This Methodology section is an excerpt from The Lymphatic System of Cattle, Pages 3-10, as I examined the canine lymph nodes, their drainage areas, and their efferent duct behaviour using the techniques described in that publication. References for this chapter are included below.

Regarding the technique for macroscopic visualization of the lymph vessels, I did not limit myself to a single method alone, although by far in most cases, and for most organs, I used the puncture injection method in principle as described by Gerota [9].

For the injection liquid, I used the following solution: Prussian blue oil paint or Berlin blue oil paint, which are available in tinfoil tubes, placed in a crucible, then rubbed with pure turpentine oil until a uniform and thin paste is formed. The amount of turpentine oil is subjective – I don’t use as much as Gerota specifies (2 g of oil paint and 3 g of turpentine oil), but rather the other way around: 3 g of paint and 2 g of turpentine oil. Then, around 3 to 5 times the amount of ether is added, and the mixture is filtered through window cleaning leather (deerskin), not through canvas. For a syringe, I use the Record^[1] syringe  (Rekordspritze) with metal cannulas as recommended by Bartels [1], however, based on my experience, I believe that the construction of the syringe does not matter, and this belief is supported by the fact that almost everyone who has dealt in depth with lymph vessel injections has either invented or constructed their own injection syringe.

What is important, in my experience, is: 1. the use of cannulas that are as narrow as possible and 2. the greatest possible practice and patience. The narrower the cannula, the more evenly the pressure exerted by the injecting force (hand) will be, and the more evenly and gradually an increase in pressure is possible. This is very important with puncture injections. The lower the pressure initially applied and the more evenly it can be exerted and increased, the lower the risk of extravasation at the puncture site, and thus the greater the likelihood of injecting the lymph vessels. I used metal cannulas that were only 0.29 mm thick, which I was unable to obtain for a long time, and which I only received after great efforts by the mechanic of the veterinary university in Dresden, Mr. Eugen Albrecht. Metal cannulas have a great advantage over glass cannulas in that they do not break off as easily when penetrating solid tissue, and in that they can still be used even with very firm tissue (e.g. the bone tissue of young animals or the cartilage tissue), while glass cannulas cannot be used at all for these tissues.

Of course, it is not only the use of narrow cannulas that lead to success, but also skill, practice, experience, and other small tricks that will be mentioned in the individual chapters – even indefinable imponderables may play a role. For instance, after my first unsuccessful attempts to inject fascia lymph vessels, I almost threw the musket into the grain^[2], because I no longer believed in success, until I finally succeeded in an injection. All at once, success was just as frequent as failure had been before, although I was unable to identify what I was doing differently.

In addition to puncture injection, I also used other methods. I successfully filled the lymph vessels of the tonsils by rubbing the coloured liquid onto the surface of the tonsils with a blunt object (scalpel handle, leather, etc.), and the lymph vessels of the joint cavities by introducing the coloured liquid into the intact joint cavity and then mimicking the natural movements of the joint for different lengths of time. The lymph vessels of the tendon sheaths can be filled even more easily, and it is only necessary to introduce the coloured liquid into the intact tendon sheath and to massage the filled tendon sheath lightly in order to inject the lymph vessels very nicely in a very short time. I filled the lymph vessels of the intestine not only by puncture injections, but also by introducing so much coloured liquid into a piece of intestine that had been tied off twice that it was completely full of liquid. The piece of intestine was then massaged until its lymph vessels were filled, which often happened very quickly after some length of massage. The lymph vessels of the uterine mucous membrane can be demonstrated using a similar method. Finally, in addition to puncture injections, the lymph vessels of the parietal pleura and the parietal peritoneum were filled by injecting the coloured liquid into both the pleural and peritoneal cavities and then using a special, albeit simple, pump to imitate the animal’s respiratory movements for a longer time. All these methods, the details of which are to be described in the respective chapters of this book, will eventually lead to the goal, and one must not be discouraged by initial failures.

I would like to draw your attention to another special method of injecting lymph vessels, which, given its great simplicity, is of particular value, because it enables injection of the lymph vessels of those sites and organs in which the other methods, specifically the puncture method, fail, such as the softer organs, adipose tissue, muscles (especially the eye muscles), etc. In such sites and organs, assuming the lymph vessels begin with small openings or equivalent structures, I placed coloured dye in the extravascular space using a low-grade injection and then lightly kneaded and massaged the area until the lymph vessels filled. In principle, this is the same method that I used in the aforementioned methods to demonstrate the lymph vessels of the tonsils, joints, tendon sheaths, intestines, pleura, and peritoneum, in that all of these methods are based on lymph vessels starting with small openings or physiologically equivalent structures, and that the coloured liquid is mechanically pressed or pushed through the openings into the lymph vessels. One could even explain the success of puncture injection in this way. As already mentioned, I have used this simple method with great success where other methods, including the puncture method, failed, especially with muscles (primarily the muscles of the globe), but above all with the renal fat, where I could only manage to fill the lymph vessels using this method. As far as I know, this method has not been published, only referred to in a statement by Oelsner [13]. Oelsner tried to inject the lymph vessels of the pectoralis major muscle by puncture injection, but did not succeed. He stated “but if small amounts of Gerotaschen dye [9] were placed extravascularly in the muscle, and then a thin spatula was used to distribute the dye by gentle stroking, the small vessels running parallel to the muscle fibers filled with the dye every time, and these vessels then filled a fine network of vessels on the sternum, confirming that these were indeed lymph vessels. These lymph vessels perforated the intercostal spaces close to the sternum on the side opposite the muscle, accompanying the blood vessels, and drained to the sternal lymph nodes”. This information came to my attention only later, after I, independently of Oelsner, had often used the method I describe with success.

I carried out my examinations only on fresh bovine cadavers, most of which I obtained from the Dresden slaughterhouse, and used for injections until putrefaction started. Based on the extraordinarily large number of injections I carried out, I agree with Bartels [1] that the injections generally work best on organs that are as fresh as possible, or still warm. In contrast to Bartels, however, I found that most organs don’t need to be warm to achieve a successful injection, and that it does not matter if the organ is fresh or a few days old. There are some individual organs that are an exception to this statement, however. Based on my observations, lymph vessel injections in the liver are much easier when the organ is as fresh as possible as opposed to when it is a few days old. In addition, when injecting the lymph vessels of muscle, one must of course not do so during the rigor mortis period. For most organs, the age of the animal also does not affect the success of the injection, that is to say, injection is just as easy in adult cows as it is in calves. There are, again, some individual organs that are an exception to this. The articular lymph vessels of young animals could be injected more easily than in old animals, and the bone lymph vessels were easy to inject only in young animals. The same applies to the cartilaginous lymph vessels, especially those of the larynx cartilage, in which injection was only successful in very young animals. If the lymph vessels were injected and their examination could not be completed quickly, the organs were then wrapped in cloths soaked with formalin solution in order to delay the onset of putrefaction. Some of the injected preparations were preserved in a 4% formalin solution.

I will use limited references in this book, since, as already mentioned, the general literature on lymph vessels is not applicable to my work. The comparative anatomical literature is also of little use, since, as will be described elsewhere, both lymph node groups and lymph vessels in the individual animal species show such great differences that comparisons between species are difficult to make, and the findings in one animal species do not apply to another species. Instead, it is necessary to examine and describe the lymphatic system of each animal species individually, if the descriptions are to be correct and valuable for practical applications. Based on this notion, only the literature that refers to the lymphatic system of cattle could be useful to my work. However, there is a limited amount of these references and almost all of them are incomplete, therefore making the existing literature of little value to my work. This even applies to information in textbooks on veterinary anatomy and meat inspection. A reference of purely historical interest is included: Duvernoy [8], Haller’s teacher, is said to have shown the entire lymphatic system of a cow. Unfortunately, however, nothing has been handed down about his technique.

Terminology and phrasing

As far as terminology, I endeavoured to retain the previous names of lymph node groups as much as possible, and for purely practical reasons have done so in isolated cases even where another name would undoubtedly have been more correct. In other cases, however, the previous names could not be retained for reasons that will be given in the relevant sections. Many lymph nodes or groups of lymph nodes had to be renamed because they had previously been described either only very inadequately and without specific naming, or not described at all. In many cases, I have had to divide the individual lymph node groups into subgroups and give them special names, because otherwise an exact description of the afferent and efferent pathways of individual lymph nodes and the relationship of lymph vessels of individual organs to the lymph nodes would not have been possible.

I have also endeavoured to name individual lymph nodes and lymph node groups as closely as possible to the corresponding lymph nodes and lymph node groups in humans and in accordance with the definitions of the Basle Nomina Anatomica (i.e. to use a valid, comparative anatomical name as a basis). However, this approach could only be followed for a relatively small number of lymph nodes and groups of lymph nodes, and not for the majority. The individual lymph node groups are so extraordinarily different in individual animal species in terms of location, drainage area, occurrence in general, number of nodes, etc., that a valid, comparative anatomical name is not possible for many. For the same reason, the classification of the lymph nodes is very uncertain and will always depend on the author’s individual opinion.

I don’t know of a single, decisive system by which individual lymph nodes and groups of lymph nodes could be named. It would undoubtedly be more correct to name the lymph nodes after their drainage area. However, even if this approach were attempted for a single animal species, it would still not be possible because, almost without exception: 1. the lymph vessels of individual organs drain to different lymph nodes and 2. the individual lymph nodes or groups of lymph nodes receive lymph vessels from different organs, as shown by the study of the lymph vessels of the organs and the drainage areas of the lymph nodes. For example, of the lymph nodes that drain the lymph vessels of the lungs, some also additionally drain the lymph vessels of the heart, while others also additionally drain the lymph vessels of the diaphragm and esophagus. Another example is the lymph vessels of the diaphragm, which drain to at least 4 different lymph node groups; these lymph nodes would be impossible to group together given the differences in the remaining areas that they drain. Therefore, one is forced to name the lymph nodes and groups of lymph nodes according to their location and, as far as possible, to take their drainage areas into account. Naturally, this classification and naming of the lymph nodes remain inadequate and unreliable, and individual lymph node groups will always be inconsistently named by different investigators, depending on whether the lymph vessels and thus the drainage areas of the lymph nodes of the particular animal species have been studied in detail or less closely examined. One can already see these differences in the description and naming of human lymph nodes, even though, at this time, the human lymphatic system is the best and most precisely known. Almost every investigator suggests different nomenclature (for example, see the chapter on the naming and classification of human bronchial lymph nodes in the work by Bartels, page 175 [1].

It is even more challenging to compare the lymphatic system of several animal species, because, as already mentioned, the individual lymph node groups in different species are not at all similar in terms of location, occurrence and drainage area. Therefore, one must describe the lymphatic system of each animal species individually, albeit while preserving the comparative anatomical aspects as much as possible. I therefore cannot agree with Bartels, pages 36, 68 and 69 [1], that in principle there is agreement in the location of the lymph vessels and lymph nodes between mammals and humans, and that the only difference is the greater number of lymph nodes in humans.

If there are 1 – 2 or 1 – 3 lymph nodes that are associated with each other, or part of a group, it can be difficult to decide whether the lymph nodes should be referred to in the singular or plural, and even after careful consideration this often remains a matter of opinion. I approached such cases in the following way: if there is only one lymph node, which can be double in the minority of cases, then I speak in the singular (e.g. mandibular lymph node, lateral retropharyngeal lymph node).  If there are 1–3 or more lymph nodes, then I speak in the plural, although such groups may often include only 1 node. I have consistently replaced the earlier, outdated, and unjustified term “lymph glands” with the more correct term “lymph nodes”; consequently, the Latin name “Lymphoglandula” should be replaced by “Lymphonodus“. However, I did not decide to change the Latin names, because the term “Lymphoglandula” has been adopted by the Basle Anatomical Nomenclature and is accordingly still used in all anatomical writing at this time.^[3]

Considering the above, I did not include the general behaviour of the lymph nodes; for this information refer to the chapter I published previously: Histology of the Circulatory Organs in Ellenberger: Handbook of the Comparative Microscopic Anatomy of Domestic Animals, Berlin 1906–1911. I have only included descriptions of behaviour when my findings contradicted previous information and views, or when the behaviour has not been previously described. My findings are supported by the numerous investigations I performed on a very large number of animals.

The size of the bovine lymph nodes fluctuates within the broadest limits; from the size of the head of a pin up to 1 meter and 20 centimeters in length, although the vast majority of lymph nodes do not exceed a size of 5 cm and the lymph nodes exceeding 10 cm in length are found almost exclusively in the mesentery of the small intestine. In calves 3 to 6 weeks in age, the lymph nodes are about half the size of those in adult animals.

The shape of lymph nodes in cattle is variable, with no consistent shape. In most cases, the lymph nodes are irregularly rounded or oval, and at the same time somewhat compressed, so that they do not appear quite as thick as they are long and wide. Frequently, the lymph nodes are long, even up to 1.2 meters in length. I found other shapes frequently; these included curved, even horseshoe-shaped lymph nodes, ring-shaped constrictions, local thickenings and other irregular shapes. A hilus is observed in approximately 70% of lymph nodes, and in elongated lymph nodes is usually in the form of a longitudinal groove on one side of the lymph node.  Occasionally there is a partial fusion of two adjacent lymph nodes; I observed this especially in the jejunal lymph nodes.

According to my observations, the number of efferent vessels of individual lymph nodes fluctuates between 1 and 12, and in most lymph nodes between 1 and 3. The largest number of efferent vessels were observed for the parotid lymph node and the long caudal mediastinal lymph node and the long mesenteric lymph nodes; for very long mesenteric lymph nodes, the number of efferent vessels can be considerably larger, for example, in the case of a 23 cm long node, 43 efferent vessels were counted.

The number of afferent vessels is very difficult to determine because the entire drainage area of ​​a lymph node cannot be injected during one examination but is certainly greater than the number of efferent vessels: on average, about twice as large. Due to limited time examining cattle, I did not try to quantify the influence of age on the macroscopic behaviour of the lymph nodes, but I believe that Merzdorf’s [12] finding in dogs, that the lymph nodes of older animals are relatively smaller and lighter than the lymph nodes of younger adult animals, also applies to cattle.

I cannot say with certainty whether individual lymph nodes can completely disappear in old age, but I do not believe that this is the case, and it certainly is not a common finding. As far as the related question, whether new lymph node formation can occur during life, I cannot provide a definite answer. Based on my work involving extirpation of lymph nodes, which I will report in detail later, I can state that after extirpation of lymph nodes, new lymph nodes can form either in the same or in another, more distant location. These observations agree with those of Bayer [2, 6] and Ritter [7, 14], who also observed the formation of new lymph nodes after extirpation of lymph nodes in dogs.

Regarding the location and number of the lymph nodes, it can generally only be stated that the lymph nodes are located individually or in groups at certain locations in the body and that the number of lymph nodes in cattle is generally small (compared to humans and horses) and that the individual lymph nodes are relatively large. While the individual lymph node groups in the horse consist of many small nodes, in the cow the groups consist of a few, relatively large nodes. General statements on the location of the lymph nodes in cattle cannot be made. It is certainly not possible to state that lymph nodes are very often located in the bifurcation angles of veins, as Bartels [1] observed for human lymph nodes; this applies to only 10% of all lymph node groups in cattle.

The lymph nodes that receive dye after injection of an organ are referred to as regional, or corresponding, lymph nodes of that organ; conversely, one can say that the organ is a tributary to these lymph nodes.  According to the prevailing view, however, a group of lymph nodes is never regional for just one organ, and rarely is an organ tributary to a single group of lymph nodes [1]. Based on my many investigations and observations, I can generally agree with this view, but not explicitly. I found that there are quite a few organs (e.g. many muscles, tendons, tendon sheaths, joints, bones, mammary glands, testes, ovaries, etc.) from which lymph vessels drain to only one group of lymph nodes; in other words, they are tributary to a single group of lymph nodes. I also found groups of lymph nodes that did drain just one organ, for example, as far as I can tell, the pericardial lymph nodes receive efferent vessels only from the pericardium.

My investigations into the general behaviour of the lymph vessels also led to both new information and corrections of previous information. This applies to the occurrence of lymph vessels. For a long time, as a result of unsuccessful injections, it was assumed that many organs were without lymph vessels (even bones, joints, tendons, tooth pulp, etc.), but we now know beyond doubt that most of these organs have lymph flow (see the relevant chapters). There are, however, certain tissues and organs in which lymph vessels have not yet been proven with certainty (e.g. epithelia, cartilage, the membranes of the eyeball and the central nervous system), while in other organs (e.g. tendon sheaths and fatty tissue) the existence of lymph vessels has not been investigated. Bartels, page 68 [1], is of the opinion that lymph vessels exist in all organs and tissues supplied by blood vessels, and that only in tissues and organs which are completely lacking blood vessels would lymph vessels be absent. Only future investigations will determine if this opinion is correct. Based on my investigations, I can only say that the cartilage of cattle (specifically the larynx and tracheal cartilage), i.e. organs without blood vessels, have lymph vessels, at least insofar as I could easily inject lymph vessels from this cartilage using a puncture injection technique. On the other hand, I have not succeeded in detecting lymph vessels in the parenchyma of hemal lymph nodes and in the parenchyma of the spleen, i.e. organs which undoubtedly have blood vessels, although that could be due to the special behaviour of the blood vessels of these organs.

An important law, which Bartels calls the circuit, or switching, law, is that all lymph vessels (with the exception of their end branches) pass through at least one, and usually several, lymph node(s) before they terminate in the venous system.  According to my investigations, this law applies to the overwhelming majority of lymph vessels, but there can be isolated exceptions to the law. In addition to the 2 to 3 such exceptions described in the literature, I can add 4 additional exceptions; I clearly saw one lymph vessel each from the heart and pericardium of the cow drain directly to the thoracic duct, and two lymph vessels from the adrenal glands drain directly to the cisterna chyli (for full details see [4]). It must be acknowledged, however, that these 4 lymph vessels were the only ones among the thousands of lymph vessels that I viewed and examined, and that they all came from organs that lie in the vicinity of the thoracic duct and the cisterna chyli.

Another related question is whether lymph vessels (apart from the large terminal trunks of the lymphatic system) can open directly into veins. This question has always been a controversial one, and for a time it appeared that authors were equally divided on the question. Recently, Bartels [1] summarized the existing literature and passed a devastating judgment on those authors who, even today, want to accept the possibility of a direct confluence of lymph vessels and veins. After the publication of Bartels’ summary, Sylvester [16] examined whether lymph vessels drain into veins. He observed the lymphatic system of 30 monkeys belonging to 6 genera of South American monkeys and found that, without exception, both the intestinal trunk and the lymphatic trunk of the pelvic limbs opened into the venous system at the level of the renal veins, namely into a renal vein or into the caudal vena cava. Huntington [10] made similar observations on Macropus rufus. Based on my extensive investigations and observations, I have also commented on this question and explained my position in detail in an article in Anatomischer Anzeiger [5]; I refer the reader to this article, and only emphasize here that I have made several impeccable observations that argue in favour of lymph vessels flowing directly into veins; I have observed that 1. rarely, lymph vessels can flow into the thoracic duct and the cisterna chyli without having passed a lymph node, and thus drain directly into the venous system (see above), and 2. that the efferent vessels of lymph nodes, including those that are far from the thoracic inlet, can open directly into veins. I have definitively observed several of these cases. Other cases that I have examined and described in the article mentioned above support at least the possibility or probability that lymph vessels can open directly into veins.

In most cases, however, it is not possible to prove beyond a doubt that an injected vessel is truly a lymph vessel, for if an injected vessel opens into a vein, the objection is always raised that it was not a lymph vessel but instead a small vein that was filled during the puncture injection. Given our current knowledge of the differences between small veins and lymph vessels, especially when they are filled with blue dye, this objection certainly cannot be refuted and therefore the question of whether lymph vessels can flow directly into veins cannot be proved using this methodology. I will not include a detailed discussion of whether a microscopic examination in each individual case would help determine if the vessel in question is a very small vein or a lymph vessel. I attempted to do this, however, when a vessel is filled with blue injection material it loses all features that distinguish lymph vessels from veins (i.e. the nucleated epithelium of the blood vessels that lymph vessels should be without [15], or the much thinner epithelium of the lymph capillaries compared to blood capillaries [11]). Even on examination of macroscopic behaviour, one cannot always decide whether the vessel is a vein or a lymph vessel with certainty, even if the observer has extensive experience in lymph vessel behaviour. Even though in most cases one can recognize injected lymph vessels by their unequal lumen, which is often constricted or bulging, their very thin walls and winding path, which is more meandering than that of comparable-sized veins, lymph vessels cannot always be distinguished from veins. Even very experienced investigators will not always be able to determine with certainty whether a vessel is a lymph vessel or a small vein.

Regarding further the behaviour of lymph vessels relative to blood vessels, most textbooks and special publications state that larger lymph vessels join blood vessels, interweaving with them to form meshes. I cannot agree with this opinion either. I believe it is one of those statements, of which there is certainly quite a number in the anatomical literature, that has been established based on limited findings and then, without being critically examined, continue for a long time in the field of anatomy. According to my observations, the statement does not technically apply to the lymph vessels of any organ, although it must be acknowledged that in some organs most of the lymph vessels run with the veins. It perhaps can be applied to the lymph vessels of the testicle, and those of the ovary and uterus, stomach, intestines, etc., but for the lymph vessels of most organs the statement does not apply. For details, see the relevant descriptions of the lymph vessels of the skin, the muscles, and other organs. The fact that some lymph vessels accompany blood vessels is obvious, but this applies only to the minority of all lymph vessels.

When I state that lymph vessels drain into a certain lymph node, there is always the possibility that in an individual case they actually bypass the lymph node and join the efferent vessels leaving the lymph node.

When assessing vaccinations, the spread of infectious substances, etc., it is important to consider that lymph vessels can pass across the midline and drain into lymph nodes of the contralateral half of the body. These midline crossings, which are particularly observed in the lymph vessels of organs adjacent to the median plane, have long been known and described in humans. I have also been able to detect them in cows, to an even greater extent than has been described in humans, and have compiled the relevant findings in a special article [3], the main content of which is as follows:

In the cow, there are lymph vessels that cross the median plane and drain to lymph nodes in the other half of the body. This applies in particular to the lymph vessels of the median parts of the outer skin, the tip of the tongue, the hard palate and gums, the lower lip, the thyroid, the trachea and esophagus, the lungs, the thymus, the kidneys, M. longus colli and the efferent vessels of the mammary lymph nodes.

Lymph vessels are known to exhibit an abundance of anastomoses and thus form networks. This applies in particular to the initial parts of lymph vessels and to the smallest lymph vessels; these form rich networks in almost all parts of the body, in almost all tissues and organs. However, the larger or coarser lymph vessels and the lymphatic trunks form fewer networks because they anastomose less frequently; in addition, the anastomoses in the larger vessels arise mainly when several lymph vessels merge, and much less often when the lymph vessels divide, and their sub-branches connect with neighbouring lymph vessels. I would particularly like to emphasize this point, because I suspect that in general it is mistakenly assumed that there is abundant anastomotic and network formation for the larger lymph vessels and the lymph vessel trunks. My illustrations, which are as true to life as possible, provide the best information about this behaviour. While larger lymph vessels divide, possibly even repeatedly, it can easily be proven that this does not occur as often as is generally assumed.

Also, the statement contained in most textbooks: “There is no communication between the lymph vessels of two anatomically separated organs” is not supported by my investigations, because such communications can occur, although they are rare (see lymph vessels of the pancreas and duodenum).

Under normal conditions, the lymph contained in lymph vessels, or the contrast injected into the lymph vessels, flows only in one direction, namely centripetally, due to the complete functioning of the valves. When injecting a cadaver, however, exceptions to this rule often occur, and retrograde filling is observed. I have often observed lymph vessels filling backward to the next lymph node, especially from the thoracic duct. I have also observed afferent lymph vessels filling backward from a lymph node after injection of contrast material into different afferent lymph vessels. It is recognized that in living animals retrograde metastasis can occur under certain circumstances.

Finally, a word about the very controversial question of whether lymph vessels begin with open stomata. The question of the origin of lymph vessels has been answered differently at different times, according to advances in our knowledge of physiology and to the perfection of technical aids. I do not want to elaborate on this question here but refer to my publication on the histology of the circulatory organs in the Handbook of the Comparative Microscopic Anatomy of Domestic Animals by Ellenberger, vol. II, Berlin 1911, as well as to special publications on histology and the work of Bartels [1]. I will also not discuss the theory that lymph vessels start with tiny openings, the so-called stomata, developed by v. Recklinghausen. I will only mention that, based on the results of my injections, especially the methods I used for injecting the lymph vessels of the tonsils, tendon sheaths, joint cavities, muscles, the pleura, the peritoneum, the intestine and the fatty tissue, I have become firmly convinced that the lymph vessels in mammals (especially in the cow) begin with open stomata or functionally equivalent devices.

Methodology References