The methodology of lymphatic anatomy studies in a cadaver model: an overview

Dissection of living animals - discovery of the lymphatic system

Discovery of the lymphatic system is credited to the Italian anatomist Aselli^[10][Figure 1]. When Aselli was asked by his colleague to demonstrate the recurrent nerve in a living dog, he noticed by chance that there were many white cords running in the mesentery. Initially, he thought that these cords were nerves, but when he cut them with scissors, he observed milky liquid (lacteal) gushing from the vessels. Aselli attempted to reproduce his findings on another day, but could not find the same type of structure. He then realised that these structures correlated with the absorption of nutrients from the small intestine, because being able to see the vessels depended on the timing of feeding^[11].

Figure 1. Aselli’s^[10] publication in 1627. This image shows white lacteal cords in the dog mesentery (right)

Aselli’s findings were magnificent and shed light on a new body system. However, the dissection of living animals had limitations for further investigation and led him to the misconception that these vessels connected to the liver, rather than the thoracic duct.

Mercury injection

Malpighi used mercury to observe the arterioles, because he knew mercury was a slippery agent that could penetrate into smaller vessels^[12]. Nuck^[13] made an amalgam by mixing mercury with tin and lead. He used this mixture to identify the lymphatics and his illustrations of the lymphatic vessels are very well detailed [Figure 2]^[13]. Mercury injection became the standard technique for anatomical investigation of the lymphatic system for the next three centuries^[14].

Figure 2. Nuck’s^[13] publication in 1696. Mercury injection was applied to the lymphatic vessels in the female reproductive organs (right)

Mascagni^[15] was one of the anatomists who used mercury injection and published his extensive studies in 1787^[16]. His contribution to the field is not only his book containing detailed anatomical illustrations, but also the life-size wax models he created. Mascagni supervised modellers Felice Fontana and Clemente Susini in creating wax models for anatomical teaching^[17,18]. These masterpiece models are well preserved and are still on display at the La Specola Museum in Florence and Josephinum Museum in Vienna. Sappey^[19] also used the mercury injection technique and published his findings in 1874 [Figure 3]. His book provided a comprehensive understanding of the human lymphatics, but his superb illustrations were more of artistic than scientific value. Sappey was probably the first anatomist to understand the idea of lymphatic territories defined by a watershed at the midline and a horizontal line crossing the umbilicus in the torso.

Figure 3. Illustration from Sappey’s^[19] book showing lymphatic vessels in the upper extremity identified using the mercury injection technique

To date, anatomical findings with mercury injection were the mainstay of our understanding of the lymphatic system. Anatomists injected mercury directly into the lymphatic vessels or indirectly into the skin in cadaveric specimens using stretched glass tubes or fine needles. The excellent illustrations made by the early anatomists led to the belief that mercury was an ideal material for demonstrating the lymphatics, but it has several downsides. Firstly, mercury is a toxic substance and its use in anatomical investigation was discontinued in the early twentieth century due to concerns about health issues. Secondly, mercury was an imperfect tool for identifying the lymphatics. A young male cadaver that had been studied using a mercury injection in the 18th century, possibly by Alexander Monro the Second (1733-1817), was stored at the University of Edinburgh^[20]. When this cadaver was later submitted to radiographic investigation, the radiographs revealed that the mercury had entered not only the lymphatic vessels, but also the veins^[21]. This finding suggests that the anatomists doing the illustrations must have spent a lot of time and effort in manually extracting the information about the lymphatics to create their drawings. Finally, the mercury injection technique was limited in the number of lymphatic vessels that could be identified in each specimen. Hence, Sappey needed to combine findings from multiple specimens to compose a single diagram.

Despite these several downsides of the technique, the painstaking efforts made by pioneer anatomists who used mercury injection succeeded in establishing the solid foundation of lymphatic anatomy for three centuries, and our current knowledge still relies heavily on their achievement.

Dye injection

As a potential replacement for mercury injection, a dye injection method for demonstrating the lymphatics was developed by Gerota^[22] in a technique known as Gerota’s method. Gerota’s original medium was composed of Prussian blue diluted with turpentine and ether and his method was used by Rouvière^[23] and Bartels^[24] and contributed to their publications [Figure 4].

Figure 4. Illustration from Bartels’^[24] book showing lymphatic vessels in the upper extremity identified using Gerota’s method

While studies using mercury injection used mainly adult cadaver specimens, Gerota’s method required the use of foetal and child cadavers. The reason for this is that the slippery nature of mercury allowed it to travel long distances inside the lymphatic vessels, but the dye used in Gerota’s method could only travel much shorter distances. To overcome this limitation, anatomists began using smaller bodies, but the difficulty of acquiring a steady supply of foetal specimens became a barrier to anatomical study and was the major factor in the decline of lymphatic anatomy research in the twentieth century. Foetal studies using Gerota’s method still continue at the Department of Anatomy at the University of Buenos Aires, where the method is combined with the Spalteholz technique to render tissues translucent^[25].

Microinjection technique with hydrogen peroxide

A new technique to investigate the lymphatic system in adult cadaver specimens without using mercury was developed by Suami et al.^[26,27]. In contrast to arteries and veins that contain red blood cells, transparent lymphatic vessels cannot be identified with the naked eye post-mortem because they collapse. Initially, patent blue violet was injected into the cadaver to identify the lymphatic vessels. The dye succeeded in demonstrating some lymphatic vessels, but it also stained the surrounding tissue and prevented any further investigation. After this, hydrogen peroxide was used and found to be an ideal substance to distinguish the lymphatic vessels from the surrounding soft tissue without any contamination. When hydrogen peroxide is injected into the skin and subcutaneous tissue around the area of investigation, its reaction with tissue enzymes produces fine bubbles of oxygen. These bubbles inflate the lymphatic vessels so they can be identified under a surgical microscope. When the location of the vessels has been determined, a fine stretched glass tube or needle is directly cannulated into them and a contrast agent (a radiocontrast medium or dye) is injected to stain them with colour. Once coloured, the vessels can easily be dissected out from the soft tissue. The whole procedure is performed under a surgical microscope and therefore has become known as the microinjection technique. Compared to both the mercury injection technique and Gerota’s method, the microinjection technique requires more dextrous skills in manipulating lymphatic vessels under the microscope.

When a radiocontrast medium is selected as the contrast agent, the prospective radiographic images are similar to those produced by lymphoangiography in live subjects. Lymphangiography is an invasive procedure requiring a small surgery consisting of cutting the skin and cannulating a fine needle into a lymphatic vessel under local anaesthesia^[1]. Usually, only one lymphatic vessel is cannulated. In contrast to lymphoangiography, the microinjection technique in cadaver specimens has no limitation on the number of lymphatic vessels that can be identified, so has the advantage of allowing the researcher to obtain a comprehensive picture of the lymphatics in each specimen. The disadvantage of the microinjection technique is that the contrast medium stops at the sentinel node, so further cannulation in the efferent vessel of the node is essential if the proximal areas are to be investigated. However, the invention of the microinjection technique enabled anatomists to undertake the study of lymphatic anatomy in adult cadavers and contributed to better lymphatic mapping of the body regions^[28-31].

Indocyanine greenlymphography

ICG lymphography was initially developed to map the sentinel lymph nodes for breast cancer treatment^[32]. Its application was then expanded to provide imaging for the diagnosis of lymphoedema^[4-6]. ICG is a water-soluble agent that emits near infrared-rays when it combines with protein in the human body. When ICG is injected into the skin, it automatically enters the lymphatic vessels. The infrared camera then scans the limb and captures the emitted signals, causing the vessels to glow green so that they can be identified in real time to a depth of 2 cm from the surface of the skin.

The authors injected ICG into fresh cadavers and confirmed that it was able to identify the lymphatic vessels in post-mortem specimens^[33,34]. ICG lymphography was found to be consistent in identifying the lymphatics and the dye was able to travel long distances from the injection site if fresh, non-frozen cadavers were used. The technique has demonstrated several advantages in anatomical study in a cadaver model. Firstly, lymphatic vessels can be identified without a skin incision, so ICG injections do not disrupt the embalming process to follow and the bodies can be reutilised for anatomical dissection after study of the lymphatics. Secondly, ICG mapping has helped make the microinjection technique more effective, as it provides a more efficient way of locating the lymphatic vessels than injecting hydrogen peroxide, the most time-consuming part of the process. Finally, it has enabled fast mapping of the course of lymphatic vessels and allowed the capture of imaging data from multiple cadaver specimens^[35].