cells of the islands of Langerhans. In a later paper(8), however, Herter states that further experiments have led him to the conclusion that no importance can be attached to the focal necroses and other changes which may result from large doses. The pancreas may appear normal in cases where glycosuria has been produced, and moreover glycosuria may be caused by painting with various reducina substances the head of the pancreas (which contains no islands in the dog) just as readily as by painting the tail (which is rich in islands). L6pine("i), also, has lately examined the pancreas in cases of experimental glycosuria produced by the injection of "lecomaines diab4togenes" prepared by the method of L6pine and Boulud. In some cases the glycosuria was maintained for weeks. Beyond slight congestion in a few instances no changes were discovered in the pancreas post mortem. Our own observations agree with the later observations of Herter. We have failed to find any special changes in the islands of Langerhans, or any constant changes in the pancreas. In some of the cases examined the pancreas was congested, in others it was not, but in all cases the acini of the gland and the islands of Langerhans appeared to be quite

3 TISSUE CHANGE AFTER ADRENALIN. normal. This is quite in accordance with what is found in at any rate some cases of diabetes mellitus. For instance in a pancreas from a case of diabetes, kindly given to the writer by Dr Bruce, the islands of Langerhans appeared to be normal on microscopic examination. Spleen. In some cases the spleen was Qf very small size as though it had become contracted. Langley (12) has noticed transient contraction of the spleen on injection of supra-renal extract. We did not find any change in its histological structure. Supra-Renal Glands. The supra-renal glands were examined in all cases, but no change, beyond slight congestion, was discovered. Kidneys. Very marked changes were present in the kidneys, which varied from congestion of the organ with cloudy swelling of the epithelial cells to a parenchymatous nephritis with desquamation of cells in the tubules. The changes in very acute cases, where the animal died after a single dose or after a couple of doses, differed a little from those seen in cases where the doses had been repeated over a prolonged period. In acute cases, in addition to congestion of the vessels throughout the organ, the convoluted tubules are seen to have suffered severely. In some the cells lining the tubules are in a condition of cloudy swelling, but in many there is a dropsical swelling of the cells, the central parts of the cells (i.e. the parts next the lumen of the tubule) being so swollen as practically to obliterate the lumen. These central portions of the cells do not stain, so that only their outlines are visible. The peripheral portion of the cells is granular, stains readily, and contains the nucleus. Here and there a tubule can be found in which the cells are desquamating. In the chronic cases the dropsical swelling of the cells in the convoluted tubules is less apparent. More often the tubules appear abnormally wide, an appearance which might be due to the dropsical portions of the cells having broken down. Desquamation of the epithelial cells may be seen in many of the tubules, and detached cells are to be seen in the lumen not only of the convoluted, but of some of the collecting tubules. The nuclei mostly stain fairly well. Some of the nuclei appear dark and homogeneous. Glomerular changes are not noticeable, beyond engorgement in some instances of the capillaries. Liver. No constant changes were found in the liver. In some cases the liver appeared perfectly normal whether the animal had been killed after a single dose, or had received several d0gep on successive

4 84 W. B. DRUMMOND. days. A moderate degree of conaestion was present in several specimens. In others more serious changes were found. The most important pathological condition discovered was necrosis of the central type-that is to say, necrosis affecting the centres of the lobules, and occurring diffusely thioughout the organ. In one animal which had received three fairly large doses on successive days the liver was found to be congested, and even under the low power a diffuse change affecting chiefly the centres of the lobules could be observed. Under a high power there was found to be a profuse exudation of leucocytes of the polymorpho-nuclear variety. This varied considerably in density, there being in some places a slight exudation throughout the lobule, but in other lobules the exudation was confined to the central zone, and throughout the specimen the density was greatest in the central zones. Where the exudation was most marked the liver cells appeared shrunken; the protoplasm was in some homogeneous, in others granular, and stained reddish with the eosin; the nuclei were smaller and more homogeneous than in normal cells, and instead of being stained blue by the ha-matoxylin they were purple or even pink. Among the leucocytes the liver cells were fewer in number than elsewhere, and from some areas they had disappeared altogether. ~~~~~~~~ ~~~~~~~~~~~~16 a- Fig. 1. Liver of Rabbit after several large injections of Adrenalin on successive days, showing central necrosis. Low Power. a. central zone;-congestion and extravasation; disappearance of liver cells. b. intermediate zone;-network appearance. c. outer zone;-liver cells normal.

5 TISSUE CHANGE AFTER ADRENALIN.. In another specimen, also from a rabbit which bad received daily injections for several days, even more striking changes were present. Under a low power the lobules were seen to be divided into three zones (Fig. 1). The cenitral zone, surrounding the hepatic veins, seemed intensely congested and was stained deep red by the eosin (in an ba-matoxylin and eosin section). The peripheral zones were normal, the connective tissue, liver cells, &c. taking on the ordinary hematoxylin staining. Between these two deeply stained zones the third, which varied considerably in breadth, stood out in sharp contrast, as it took on practically no stain at all, '~~~ but appeared to be made up of spaces like large clear cells with fine connective tissue strands between. Under a high power (Fig. 2) the central zone was seen to contain no liver cells 85 Fig. 2. Liver of Rabbit after several large injections of Adrenalin on successive days, showing central necrosis. High Power. at all. The entire area was occupied by red corpuscles, with a few connective tissue cells, and leucocytes. Whether these blood corpuscles were confined to capillaries or not could not be made out with certainty, but the appearance presented resembled extravasation rather than congestion. The liver cells in the peripheral zone were quite normal. If a column of liver cells was traced from the portal space towards the clear middle zone the transition between the healthy liver cells and the clear spaces of the middle zone was often quite abrupt, and in all cases was rapid, only the last one or two cells being altered in any way. These

6 86, W. B. DR UMMOND. latter appeared swollen and vacuolated, and were followed immtediately by a clear space about twice as large as a normal liver cell, which might or might not contain a degenerated nucleus. In this specimen little clumps of leucocytes were present here and there, but the leucocytes were not nearly so abundant as in the last specimen. Another animal, which received the same injections as the last, showed similar central necrosis of the lobules, but not to quite such a degree. Several specimens were found to contain small areas of focal necrosis, that is to say, areas of necrosis irregularly scattered tlroughout the organ, and confined to no particular part of the lobule. Necrosis of the liver has been carefully studied by Mallory(13), and it is of interest that he found the condition of central necrosis, similar to what has been described, chiefly in toxic and infectious conditions; for instance, in diphtheria; in acute endocarditis; in lobar pneumonia; in acute peritonitis. He found the same condition in two rabbits killed by the injection of diphtheria toxine, one of the rabbits dying on the third, the other on the seventh day. As regards the condition of focal necrosis it must be remembered that this condition may occur in normal rabbits, but it was certainly more common in the experimental than in the check rabbits examined. Still one cannot lay much stress on its occurrence, as it probably results very frequentlyfrom accidental circumstances. Malloryexamined the livers of sixteen normal guinea-pigs and found focal necrosis in no less than nine of them. Another appearance noticed in the liver cells may be referred to briefly. In several specimens the liver cells were swollen and vacuolated, and in some the change was so marked that the entire protoplasm appeared clear and colourless except that the outlines of the cells were remarkably clear and that the cell body contained a number of granules of variable size. This change was most marked, in some instances at least, in the central zones of the lobules, the zones that are affected in cases of central necrosis. The question arose whether this change was an early stage of the central necrosis above described; whether the vacuolation was due to a fatty degeneration of the cells; or whether the appearance was not simply due to the cells having contained abundant glycogen at the time of death. Finally the question had to be considered whether, if these appearances should be due to glycogen, the injection of adrenalin had any effect in increasing the amount of glycogen in the liver. These questions could not be answered from the specimens

7 TISSUE CHANGE AFTER ADRENALIN. which had been preserved, as none had been prepared for examination for glycogen, and accordingly some fresh experiments were undertaken by Dr Paton, and the results will be found in another paper. Lungs. Swale Vincent (3) mentions that before death animals killed by extract of supra-renal gland may suffer from convulsions resembling those of asphyxia, and Battelli (9) refers the cause of death in most instances to an acute cedema of the lungs. Brodie (14), also, has shown that the injection of supra-renal extract produces a distinct congestion of the lungs, the nature of which he has recently studied more fully in conjunction with Dixon ("5). The congestion in the lungs was usually very intense, and sometimes apparently hemorrhagic in character, considerable areas of the organs being of a dark red colour, and more solid than normal. The distribution of the congestion was not uniform throughout the lungs. As a general rule parts of the organs were congested or even hamorrhagic, while the remaining parts, a very variable proportion of the whole, were unusually pale. The appearances on section vary a good deal with the intensity and 87 Fig. S. Lung of Rabbit. Acute Adrenalin Poisoning. Extreme congestion.

8 88 W. B. DRUMMOND. the duration of the process. In animals which have died shortly after a single large dose there may be nothing but a most intense congestion of the organs (Fig. 3). The arteries, veins, and capillaries are all full of blood. In places the engorgement may be so great that the walls of the air cells appear to occupy more space than the lumen of the vesicles. The air vesicles are small, collapsed, and empty, or contain a very few red blood corpuscles. A slightly later stage of this condition may show the vesicles in some areas actually filled with red corpuscles and with fibrin. In such haemorrhagic patches the walls of some of the small vessels seem to have undergone a colloidal (?) change, and rupture of such vessels has been seen in a section. Among the contents of these air vesicles are a few nucleated cells. Of these a very few are polymorpho-nuclear leucocytes. The majority are large rounded epithelioid cells with dark granules of pigment in KA Fig. 4. Lung of Rabbit. Adrenalin daily. Death on third day. Exudation with epithelioid cells in air spaes. their protoplasm (Fig. 4). These are found chiefly close to the wall of the vesicle and are evidently desquamated epithelial cells. In the case of animals which have lived for a day or two after injection, or which have received a number of injections extending over some days, nucleated cells are much more abundant in the exudation in the air-cells. The epithelioid cells are more numerous, and the

9 TISSUE CHANGE AFTER ADRENALIN. polymorpho-nuclear leucocytes may have increased to an even greater extent so as to constitute the majority of the cells in the exudate. A moderate number of eosinophil cells may also be present. The proportion of polymorpho-nuclear cells, however, varies greatly in different cases and may be larger in an animal which has died some time after a single in*jection than in one that has received several on successive days. In one animnal which died about twelve hours after a single dose there was a copious exudation of leucocytes in the lung. Sections of this (and of other) lungs were stained for organisms, but none could be discovered. DISCUSSION OF RESULTS. The histological changes produced by the injection of adrenalin appear to be due to at least two factors. The first is the well-known influence of adrenalin upon blood-pressure, and the second is its direct toxic action. The action of adrenalin upon the vascular system is seen after death in the congestion of the viscera, and in the occasional presence of hbemorrhages and of serous effusion. Congestion due to the action of adrenalin on the blood vessels may result either from the increase in the blood-pressure in those large arteries whose arterioles do not contract or from a rise of pressure in the large veins. The latter, which is a recognised result of the injection of adrenalin, wvould increase the pressure, for instance, in the central parts of the liver lobules and in the convoluted tube region of the kidney. In these regions, therefore, congestion, when present, may be due to the rise in venous pressure. Battelli ascribes death to an acute cedeina of the lungs, and we agree with him that in most cases, if not in all, asphyxia is the cause of death. The changes in the lungs, however, are much more than cedema. In all the cases wve examined, with a single exception, even in cases where the abdominal viscera were not distinctly congested, there was distinct congestion of the lungs, and in most instances this congestion was very great, so great as to be associated with more or less haemorrhage into the air vesicles. These changes may be explained by the influence of the adrenalin upon the blood-pressure. But these changes are not all. As we have already described, the congestion, which probably occurs almost immediately after the in.jection, is rapidly followed, if the animal lives, by a distinctly inflammatory reaction. This may be due simply to the presence of effused blood in the air cells, or to the invasion of micro- 89

10 90 W. B. DR UMMOND. organisms from the respiratory passages. But we are inclined rather to regard these inflammatory changes as due to a toxic influence of the adrenalin, and in support of this view we would cite the degenerative changes observed in the blood vessels (here and there); the comparatively short time which may elapse between the injection and the appearance of inflammatory changes; and the absence or practical absence of organisms in specimens where distinct inflammnation is present. As' regards the primary cause of the congestion of the lungs the observations of Brodie are of importance. Brodie found that the injection of supra-renal extract was followed by a rise in the pulmonary blood-pressure to nearly twice its original value, this rise commencing with, but lasting longer than, the rise in aortic pressure. He conctludes that these effects are mainly due to the action of the peripheral blood vessels in causing the large rise in aortic pressure. The adrenalin appears to act upon the blood vessels through the vaso-motor nervous system, for Brodie found that if he made the injection directly into the systemic vessels there was an immediate contraction amounting almost to complete closure, while, on the other hand, injection directly into the pulmonary vessels resulted in an increased flow. These facts have an important bearing upon the use of adrenalin in cases of heemoptysis, and we agree with Brod ie that experimental results " strongly contra-indicate its employment for such a purpose." The toxic influence of adrenalin is most distinctly seen in the glandular organs, and particuilarly the liver and kidney. Upon these organs it appears to act as a protoplasmic poison. As such it does not act indiscriminately. Not only are the changes which it produces more marked in these organs than in others examined, but within the organs themselves some parts are affected more than others. In the kidney the brunt of the toxic action falls upon the cells of the convoluted tubules, and in the Jiver the cells of the central zone are the first to succumb. The changes found in the liver are very similar to those which other observers have described in various toxic conditionis (see 13 and 16). The changes in the liver are of interest in connection with the marked fall of urea nitrogen described by Paton (12) [Exp. IX and X], seeing that it is in the liver that the formation of urea is chiefly carried on.

11 TISSUE CHANGE AFTER ADRENALIN. 91 REFERENCES. 1. This Journal, xxix. p This Journal, xix. p This Journal, xxi. p Deutsch. Arch. f. klin. Med. LXXI. p Berlin. klin. Woch. 1901, p American Medicine, Jan. 18, New York Med. News, 1902, p New York Med. News, 1901, p Also Virchow's Archiv, CLXIX. p Compt. Rend. de Biol. LIV. p Journ. of Exper. Med , p Compt. Rend. de Biol. LV. p This Journal, XxVII. p Journ. of Med. Research, vi. pp Lancet, 1902, p This Journal, xxx. pp Tischner, Virchow's Archiv, F. xvii. Bd. v. pp Also Pat on and Ea s on, On a method of estimating the interference with the Hepatic Metabolism produced by drugs, This Journal, xxvi. p