ABSORPTION FROM THE PERITONEAL CAVITY. *

By CHARLES BOLTON, M.D., F.E.S. From tJLe Graham Laboratories, University College IIospital Jfedical

School, London.

FOR a considerable time I have been engaged in the study of the pathology of ascites produced in cats by narrowing of the inferior vena cava in the chest, the results of which have been published a t intervals (1903, -09, -16l). At the present point in the work it has become necessary to investigate the rate of absorption in ascitic cats as compared with the normal, but on looking up the subject I have found that the normal absorption of fluids and particles is by no means clearly established, more particularly with regard to the part played by the thoracic duct in draining the peritoneum. It is not only from the point of view of the absorption of normal body fluids and cells that the subject is of importance, but also from that of the absorption of bacteria and their products, and of other chemical substances, whether they are so-called immune products or altogether foreign to the animal body. This paper deals only with the mechanical and physical factors concerned in the process of absorption, and with the paths by which this is accomplished. I t has nothing whatever to do with tissue reaction and the vital factors which are called into play. An animal absorbs substances from its peritoneal cavity in two ways: (1) by its subperitoneal capillary blood vessels ; (2) by its lymphatic vessels.

1. Subperitoneal Capillary Blood Vessels.--It was first clearly demonstrated by Starling and Tubby (1894 2) that salt solution coloured with a diffusible dye was directly absorbed into the blood from the peritoneal cavity, the urine being coloured within five minutes and the lymph in about half an hour. They found that the residue of the peritoneal fluid contained protein and concluded that the process a t work 'was one of interchange between this fluid and the blood, each taking from the other that constituent which it did not possess. Leathes and Starling (1895 3, subsequently showed that the water was absorbed by osmosis and the sodium chloride by diffusion, and that, there was no active absorption by the cndothelial cells, since there was no difference in the process when the cells were injured by sodium fluoride or by scalding.

* Received June 24,1021. The expenses of this researcli have been defrayed by a grant from the Graham Rcsearch Fund.

420

430 CHARLES BOLTON

Osmosis and diffusion will thus account for the absorption of saline solutions by the blood vessels up to the point a t which there is equalisation of the amounts of the various salts on each side of the membrane, when equilibrium will be established. The osmotic pressure of the blood proteins, as Starling has pointed out, must also be taken into account, so that absorption of fluid by the blood vessels will still continue until increasing protein concentration of the peritoneal saline solution reduces the absorbing force to the level of the hydrostatic pressure in the capillaries, when absorption will cease. If further absorption by the blood vessels occurs i t must be due to an active absorption by the endothelium, of which there is no evidence ; indeed the experiments to be described lend support to the view that there is no absorption of ascitic fluid by the blood vessels.

The principle of direct absorption by the blood vessels by osmosis and diffusion is thus established ; all parts of the peritoneum naturally take part in this process, and the only necessary conditions for absorption in this way are that the substances to be taken up must be diffusible through the capillary wall, and must not exist in the same concentration on both sides of the membrane. Further papers published have added nothing new to this principle.

2. Lymphatic Paths of Absorption.-The peritoneal cavity is not, strictly speaking, a lymph space, an increase in the lymph pressure of which will produce a corresponding increase in the flow of lymph from the lymphatics draining it, and Orlow (1895 4, has shown that the presence of serum or salt solution in this cavity does not increase the lymph flow from the thoracic duct. Some special mechanism is there- fore requisite for draining this cavity, and for many years it has been recognised that the contractions of the diaphragm and the alterations in pressure so produced in the abdomen and thorax during respiration are the means whereby the necessary force is supplied to produce a flow of fluid into the lymphatics.

According to the anatomists the diaphragm has two main sets of efferent lymphatics:-(1) the lymphatics of the anterior portion of the diaphragm which pass to the sternal and anterior mediastinal lymphatic glands and thence to the right lymphatic duct, communications being established in the chest with the thoracic duct; (2) the lymphatjca from the posterior portion of the diaphragm which pass to the cisterna chyli and thoracic duct running on their way through one or two upper lumbar and posterior mediastinal glands. This general arrangement is probably the same in most mammals, the details of distribution differing somewhat in different animals.

It is well established that both fluids and particles readily pass by the former path, but it is not so clear what is the importance of the latter,

Von 1Xeclrlingliausen (1863 j) was the first observer to show that various substances and particles (milk, Chinese ink, egg yolk, cinnabar, oil, blood) were able to pass into the diaphragmatic lymphatics from the peritoneum. He

ARSORPTZON FROM PERZTONEAL C A VIT I’ 431

endeavoured to show that the particles passed through definite openings in the peritoneum, and by ingenious experiments on dead animals that this passage occurred when the pressures on the two sides of the diaphrsgm were equalised. These conclusions were not altogether accepted, but Schweigger-Seidel and Dogie1 (1866 6) definitely described openings in the peritoneal membrane of the frog communicating with the lymphatic system. Further, Ludwig and Fkhweigger-Seidel (1866 7), by hanging up an eviscerated dead rabbit head downwards, placing a coloured gelatinous solution in the concavity of the diaphragm, and performing artificial respiration, showed that the lymphatics of the diaphragm could be filled, and they also found openings in the region of the central tendon of the diaphragm between the endothelial cells. The presence of stomata was considered to be proved for many years, although Tourneux (18748) was unable to find them in frogs and maintained that they were artifacts and the result of the method of preparation used, and Ranvier (1875 9, stated that they could not be found if the membrane was washed free from albumin before staining with the silver solution. Some years after this Kolossow (1892 10) carefully described the structure of the peritoneum and denied the existence of stomata, this result being confirmed by Muscatello ( 189511).

Hertzler (1901 lz), by injecting silver nitrate solution into the peritoneal cavity of living animals, came to a similar conclusion. Finally MacCallnm (1903 13) was unable to find openings and considered that granules were able to find their way between the cells at points, but that they were chiefly carried to the mediastinal glands by leucocytes. He found that the process was only slightly affected by opening the chest, but considered that Ludwig had proved that the movements of the diaphragm were a factor in their absorption into the diaphragmatic lymphatics.

With regard to the site of absorption of the particles, other observers had stated that these entered a t many points in the peritoneum, and even passed into the blood vessels, but Muscatello’s work clearly shows that the region of the diaphragm is the only locality where this occurs, tlie particles being always found in the mediastinal glands, and in these alone, before obtaining access to the blood and organs: also when the animal was placed vertically upright the absorption was almost a t a standstill. The particles passed through the diaphragm very quickly.

Beck (1893”), however, injected bird’s blood into the peritoneum, and states that he found red corpuscles microscopically in the lymph of the thoracic duct in one to two hours.

In an important work upon the ‘‘ Mechanism of Reaction to Peritoneal Infection,” Durham (1897 15) found that bacteria were rapidly absorbed through the diaphragm. In half to three-quarters of an hour after microbe injections he found large numbers of organisms in the anterior mediastinal glands, mostly free, some however ingested by cells. I n a guinea-pig, killed six minutes after an intraperitoneal injection of B. prodigiosus, the lymph vessels of the falciform ligament were found filled with the bacilli. He found that Indian ink coloured the anterior mediastinal glands in eight minutes, whilst all the other glands were devoid of colour, and that a solution of carmine coloured the lymph paths of the anterior niediastinum in three minutes. The thoracic duct was coloured in half an hour, but this result was no doubt due to diffusion of the colouring matter into the blood. The cxperiments of Buxton and Torry (190611i) agree with those of Durham in the rapidity of passage of particles through the diaphragm. They found that anthrax bacilli and chicken’s red blood corpuscles were present in the anterior mediastinal glands in fifteen minutes after injection into the peritoneum of the guinea-pig. On the other hand Dandy and Rowntree (191317) came to a different conclusion. They injected a solution of carmine, having also particles in suspension into the peritoneum of dogs, which were

There is very little mention of the thoracic duct.

432 CHARLES BOLTON

killed in two to three hours. The lymph and lymph glands were stained, but only very few particles were found and quite as many in the abdominal as in the anterior inediastinal lymphatic glands, for which the particles had no special predilection. These observers also repeated Starling’s experiment, using phenolsulphonphthalein, an easily diffusible substance, which they found to pass into the blood in all positions of the animal in about six minutes and into the lymph in thirty-seven minutes. They drew the general conclusion that the diaphragm plays no special rOle in absorption, which takes place at all parts of the peritoneum directly into the blood. These conclusions appear to apply to all kinds of “toxic materials.”

Thiele and Embleton (1914l8) regard the lymphatic path by the superior mediastinal glands as being quite secondary, as they found that infection and pigmentation of those glands was later than the appearance of bacteria in the blood stream and of pigment in the urine. They injected a suspension of carmine intraperitoneally into guinea-pigs and found that there were no particles in the mediastinal glands until twelve hours had elapsed, whilst the urine was pigmented in twenty minutes. With regard to organisms, they found after intraperitoneal inoculation into rabbits that the superior mediastinal glands contained no bacteria until after eight hours, whilst the blood was infected in five to twenty minutes. I n the cat they found bacteria in the lymph from the thoracic duct in from two to ten minutes after intraperitoneal inoculation. I n connection with this statement it should be remembered that a diffusible dye does not colour the lymph before half an hour. The bacteria were detected by cultivating the fluid or organ in question, which should be a reliable method, but introduces the risk of contamination.

It is thus apparent that the results of the various observers who have worked at this subject are by no means uniform. There are certain experimental conditions which must be fulfilled, and the follow- ing considerations will perhaps help to explain these discrepancies.

1. The fluid or suspension, which is injected into the peritoneum, must be in such an amount that it can flow freely about so as to reach the diaphragm in a thorough manner. If it is in small amount and particularly if injected into the hinder part of the peritoneum, it is held up by the intestines and other organs, and has to gradually find its way between them to reach the diaphragm, so that its absorption by the lymphatics is delayed. Similarly different positions of the animal produce different results. All these effects must come into play in the human being. On the other hand, i f the fluid is diffusible into the blood it cannot matter where it is nor in what amount.

2. The suspension should contain a sufficient number of particles, and the latter must be well suspended and not aggregate and sediment too quickly, otherwise their chance of passing through the diaphragm is greatly diminished. If a coloured fluid is injected it must be of sufficient strength to colour the lymph glands and lymphatics easily, and should be made up just before use.

3. Dyes which directly diffuse into the blood should not be used for studying the lymphatic paths of absorption, because, if so, the lymph passing out of the capillaries is stained and colours the thoracic duct, so vitiating the result. Conclusions drawn from the absorption of these dyes must not be applied to that of substances in general,

ABSORPTION FROM PERITONEAL CA VITY 433

~~~

Introducorl.

C.6.

24

30

30

4. The distiiiction between the purely mechanical factors of absorption and those depending upon the activities of cells must be borne in mind. Particles are mechanically carried aloag the normal lymphatic paths by the lymph flow, but this is not necessarily the case after they have been ingested by cells. Mechanical absorplion commences a t once, and experiments dealing with this subject should not be too prolonged, otherwise they are complicated by the factors due to tissue reaction.

5. The mode of death and the condition of the respiration should be mentioned.

My own experiments will now be considered under two headings- (1) the absorption of fluids; (2) the absorption of parlicles.

I. ABSORPTION OF FLUIDS. Ascitic $fluid is absorbed as a whole, the total solids contained in

it remaining of the same percentage during the whole process. I1 is aIso absorbed a t an aImost uniform rate. These points are illustrated by the three following experiments, in which the fluid from ascitic cats was introduced into the peritoneum of three normal animals, and removed after certain intervals. The animals in this and the following two series of experiments were allowed to move about as they chose.

Absorption of Ascitic Fluid-Normal Cats.

Solids. Time.

I’or cmt. Hours. 6 5 14

7.9 7

7.5 11)

Iiitruduced. Roliils. Tinio. llciix~i~rit~g. Solids. ~

C.C. l’cr ccut. Hours. C.C. 1’r:r cw t . 50 0 ’9 1 32 1.2

70 0 ’9 8 3 4 -7

I~emaiiiiiig. 1 Solids. Absorbed. I

Absurbed.

C.C.

18

67

C.C. Pcr cent. U.C.

19

10

This ascitic fluid is evidently entirely absorbed by the lymphatics, because, as shown by Starling, the rate of absorption of salt solzctioqb by the peritoneal blood capillaries falls off during the process owing to accumulation of protein and other salts in it, so that the force -

tending to osmosis diminishes, as the percentage of total increases. This is illustrated by the two following experiments.

Absorption of 0.9 per cent. Salt Solution--Normal Cats.

solids

During the first hour 15 C.C. were absorbed; after this the average was about The total solids gradually increased, but the figure 4.7 is too high as 7 C.C. per hour.

cells were present in the fluid. J O L R N . Ok P A l H - V O L . XXIV. 2 E

434 CHARLES BOLTON

Ascitca Iteinoved.

C.C.

11

45

30

If osmotic equilibrium becomes established before the fluid is absorbed, the remainder is taken up by the lymphatics entirely. This point was tested by observing t81ie absorption of salt solution in ascitic cats, in which this solution was substituted for the ascitic fluid; the output of lymph into the peritoneum of the ascitic cat is of course rapid, and osmotic equilibrium is comparatively soon established. The following three experiments were performed :-

Saliiie '*lids. liitroclucetl.

€'or coiit. C.C.

... 50

7 '9 70

6.5 49

Absorpt ion of 0'9 per cent. Sult Solution-Ascitic Cats.

3.37 p.m. . . 1.0 C.C.

3.47 ,) . . 1.3 ,, 3.57 ,, . . 0'9 ),

4.7 ,, . . 1.0 ,,

4.17 p.m. . . 1.4 C.C.

4.27 ,, . . 1.5 ,) 4.37 1, . . 1.3 ,,

Timc. Absorbed.

c.c. 19

25

a7

434 CHARLES BOLTON

If osmotic equilibrium becomes established before the fluid is absorbed, the remainder is taken up by the lymphatics entirely. This point was tested by observing t81ie absorption of salt solution in ascitic cats, in which this solution was substituted for the ascitic fluid; the output of lymph into the peritoneum of the ascitic cat is of course rapid, and osmotic equilibrium is comparatively soon established. The following three experiments were performed :-

Absorpt ion of 0’9 per cent. Sult Solution-Ascitic Cats.

The rate of absorption during the first hour is practically the same as in the normal, but falls off much more rapidly, owing to the more rapid accumulation of solids in the peritoneal fluid. After eight hours osmotic equilibrium is almost established and the fluid has become almost of the same composition as the ascitic fluid originally removed from the animal. Only 25 C.C. were absorbed in the second experiment during eight hours as compared with 67 C.C. in the former aeries of experiments, and after this time very little fluid was absorbed at all. The peritoneal blood vessels thus regulate the quality of the ascitic fluid, which is maintained of the same composition, but apparently play no part in the absorption of the fluid when of the requisite quality. The amount of salt solution absorbed during the first hour by the blood vessels of the normal animal can be more than doubled if large amounts are introduced into the peritoneum under pressure. During this time the lymph flow is not affected. The following experiment proves this point.

Cat, wt. 4250 grms. Ether. Artificial respiration. Canula introduced at 3.7 p.m. Lymph Flow. Tliorauic Iluct.

Tlnir. Amuunt. 3.17 p.m. 2’8 C.C.

3.27 ,, 2.3 3 9

8.27 to 3.37 p.m.-740 C.C. salt solution (0.9 per cent.) run into peritoneum, pressure being 90 mm. saline solution, gradually falling to 40 inm. during the experiment.

Killed, 4.45 pin. 670 C.C. removed, CoiihiIiing 1.01 per cent. dids-trace of Allowing for the loss of 20 c.c., a liberal amount, the quantity albumin on boiling.

absorbed was GO C.C.

ABSORPTZON FROM PERITONEAL C A VZT I/ 435

Absorption of Colloids by the Blood Vessels. It is an important point to determine whether substances which

will not diffuse through membranes are able to pass into the blood through thc capillary wall, and if so what are the relative sizes of the molecules of such substances, from the point of view of the absorption of pathological products, many of which on intraperitoneal injection cause symptoms in a few minutes, showing that they must diffuse directly into the blood. This question of determining the relative size of the molecules which will pass through the capillary wall is one of great difficulty, because the salt solution with which the colloids are mixed for injection causes aggregation of the molecules and there is no means of determining what degree of aggregation occurs, nor what is the size of such aggregates. It is supposed at the present day that the blood colloids are unable to pass through the capillary wall unless aided by the blood pressure within the vessels, but I have not found any literature dealing with the diffusion of colloids of smaller molecular weight into the blood vessels. I therefore used three colloidal dyes, Congo red, cloth red, and Congo blue, kindly supplied to me by Prof. Bayliss for this purpose, and found that each of them diffused directly into the blood vessels, but a t a slower rate than in the case of crystalloids. The capillary wall does not therefore react as do some animal membranes outside the body, and is probably more permeable than is usually supposed. The solutions were made up in 0.9 per cent. salt solution and also in Ringer's fluid, in strengths varying from 0.5 per cent. to 1 per cent. Samples of blood were withdrawn from the femoral artery a t intervals into oiled tubes, and the colour of the serum read off on the following day. One or two cubic centimetras of blood were withdrawn each time and the canula cIeared by injecting the same arnount of salt solution. The lymph was collected from the thoracic duct. It is possible to put a small canula into the thoracic duct of a cat, but this is not a workable method for constant use. The best method is to tie a specially shaped canula into the left external jugular vein, having ligatured all the communicating veins. For this purpose the lower end of the external jugular vein, the common jugular and its junction with the subclavian vein are exposed just above the first rib by division of the pectoral muscles. The fascia is dissected off the veins, and the outer margin of the left sternomastoid muscle defined and the muscle reflected. Some lymphatics passing across the junction of the subclavian with the common jugular from the mediastinal glands over the apex of the lung are ligatured and divided. The innominate vein is followed down behind the pleura and ligatured above a large vein which joins it. The subclavian and internal jugular veins are tied, and also the external jugular about the level of the anterior border of the pectoral muscles. The external jugular is now incised below the ligature and the blood evacuated from it with a seeker. Pure lymph will shortly flow out ; if not, one or two small irregular venous trunks may have to be ligatured, but often the blood in these clots and causes

436 CHARLES BOLTON

Lympli Flow Tliorncic DhA.

Timr. Amonnt.

3.40 p.m. 1.1 C.C. 3.50 ,. 1’2 ,, 4.0 ,, 1’5 ,, 4.10 ,, 1.1 ,, 4.20 ,, 1.7 ,, 4.30 ,, 2.0 ,, 4.40 ,, 2’2 ,, 4.50 ,, 1‘5 ,, 5.0 ,, 1.0 ..

______-

no further trouble. in a watch glass and measured every ten minutes.

The following experiments were performed :- Congo Red, C,,H,,N,O,S,Na, - Mol.wt. = 696.-Seven experiments

were done using different strengths of solulions. Some aggregation of the molecules is caused by the saline solution, the solutions having the appearance of whipped blood, and a slight precipitate settling in places on the glass if left in a bottle.

All the experiments ngreed in the fact that the blood wa5 stained before the lymph. The blood serum was coloured in from ten to twenty minutes and the lymph in from twenty-five to forty-seven minutes, the time varying a little in different animals, not definitely with the strength of the solution. Towards the end of the first hour the lymph became darker in colour than the blood. The dye passes directly into the blood, the lymph being stained as a result, and the depth of colour of the lymph being in keeping with this; the final deeper colour of the lymph is due to lymphatic absorption, which is a very slow process compared with direct absorption by the blood vessels. The diaphragmatic lymphatics and those along the internal mammary vessels were always deeply stained, as were also the sternal and anterior mediastinal lymph glands and their efferent lymphatics, also one lumbar gland lying on the cisterna chyli. The following is an illustrative experiment :-

The canula is introduced and the lymph collectd

C n f , wt. 2350 grms. Ether and 3rtificial respiration. Canula in thoracic duct, G O e.c. 0.5 per cent. Congo Red in 0.9 per 3.30 pm. , and canula tied in peritoneum.

rent. Saline introduccd into peritoneum a t 3.40 p.ni.

Blood, Fcmoral Artcry.

Coloiir.

Time.

Colourlcss. 3.40 p m . 3.44 ., 3.48 ,,

Very faint pink. 3.52 ,, Faint pink. 3.56 ,, Darker. 4.0 ,,

4.4 ,. 4.8 ,, 4.12 ,, 4.20 ,,

The blood was stained in sixteen minutes, and the lymph in thirty minutes. Finally the colour of the lymph was darker than that of the blood.

Coloiir.

4.27 ,, 4.37 ,, 4.47 ,, 4.57 ~ i l l ~ l . ’

Amouut. 1 I \ -I

2.0 c.c. 2.0 ,. 2‘0 7.

2.0 3 .

2.0 ,, 2’0 ,, 2.0 ,, 2‘0 ,, 2‘0 .. 2’0 1.

2.0 ,. 2’0 .. 2.0 7.

2’0 7.

Colourless.

Very faint pink. Faint pink. Darker.

Cloth Red, C,,H,,N,O,S,Na~,, 3101. wt. = 584.-Four experiments were done. There is more aggregation of molecules in the caae of cloth red and a precipitate eventually forms at the bottom of the bottle. The blood in all cases was stained before the lymph, and the blood serum was coloured in from twenty to sixty minutes and the lymph in from fifty to eighty-six minutes. Ringer’s solution caused more

ABSORPTION FROM PERZTONEAL CA VITY 437

3.16p.m. 3.26 ))

3.36 ,, 3.46 ,. 3.66 ,) 4.6 ,, 4.16 ,, 4.26 ., 4.36 ) )

aggregation than salt solution and the time of absorption was longer. These effects are due to the greater degree of aggregation of molecules than in the case of Congo red, although the molecule is smaller than that of Congo red. The post-mortem appearances were the same as in the Congo red cats, but the staining less marked. The following is an illustrative experiment.

Cat, wt. 2670 grms. Ether and artificial respiration. Canula in thoracic duct, 60 c.c. 1 per cent. Cloth Red in 0‘9 per cent. 3.6 p.m., and canula in peritoneum.

Saline, introduced into peritoneum, 3.16 p.m.

1.3 c.c. 1.3 ,, 1.6 ,, 1’1 ,, 1’0 ,, 0.6 ,, 1.1 ,,, 1.0 ,, 1‘3 1,

Lyniyll Flow, Tlioracic Duct.

Time. I Amount.

Colourless. 11

,,

Very faint pink.

Pink. 79

3.16p.m. 2’0 c.c. 3.21 ,, 2’0 ,, 3.26 ,, 2.0 ,, 3.31 ,, 2.0 ,, 3.36 ), 2.0 ,, 3.41 ,. 2.0 ,, 3.46 1, 2’0 ), 3.51 ,, 2.0 ,, 4.1 ,, 2’0 ,, 4.11 ,, 2.0 ,, 4.21 ,. 2’0 ,, 4.31 2.0 ,, Killed’

Colour.

3.41p.m. 3.51 ,, 4.1 ), 4.11 ,, 4.21 ,, 4.31 ,, 4.41 ,, 4.51 ,, 5.1 ,, 5.11 ,,

Dloud, Fcmoral Artcry.

Tiine. ‘ 1 Amount.

1’0 c.c. 0.5 1,

0’4 ,, 0.3 ,, 0’3 ,, 0‘4 .. 0‘4 ,, 0’4 ,, 0 5 ,, 0-7 ,,

Colour.

3.43p.m. 3.53 ,, 4.3 ( (

4.1; ,, 4.23 ,( 4.33 ), 4.4; ,, 4.53 ,, 5.3 Killed’

2‘0 C.C. 2.0 ,) 2.0 ,, 2.0 ,, 2.0 ,, 2.0 ,, 2.0 ,, 2.0 ,. 2’0 ,,

Colourless.

3,

The blood was stained in twenty minutes, and the lymph in fifty minutes. The experiment was kept going for three hours, and a t the end of this time the tint of the blood was about the same as that of the lymph.

Congo Blue, C,,H,,N,O,,S,Na,, Mol. Wt. = lll6.-This dye forms a deep blue solution, which is not so muddy as that of Congo red, the molecules being less aggregated. The blood serum was stained in twenty minutes in the two experiments that were done and the lymph in fifty-eight minutes in one and seventy-five minutes in the other. The post-mortem appearances were the same as in the case of the two former dyes. The following is one of the experiments :-

Ether and artificial respiration. Canula in thoracic duct, 3.31 p.m., and canula in peritoneum. 47 C.C. 0’54 per cent. Congo Blue in Ringer, introduced into peritoneum 3.43 p.m.

Cat, wt. 2250 grms.

Lymph Flow, Thoracic Duct.

Timc. I Amount.

I-

Colour.

Colourless.

Faint mauve.

Blue mauve. 9 ,

Ulood, Fcmoral Artery.

Timc. 1 Amount.

Golour.

CoIourless.

Faint mauve. Mauve. Blue mauve.

,,

The blood was stained in twenty minutes, and the lymph in fifty-eight minutes. JOURN. OF PAT€I.-VOL. XXIV. 2 E 2

438 CHARLES BOLTON

I was unable to obtain any other colloidal dyes so used colloid silver for the next series of experiments.

Colloid Silver, when made up in salt solution of the strengths named, forms a black solution which on dilution becomes canary yellow. The particular sample used was stated by the manufacturers to be kept in the colloidal state by the presence of albumin. Three experiments were done as above described, and in no case was the blood serum stained a t all. The lymph flowing from the thoracic duct was not stained yellow till over one and a half hours in each case. In each caso the lymph in the cisterna chyli was yellow, showing that the colloid silver had passed directly from the peritoneum into the cisterna, and had not coloured the lymph in the thoracic duct merely through the anastomoses in the chest. The following is one of these three experiments :-

Cat, wt. 2270 grms. Ether and artificial respiration, dorsal decubitus. Canula in thoracic duct, 3.43 p.m., and canula in peritoneum. 50 C.C. 1 per cent. Colloid Silver in 0.9 per cent. Saline, introduced into peritoneum a t 3.63 p.m.

Lymph Flow, Thoracic Duct.

Time.

3.43 p.m. 4.3 ,. 4.13 ,, 4.2:: ,, 4.33 ,, 4.43 ,, 4.63 1,

5.3 ,, 5.13 ,, 5.23 ,, 5.33 I ,

5.43 ,,

Amount.

Dlood, Femoral Artery.

Colonr.

Colourless. 7 .

Slight yellow. Yellow.

I[ Time.

4.3 p.m. 4.13 ,, 4.23 ,, 4.33 ,, 4.43 ,, 4.53 1,

5.3 ,, 5.13 ,, 5.23 ,, 5.33 1 ,

8.43 Killed

Amouiit.

2.0 C.C. 2.0 ,, 2.0 ) )

2.0 ,, 2.0 ,, 2 0 ,t

2.0 ,, 2.0 ,, 2’0 ,, 2.0 ,, 2.0 ,,

Colour.

Colourless.

,.

The lymph was stained in one hour forty minutes ; the blood was not stained.

One has no idea whatever what is the size of the colloid silver It seems improbable that the capillary wall will admit

However, this molecule. molecules much larger than those of the colloidal dyes. portion of the research has been discontinued for the present.

Zymphatic Paths of Absorption. In order t o study this question it was necessary to use for injection

into the peritoneum a fluid which would colour the lymph and lymphatic glands and yet would not be absorbed by the blood vessels. C’olloid Silver was obviously a suitable substance to employ. It stains the lymphatics brown, the glands in their path brown or black, and the lymph in the thoracic duct yellow.

The appeayances presented after death were uniform in all the animals. The time of absorption varied a little in different animals, but the ordsT in which the lymphatics and glands were staiiied was invariable.

ABSORPTION FROM PERITONEAL CA VlTY 439

Lymph Glands.-There were two groups of glands only which were stained : (1) anterior mediastinal; (2) lumbar.

The anterior rnediastinal glands were always the first to be stained and the most deeply stained. They were stained brown in about five minutes, and after this time were black.

There were two lumbar glands which were stained, (a) a single gland usually lying directly on the cisterna chyli, (a) a gland or glands farther back at the brim of the pelvis. The anterior lumbar gland was usually stained a light brown colour in about half an hour, and was never found to be black: the posterior lumbar glands were stained inuch later, not usucllly under an hour, and were always much lighter in colour than the anterior. If all these glands were stained in any given case the anterior mediastinal would be black, the anterior lumbar brown, and the posterior yellow or yellowish-brown.

Lymphatics.-The first lymphatics to be stained brown were those of the diaphragm, and they communicate with the anterior inecliastinal glands by well-stained lymphatics passing up with the interim1 mammary vessels. Other brown lymphatics pass from the anterior mediastinal glands up to the right lymphatic duct and others to the thoracic duct. lZrowii streaks also pass from them backwards into the mediastinum. The second group of lymphatics to be stained are those behind the peritoneum which connect with the posterior lumbar glands mentioned above. They are stained later than the diaphragmatic lymphatics, and about the same time as the posterior lumbar gland. Finally, the cisterna chyli is stained yellow but not till about an hour has elapsed.

Absorption appears to be quicker with normal than with artificial respiration, but it does not matter whether the animal is anesthetised or not. I n the following three experiments the cats received intra- peritoneal injections of 50 C.C. 2 per cent. colloid silver solution in saline, and were then allowed to move about as they wished, and were killed by bleeding :-

1. Anterior mediastinal glands, black. Anterior lumbar gland, brown. Posterior lumbar gland, nil. Retroperitoneal lymphatics, nil. Cisterna chyli, nil.

2. Anterior mediastinal glands, black. Anterior lumbar gland, brown. Posterior lumbar gland, nil. Retroperitoneal lymphatics, nil. Cisterna chyli, nil.

8. Anterior mediastinal glands, black. Anterior lumbar gland, brown.

Retroperitoneal lymphatics, slight yellow. Cisterna chyli, yellow.

Posterior lumbar gland, slight yellow.

440 CHARLES BOLTON

I n the following seven experiments the animals were in exactly the same position, and under identical conditions during the time of experiment.

No?mel Cnts, Ether, Norinnl resp., Dorsal deciibitns. Killed by bleeding. 50 C.C.

2 per cent. Colloid Silver in 0.9 per cent. Saline-intraperitonetll.

I t I I I I 1 5 mins. Dirk brown Nil Nil

I3I:lC.k I 1 11 1 :: 1 Lightbrown 1 :: I Yellow tint

6 90 ,, Brown 120 ,, I ::

It will be observed that the cisterna was coloured in an hour, rather quicker than in the experiments in which artificial respiration was employed, and the lymph collected from the thoracic duct. This is no doubt due to the influence of the respiratory movements. It is thus evident that the staining of the lymph in the thoracic duct is due to the passage of coloured fluid from the diaphragmatic peritoneum directly t o the cisterna chyli, and not simply through anastomoses from the anterior mediastinal glands t o the thoracic duct. This direct flow of fluid from the peritoneum into the cisterna chyli is apparently a very slow process. A much more rapid 00w occurs through the diaphragm into the lymphatics running along the internal mammary vessels to the sternal and anterior mediastinal glands. The dia- phragmatic lymphatics and those afferent to and efferent from the sternal glands constituted a definite and well-marked path of absorption from the peritoneum. No other lymphatic glands were stained within the limits of time of these experiments. It seems quite clear that there are two paths of lymphatic absorption from the peritoneum corresponding to the distribution of the diaphragmatic lymphatics mentioned at the beginning of this paper: (1) the chief path; through the diaphragmatic lymphatics to the sternal and anterior mediastinal glands, and thence to the right lymphatic duct, and also to a moderate degree through anastomoses in the chest to the thoracic duct: (2) the subsidiary path ; through the diaphragmatio lymphatics to the anterior lumbar gland and thence to the cisterna chyli and thoracic duct. Absorption occurs most rapidly and chiefly through the former set of lymphatics, and more slowly and to a more moderate degree through the latter set. The force giving rise to the flow of the lymph is derived from the movements of the diaphragm and the negative pressure within the chest. There is probably also a very slow flow into the retroperitoneal lymphatics, since these are stained, the exact mechanism of which is not quite clear, but possibly depends on the

ABSORPTlON FROM PERZTONEAL C A VZT Y 441

respiratory alterations of pressure in the abdomen. The effect of respiration upon the rate of absorption is more easily demonstrated by the experiments dealing with the absorption of particles.

11. ABSORPTION OF PARTICLES.

The particles used in these experiments were lamp black, bacteria, and red corpuscles of the cat and of man. They were suspended in 0.9 per cent. salt solution and injected into the peritoneal cavity. I t is very dificult to identify these particles in the lymph in small numbers, but quite easy to see them niacroscopically and microscopi- cally in the lymphatic glands on the course of the two sets of lymphatics which have been described above-namely, the sternal and anterior mediastinal glands and the anterior lumbar gland. Sections of the lymph glands were cut and examined microscopically for the particles in question. As in the case of colloidal silver, these were the only lymphatic glands containing particles within the limits of time of these experiments.

Lamp Black.-A simple suspension of this very fine powder in 0.9 per cent. saline solution was found to answer the purpose quite satisfactorily. The amount of lamp black pat into the fluid was such that on settling it appeared to occupy about one-third of the total volume of the emulsion. This is by no means a perfect sus- pension, but it takes very little trouble to make, and gives quite definite results.

The particles pass through the diaphragm and collect in black patches in the peripheral lymph channel of the lymphatic gland. Finally, the whole peripheral lymph channel is choked with densely- packed particles, and outlined in the section as a dense black ring. From this peripheral ring branches penetrate to the centre of the gland in lines following the lymph channels and outlining the lymph nodes.

The rate of passage was determined by killing the animals by bleeding a t definite intervals after injection of the lamp black.

Sternal Glands.-After one hour the peripheral lymph channel contained small black patches, and after one and a half hours it formed rz black ring containing scattered black particles throughout its whole extent. Up to twenty-five minutes after injection nothing whatever could be seen in the glands.

Lumbar Glands.-Nothing was seen in these glands in the above experiments, so one experiment was kept going for four and a half hours, and at the end of this time the anterior lumbar gland contained at one end a few scattered black particles.

These experiments demonstrate with what ease particles pass through the diaphragm t o the sternal lymph glands as compared with their passage to the lumbar glands ; and afford further evidence

442 CHARLES BOLTON

of the fact, shown by the colloid silver experiments, that the chief path of lymph absorption from the peritoneum is by the lymphatics running along the internal mammary vessels to the right lymphatic duct, and the subsidiary path by the lymphatics directly into the cisterna chyli.

The efects of respiration upon the absorption of the particles was tested by killing four animals by asphyxia. I n this way the force of contraction of the diaphragm, together with the negative pressure in the chest, were greatly increased.

It was found that after fifteen minutes the peripheral lymph channel of the sternal glands contained small black patches, similar to the condition occurring after one hour in animals killed by bleeding. In thirty-five minutes the whole gland was surrounded by a black ring with branches penetrating deeply into the gland. In one case the respiration was partially obstructed for fifteen minutes and the animal killed by bleeding, with the result that black patches were present in the peripheral lymph channel. I n another case the respiration was obstructed for fifteen minutes and the animal killed by asphyxia, with the result that the whole peripheral lymph channel was black with branches penetrating the gland deeply. The following table shows twelve experiments set out in order. In each case the animal was under ether and lying on its back. The conditions were therefore exactly the same, the only variation being in the respiration.

LAVV BLACK IN 0'9 PER CENT. SALINE-INTRAPERITONEAT.,

Normal rmpiration. ICilLed by bleedinq. Cat. 'J'imr. Sternal Glands. 1. 5 mins. Nil. 2. 15 ,, 3. 25 ,, ,t

4. G O ,, Peripheral lymph channel = small black patches. <I. 90 7 , Whole ,, ,, = scattered black patches.

G . 43 hours. Antcrior 1;unibar Gland.

Scattered black particles at one end of the gland.

Normal respiration. Killed by asphyxia. Cat. Timc. Steriial Glands. 7. at once. Nil. 8. 15 mins. Peripheral lymph channel = small black patches. 9. 35 1. Whole ,, ,, = denselyblack,penetrating

gland deeply. - 10. 120 f , 1, I 3 -

Obstructed respiration. IZilIerl by bleeding. Cat. Timp 11. 15 mins. Peripheral lymph channel = small black patches.

Obstricetetl respiration. ICiilled by asphyxia. c a t . Time 12. 15 mins. Whole peripheral lymph channel = black, penetrating

gland deeply.

The rate a t which these particles pass from the peritoneum into the lymphatics of the chest is therefore proportional to the violence of the

dBSORPTlOiV FROM PERZTONEAL Cd VZT Y 443

respiratory movements, and there can be no reasonable doubt that the force causing the lymph to flow, and particles to pass, from the peritoneal cavity is derived from the movements of the diaphragm and the negative pressure in the chest. The absorption is therefore purely mechanical, depends upon a definite force which is constantly acting, and commences at once.

Size of Particles.-The size of the particles that will mechanically pass through the diaphragm was tested by using bacteria, which are considerably larger than lamp black particles, red blood corpuscles of the cat and man, and starch granules. In each case the emulsion was injected into the peritoneum, and after two hours, respiration being normal, the animals were killed by asphyxia. It was found that the bacteria used passed through but to nothing like the extent of lamp black, and that the red corpuscles of the cat also passed through but in smaller numbers, but the one experiment done with human corpuscles was negative. Human red corpuscles are distinctly larger than those of the cat. According to Price-Jones the mean diameter of cats’ corpuscles is 5 . 2 5 ~ ) that of human corpuscles being 7 . 2 2 ~ . Starch grains were also used, as stated, but all the attempts to find them by specific colour reactions in the glands after the intra-peritoneal injection with a suspension were quite without results. With the other two classes of bodies-micro-organisms and corpuscles- it was obviously necessary that they should be stained in such a way as to be recognisable in the glands without further treatment-the gland sections being stained a complementary colour to facilitate recognition. It was also necessary that the staining should be such that no colouring matter diffused into the tissues after introduction into the peritoneal cavity, and that it should be carried out in such a manner as to keep the suspension as homogeneous as possible and to avoid any aggregation of particles. Dr H. G. Rutterfield very kindly undertook the staining for me, and his account of the method he employed is as follows :-

“It was found in experiments carried out in v i k o that, while brilliant and distinctive stainings were easiiy produced in bulk with dyes of the aniline class (methyl green, methyl violet, eosin, nigrosin, etc.), none of these results were of use for the purpose in view, because when an apparently clean-washed suspension was transferred from distilled water to normal saline preliminary to filling up the peritoneal cavity, large quantities of dye immediately diffused out, and as this would have stained the tissues generally, including the groups of glands under investigation, the identification of the foreign particles would have been impossible. Aggregation of the particles into masses and too rapid sedimentation after introduction into the peritoneal cavity were also difficulties encountered, particularly in the case of the corpuscles.

The “ The micro-organisms used were saroina and staphylococcus.

444 CHARLES BOLTON

growth from twenty agar slops was used to make a suspension of about 80 c.c.-the bacteria were killed by heating to a temperature of 60" C. for two honrs on each of three days after being thoroughly washed in many changes of distilled water. They were then stained in the same way as the blood corpuscles, To avoid the formation of clot the blood was collected in a mixture of saline and sodium citrate. Formalin was then added. Alcohol could not be used at any stage of treatment after fixation, nor could heat be employed in staining. The staining was carried out in bulk with an excess of Ehrlich's acid haematoxylin for three days in the cold. The suspension was then washed repeatedly in successive changes of distilled water till the supernatant fluid was quite clear and colourless, finishing the process with a t least three changes of normal saline before use. Stained in this manner, both bacteria and corpuscles could be recognised as indigo-coloured bodies of definite shape in the glands, while both blood and tissues remained free of stain. The difficulty of preventing aggregations and sedimentation in the peritoneal cavity, two hours being allowed for each experiment, was overcome by heating the suspension to 3'7.5" C., just before the experi- ment, and adding a sufficient volume of a 20 per cent. gelatine solution to bring the gelatine content of the suspension as used up to 2 per cent. With this method it was found that a t the end of two hours the suspension was still quite good. A certain amount of fine amorphous blue precipitate was always present with the corpuscles, but this in no way interferes with the result. The glands were counterstained with neutral red."

The micro-organisms passed through the diaphragm in smaller quantity than the lamp black and did not choke up the peripheral lymph channel of the lymphatic glands, but were found uniformly scattered about the periphery of the gland in fair numbers. The blood corpuscles occurred in small masses here and there in the peripheral lymph channel and in places in the lymph sinuses.

CONCLUSIONS.

1. The peritoneal cavity is drained principally by the diaphragmatic lyiiiphatics into the niediastinal lymphatics passing through the sternal and anterior mediastinal lymphatic glands to the right lymphatic duct, and also through anastomoses in the chest to the thora.cic duct.

2. It is drained also by the diaphragmatic lymphatics into the cisterna chyli, but this path is quite subsidiary to the former.

3. It is probably also drained to a small extent very slowly by the retroperitoneal lymphatics into the cisterna chyli.

4. Particles easily pass between the endothelial cells with the lymph, the limit of size of such particles being approximately that of the red blood corpuscles of the animal used.

ABSOlZPTlON 3 R O M PERlTONEAL CA V l T Y 445

Probably only the finest particles pass directly into the cisterna chyli and then very slowly.

5. The drainage is accomplished by a purely mechanical process, the Iorce being supplied by the respiratory movements.

6 . Colloidal dyes, which are indiffusible outside the body, pass through the peritoneum and capillary wall by diffusion directly into the blood, but slower than crystalloids. If colloids o€ a larger molecular weight are able to pass through, they must do so very slowly and in small quantity, but it is probable that molecules of the complexity of those of albumins are unable to do so.

7. Poisonous or other substances, whether formed by bacteria or otherwise, which are indiffusible through an artificial membrane, may still be directly absorbed into the blood from the peritoneum, provided that they are not of great molecular complexity, otherwise they will be slowly absorbed by the lymphatics in accordance with their position in the peritoneum.

BEFEIXENCES.

1. BOLTON . . . . . . .

2. STARLINGANDTUBBY . .

4. ORLOW. . . . . . . . 3. LEATIIES AND STARLING .

5. v. RECKLINGHAUSEN . . . 6. SCHWEIGGER - SEIDEL AND

DOGIEL

SEIDEL 7 . LUDWIG AND SCHWEIGGER-

8. TOURNEIJX . . . . . . 9. RANVIER . . . . . . .

lo. KOLOSSOW . . . . . . 11. MUSCATELLO . . . . . 12. HERTZLER . . I . . . 13. MACCALLUM . . . . . 14. BECK . . . . . . . . 15. DURHAM . . . . . . .

17. DANDY AND ROWNTREE . . 16. BUXTON AND TOILRY . . .

18. ’FBIELE AND EMULETON . .

Journ. of Path. and Bact., 1904, vol. ix. p. 67 ; 1910, v d . xiv. p. 40 ; 191G, vol. xx. p. 290.

h w n . P/~.?/siol., 1894, vol. xvi. p. 140. .7ozcr.n. Physiol., 1895, vol. xviii. p. 106. Arch. f. d. Ges. physiol. (Pfliiger), 1895, Rd. lix.

Vi~ch. Archiw., 1863, Ed. xxvi. S. 172. Arbeiten a. d. ph?lsiol. Anst. z u Leipig, 18GG,

S. 170.

s. 68. Arbeiten a. d. physiol. Anst. zu Leipig, 1866,

8. 174. Jozcrn. de l’anat. et de la physiol., 1874, vol. x.

‘‘ Trait@ Technique d’Histologie,” 1875, p. 385. Biolog. Centralbl., 1892, Bd. xii. S. 87. Virch. h-chiw., 1895, Bd. cxlii. S. 337. Trans. Am. Micr. SOC., 1901, vol. xxii. p. 63. Anat. Am., Jena, 1903, Bd. xxiii. S. 157. TVien. lclin. Wchnschr., 1893, Bd. vi. 5. 823-24. Journ. Path. and Bact., 1897, vol. iv. p. 338. Journ. M P ~ . Rfsearch, 1906, vol. xv. p. 3. Xeitr. z . Iclin. Cl~ir., 1913, Ed. lxxxvii. S. 539. l’roc. Roy. Soc. Mfd. (Path), 1914, vol. vii. p. 69.

p. 66.